乳蛋白与CMV复合启动子驱动hLF cDNA乳腺特异性表达
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摘要
由于高质量生物药品和医学诊断的需求,转基因动物乳腺生物反应器的研究已经成为生物工程领域研究的热点之一;成为高效、高质量生产昂贵生物活性蛋白的有效途径之一。应用乳腺生物反应器生产人的重组蛋白较其他的生物学制备系统有产品活性高、成本低的优点,因而某些人的重组蛋白更适宜用乳腺生物反应器来生产。
外源基因在转基因动物乳腺中特异性表达是转基因动物乳腺生物反应器的根本目的。有许多生物活性蛋白基因在乳蛋白调控序列的指导下已经在转基因动物乳腺中表达,其中应用较多的调控元件有:牛αs1-酪蛋白(bovineαs1-casein)、山羊β-酪蛋白(β-casein)、绵羊β-乳球蛋白(BLG)、小鼠的乳清酸蛋白(WAP)等。尽管已有应用这些调控序列实现乳腺特异性表达成功的事例,但大多数实验研究中,乳腺特异性表达的成功率低、乳汁中的表达水平低。
已经发现某些非乳蛋白调控元件对乳腺特异性表达有明显的作用,如绝缘子、染色质开启子、增强子、核基质附着区和内含子等,其中起到重要作用的是转录和翻译调控因子。近年来,尽管乳蛋白启动子及其它表达调控元件已广泛应用于乳腺特异性表达,但相对低的表达水平和不稳定表达案例也较多。
人巨细胞病毒(CMV)启动/增强子是广泛用于真核细胞表达的调控元件,早期的研究证明有提高转录水平的作用,但未见报道用于激活乳腺特异性表达。本研究应用人巨细胞病毒启动/增强子(Pcmv)与乳蛋白调控元件构建复合启动/增强子和人乳铁蛋白cDNA组成乳腺特异性表达载体。
为了鉴定复合启动/增强子对转基因动物乳腺特异性表达水平的作用,我们构建了单一乳蛋白启动子与乳蛋白/Pcmv复合启动子两种不同类型的乳腺特异性表达载体。应用山羊β-酪蛋白、山羊β-乳球蛋白、αs1-牛酪蛋白为乳蛋白调控序列为乳蛋白调控序列;鸡β-珠蛋白(β-globin,隔离元件)、CMV启动/增强子和SV40polyA为非乳蛋白启动/增强子。以不同的乳蛋白调控序列与启动/增强子组合构建乳腺特异性表达复合启动/增强子(chimeric promoter/enhancer),单一的酪蛋白启动子作为对照,与人乳铁蛋白cDNA组成乳腺特异性表达载体。共构建了7个乳腺特异性表达载体(BnF95,pBnCL14,pBnLC2G,pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36),其中4个为复合启动/增强子型载体,另3个为单一乳蛋白启动子型载体。BnF95、pBnCL14和pBnLC2G载体用于转染山羊乳腺上皮细胞(goat mammary epithetical cells,GMECs);pgCNCG/LF25和pbCNCS/LF36为复合启动/增强子型,其中pgCN/LF/g和pbCN/LF为单一酪蛋白启动子作为对照,用于制备转基因小鼠。
泌乳山羊乳腺组织经Ⅰ型胶原酶消化分离乳腺上皮细胞,DMEM/F_(12)培养液(10%FCS)传代培养,获得的乳腺上皮细胞最多可培养25代以上。采用分步胰酶消化法去除上皮细胞中所含的成纤维细胞,从而乳腺上皮细胞得到纯化。通过电转染法将含NEO~τ标记基因的BnF95、pBnCL14和pBnLC2G外源基因导入23个山羊乳腺上皮细胞系,经G418筛选三周后,得到有抗性的转染细胞株,挑取单克隆细胞株进行增殖培养。共获得242株转染外源基因的细胞,有118株转染pBnCL14,82株转染BnF95,42株转染pBnLC2G基因构件。有3株转染pBnCL14基因的细胞进行nestedPCR整合检测,结果均为外源基因整合阳性。初步认为经G418抗性培养后获得细胞为整合细胞。对抗性培养阳性的细胞株应用催乳素进行诱导表达,收集48和72小时的诱导细胞培养液进行ELISA检测,其中有30株细胞的诱导液中检测出重组人乳铁蛋白,有4株转染pBnCL14基因细胞的表达水平达到10-50mg·L~(-1);另有22株细胞(9株/BnF95,7株/pBnLC2G)表达水平低于10 mg·L~(-1)。通过G418筛选和诱导表达和ELISA检测验证具有表达功能的乳腺上皮将用于山羊体细胞克隆的供核细胞。
pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36四个构件应用胚胎显微注射的方法制备转基因小鼠。应用PCR初步筛选整合小鼠,获得14只原代转基因小鼠,PCR产物序列分析显示转基因小鼠PCR检测产物与模板的同源率为99.95%～99.75,在PCR正常正确率范围以上。原代鼠与C57或ICR正常鼠交配,在出生的后代鼠中检出37只F1代转基因小鼠。获得的转基因母鼠用ELISA检测乳汁中重组人乳铁蛋白,并繁殖传代,获得F1、F2代转基因小鼠。
复合启动/增强子构件指导人乳铁蛋白cDNA在原代转基因小鼠乳腺中重组人乳铁蛋白(rhLF)表达水平(2～8.1g·L~(-1))明显高于单一酪蛋白启动子构件(0～12mg·L~(-1)),约10000倍。在所有转基因小鼠的血清和唾液中未检测(ELISA)出重组人乳铁蛋白(rhLF)。研究表明,酪蛋白/Pcmv复合启动/增强子不仅可提高转基因小鼠乳腺特异性表达水平,而且具有良好的乳腺组织表达定位性。
近年来,对基因表达调控机制提出了一些新的看法,认为几乎所有的哺乳动物基因表达是由多个环节调控的,其中包括了转录、转录后加工、核输出和定位、mRNA分子的成熟、翻译和稳定性等。生物信息学分析显示Pcmv含有多个uORF和CCAAT增强子结合位点。Lodhi等(2003)认为uORF能介导编码mRNA翻译的作用,而CCAAT序列是增强子结合蛋白结合的位点。有研究报道,CCAAT位点结合增强子结合蛋白也可介导功能基因的表达,这一过程可能意味着在5'-UTR顺式调控元件,包括复合启动子中乳蛋白和CMV启动子序列能激活人乳铁蛋白cDNA在培养的山羊乳腺上皮细胞和转基因小鼠乳腺中表达。
本领域的研究报道显示,尽管已有一些利用长片段调控元件获得高表达的事例,但更多的低表达案例使开发那些构建方便、片段长度短小、表达效率和表达水平高的乳腺特异性表达载体显得十分迫切。本研究的结果不仅实现了cDNA功能基因的高水平、高效乳腺特异性表达,而且其载体的构建简单方便。到目前为止,尚未见有应用乳蛋白/Pcmv复合启动子激活cDNA功能基因实现乳腺特异性高效表达的报道。
The use of transgenic animals for mammary bioreactors is a rapidly developing and intensely interesting field due to the increased demand for high-quality pharmaceuticals and medically diagnostic preparations.The manufacture of recombinant human proteins is more profitable by mammary gland bioreactor than that by other biological systems.
The mammary gland-specific expression of a transgene is a major objective for producing heterogenous proteins in the milk of transgenic animals.Bio-active foreign proteins have been expressed specifically in the mammary under the direction of particular milk regulatory sequences,such as:goatβ-casein,bovineαs1-casein,ovineβ-lactoglobulin (BLG) and rat whey acid protein(WAP).However,much of the evidences regarding the mammary as bioreactor indicated that only a small percentage of the mammary glands of transgenic animals expressed functional protein,and most of them were found at low levels in milk.
Several major regulatory elements are required in order to obtain satisfactory transgene expression,such as essential gene insulators,chromatin openers,enhancers,matrix attached regions,and introns.One of the most important elements may be the factor controlling the transgene's transcription and translation.In recent years,although lactoprotein gene promoters were widely used,it should be noted that their expression levels in milk were relatively low and unstable in many instances.
As an alternative to the widely used cytomegalovirus(Pcmv) enhancer for expressing transgene in eukaryon,there was no report for promoting transgenic expression in mammary gland,our aim was to build a series of promoters bearing at least one,or if possible,all of the following characteristics:promoters that would increase high levels of expressing transgene in milk,and expression specifically located in the mammary organs. We used the chimeric promoters/enhancers combining sequences of lactoprotein promoters from cow and goat,with the Pcmv or mono-milk-promoter without Pcmv or SV40ployA to construct vectors for expressing human lactoferrin(hLF) cDNA,their ability to drive high levels of specific recombinant human lactoferrin(rhLF) expression was determined in transgenic mice or goat mammary epithelial cells(GMECs) in vitro.Vectors of mono-promoters of lactoprotein elements were constructed as a control.Seven vectors (BnF95,pBnCL14,pBnLC2G,pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36) with different promoters/enhancers were constructed.Three vectors of BnF95,pBnCL14 and pBnLC2G were constructed by using milk regulatory elements of goat BLG and neomycin resistance marker gene(NEO~r) for transfecting of GMECs;pgCNCG/LF25,and pbCNCS/LF36 were constructed by using promoter enhance sequences of goat casein, bovineαs1-casein,chickenβ-globin insulators,Pcmv and SV40polyA for producing transgenic mice.pgCN/LF/g and pbCN/LF contained mono-promoters of casein as controls.
Primary GMEC lines were derived from biopsy tissue from artificial lactating goats. The cells were cultured in DMEM/F_(12) containing 10%fetal calf serum(FCS).GMECs could keep proliferate vigorously even 25 passages in vitro.The pure GMECs lines could be obtained within 2-3 passages by multi-alternation of digesting and adhering repeatedly. By electroporation,BnF95,pBnCL14 and pBnLC2G encoding neomycin resistance for selecting the positive cells were transfected into GMECs in vitro.After three-weeks-culture by G418(400ng·L~(-1)),the transfected cell colonies grew and proliferated in G418 medium. Single colonies were picked into another 24-well plate for continue culture,and 242 colonies were generated from 118 clonies of GMECs by neo-resistant culture.Three of cell clones from 242 colonies were respectively screened by PCR and each was confirmed to be transgenic colonies corresponded with NEO~r ones,the total 82,42 and 118 colonies were transfected transgenes of pBnCL14,BnF95 and pBnLC2G respectively.These colonies were induced with luteotropic hormone,and then the media were gleaned respectively at 48h or 72h.It was found that there were 30 colonies expressing recombinant human lactoferrin(rhLF) by ELISA at 72h.4 colonies of higher expression level of rhLF were about 50mg·L~(-1) from transgene of pBnCL14.These GMECs of expressing rhLF would be used as donor cells for hircine somatic cell nuclear transfer(SCNT) to produce the transgenic goats.
Four vectors(pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36) were used to generate transgenic mice.A total of 14 lines were selected out of 160 candidates.To verify transgenic integration,these PCR products were sequenced,and the sequences were analyzed using DNAStar software.Compared with correlative transgenes,the percentage of sequence identity was 100 except 99.75 for pgCNCG/LF25 founder line.Founder mice bearing the transgene were mated to C57 males or female and delivered 110 pups,of which 52 progeny were identified as being transgenic.
Using ELISA detection,results showed that the recombinant human lactoferrin rhLF expression was significantly increased in the milk of transgenic mice with chimeric promoter transgenes.The expression level was 2-8.1g·L~(-1) in the milk using chimeric promoter.In contrast,only 0～0.012g·L~(-1) was found in the milk using mono-casein promoter.In two of the highly expressing lines,pbCNCS/LF36 and pgCNCG/LF25,there was approximately a 10,000-fold increase in target protein in the milk of transgenic mice (P＜0.001).In addition,we have also shown that the expression of rhLF was strictly limited to the mammary organs in both founders and F1 offspring,because no rhLF was detected in the saliva or blood of transgenic mice expressing high levels rhLF in milk.This result suggested that the constructs with chimeric promoter/enhancer could increase the expression of heterogenous protein in the mammary gland of transgenic animals with high specificity.
Recently,continuing discoveries of new and surprising mechanism of gene regulation suggest many and perhaps all genes are regulated at multiple steps including transcription, post-transcriptional processing,nuclear export and localization,stability,and translation of mature mRNA molecules.Chimeric promoter/enhancer contains both mammary gland specific elements and Pcmv for mammary expression.Bio-informatics analysis shows that Pcmv sequence contains 10 uORF(upstream open reading frame) and 4 CCAAT/enhancer sites.Lodhi et al(2003) considered that the uORF could mediate constitutive effects on translation of mRNA from AUG codon.CCAAT sequence is an enhancer-binding site having been described in being linked to CCAAT-binding protein,some research show the increasing of the binding would mediate expression of functional gene,which might underlie observation that some cis-acting elements within the 5'-UTR including chimeric promoters of lactoprotein and CMV can activate initiation of translation.
From overall results,pgCNCG/LF25(composed of Pcmv chickenβ-globin and goatβ-casein promoter),pbCNCS/LF36(Pcmv,SV40polyA and bovineαs1-casein promoter),pBnCL14(Pcmv goatβ-BLG promoter AND SV40polyA) and pBnLC2G (Pcmv,2×chickenβ-globin and goatβ-BLG) were the most efficient under in vivo and in vitro conditions.The pBnCL14 was the most efficient in BnF95 in the expression of GMECs in three of pBnCL14,pBnCL14 and pBnLC2G,while the PgCNCG/LF25 was more efficient than the pbCNCS/LF36 in mammary gland of transgenic mice.It is worth mentioning that the binary promoter of Pcmv can improve mammary gland-specific expression,while ternary promoter of CMV,chickenβ-globin and lactoprotein may be more active than the binary one in mammary gland of transgenic mice.
Conclusively,although many long regulatory sequence can be useful for driving expression of targeted transgene in milk,their low efficiencies and uncertain expression,in most transgenic individuals,caused researchers to concentrate much time and effort on constructing over-expressing vectors for mammary specific-gland expression.Compared to constructing large size regulatory elements,the chimerc promoters/enhancers for conveniently constructing mammary-specific expression vectors,were shown to ascribe a high level of stability and efficiency of expression.We describe for the first time the synergistic effect achieved by the use of binary or ternary promoters/enhancers for mammary specific expression of rhLF in transfected GMECs and the milk of transgenic mice.
外源基因在转基因动物乳腺中特异性表达是转基因动物乳腺生物反应器的根本目的。有许多生物活性蛋白基因在乳蛋白调控序列的指导下已经在转基因动物乳腺中表达,其中应用较多的调控元件有:牛αs1-酪蛋白(bovineαs1-casein)、山羊β-酪蛋白(β-casein)、绵羊β-乳球蛋白(BLG)、小鼠的乳清酸蛋白(WAP)等。尽管已有应用这些调控序列实现乳腺特异性表达成功的事例,但大多数实验研究中,乳腺特异性表达的成功率低、乳汁中的表达水平低。
已经发现某些非乳蛋白调控元件对乳腺特异性表达有明显的作用,如绝缘子、染色质开启子、增强子、核基质附着区和内含子等,其中起到重要作用的是转录和翻译调控因子。近年来,尽管乳蛋白启动子及其它表达调控元件已广泛应用于乳腺特异性表达,但相对低的表达水平和不稳定表达案例也较多。
人巨细胞病毒(CMV)启动/增强子是广泛用于真核细胞表达的调控元件,早期的研究证明有提高转录水平的作用,但未见报道用于激活乳腺特异性表达。本研究应用人巨细胞病毒启动/增强子(Pcmv)与乳蛋白调控元件构建复合启动/增强子和人乳铁蛋白cDNA组成乳腺特异性表达载体。
为了鉴定复合启动/增强子对转基因动物乳腺特异性表达水平的作用,我们构建了单一乳蛋白启动子与乳蛋白/Pcmv复合启动子两种不同类型的乳腺特异性表达载体。应用山羊β-酪蛋白、山羊β-乳球蛋白、αs1-牛酪蛋白为乳蛋白调控序列为乳蛋白调控序列;鸡β-珠蛋白(β-globin,隔离元件)、CMV启动/增强子和SV40polyA为非乳蛋白启动/增强子。以不同的乳蛋白调控序列与启动/增强子组合构建乳腺特异性表达复合启动/增强子(chimeric promoter/enhancer),单一的酪蛋白启动子作为对照,与人乳铁蛋白cDNA组成乳腺特异性表达载体。共构建了7个乳腺特异性表达载体(BnF95,pBnCL14,pBnLC2G,pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36),其中4个为复合启动/增强子型载体,另3个为单一乳蛋白启动子型载体。BnF95、pBnCL14和pBnLC2G载体用于转染山羊乳腺上皮细胞(goat mammary epithetical cells,GMECs);pgCNCG/LF25和pbCNCS/LF36为复合启动/增强子型,其中pgCN/LF/g和pbCN/LF为单一酪蛋白启动子作为对照,用于制备转基因小鼠。
泌乳山羊乳腺组织经Ⅰ型胶原酶消化分离乳腺上皮细胞,DMEM/F_(12)培养液(10%FCS)传代培养,获得的乳腺上皮细胞最多可培养25代以上。采用分步胰酶消化法去除上皮细胞中所含的成纤维细胞,从而乳腺上皮细胞得到纯化。通过电转染法将含NEO~τ标记基因的BnF95、pBnCL14和pBnLC2G外源基因导入23个山羊乳腺上皮细胞系,经G418筛选三周后,得到有抗性的转染细胞株,挑取单克隆细胞株进行增殖培养。共获得242株转染外源基因的细胞,有118株转染pBnCL14,82株转染BnF95,42株转染pBnLC2G基因构件。有3株转染pBnCL14基因的细胞进行nestedPCR整合检测,结果均为外源基因整合阳性。初步认为经G418抗性培养后获得细胞为整合细胞。对抗性培养阳性的细胞株应用催乳素进行诱导表达,收集48和72小时的诱导细胞培养液进行ELISA检测,其中有30株细胞的诱导液中检测出重组人乳铁蛋白,有4株转染pBnCL14基因细胞的表达水平达到10-50mg·L~(-1);另有22株细胞(9株/BnF95,7株/pBnLC2G)表达水平低于10 mg·L~(-1)。通过G418筛选和诱导表达和ELISA检测验证具有表达功能的乳腺上皮将用于山羊体细胞克隆的供核细胞。
pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36四个构件应用胚胎显微注射的方法制备转基因小鼠。应用PCR初步筛选整合小鼠,获得14只原代转基因小鼠,PCR产物序列分析显示转基因小鼠PCR检测产物与模板的同源率为99.95%～99.75,在PCR正常正确率范围以上。原代鼠与C57或ICR正常鼠交配,在出生的后代鼠中检出37只F1代转基因小鼠。获得的转基因母鼠用ELISA检测乳汁中重组人乳铁蛋白,并繁殖传代,获得F1、F2代转基因小鼠。
复合启动/增强子构件指导人乳铁蛋白cDNA在原代转基因小鼠乳腺中重组人乳铁蛋白(rhLF)表达水平(2～8.1g·L~(-1))明显高于单一酪蛋白启动子构件(0～12mg·L~(-1)),约10000倍。在所有转基因小鼠的血清和唾液中未检测(ELISA)出重组人乳铁蛋白(rhLF)。研究表明,酪蛋白/Pcmv复合启动/增强子不仅可提高转基因小鼠乳腺特异性表达水平,而且具有良好的乳腺组织表达定位性。
近年来,对基因表达调控机制提出了一些新的看法,认为几乎所有的哺乳动物基因表达是由多个环节调控的,其中包括了转录、转录后加工、核输出和定位、mRNA分子的成熟、翻译和稳定性等。生物信息学分析显示Pcmv含有多个uORF和CCAAT增强子结合位点。Lodhi等(2003)认为uORF能介导编码mRNA翻译的作用,而CCAAT序列是增强子结合蛋白结合的位点。有研究报道,CCAAT位点结合增强子结合蛋白也可介导功能基因的表达,这一过程可能意味着在5'-UTR顺式调控元件,包括复合启动子中乳蛋白和CMV启动子序列能激活人乳铁蛋白cDNA在培养的山羊乳腺上皮细胞和转基因小鼠乳腺中表达。
本领域的研究报道显示,尽管已有一些利用长片段调控元件获得高表达的事例,但更多的低表达案例使开发那些构建方便、片段长度短小、表达效率和表达水平高的乳腺特异性表达载体显得十分迫切。本研究的结果不仅实现了cDNA功能基因的高水平、高效乳腺特异性表达,而且其载体的构建简单方便。到目前为止,尚未见有应用乳蛋白/Pcmv复合启动子激活cDNA功能基因实现乳腺特异性高效表达的报道。
The use of transgenic animals for mammary bioreactors is a rapidly developing and intensely interesting field due to the increased demand for high-quality pharmaceuticals and medically diagnostic preparations.The manufacture of recombinant human proteins is more profitable by mammary gland bioreactor than that by other biological systems.
The mammary gland-specific expression of a transgene is a major objective for producing heterogenous proteins in the milk of transgenic animals.Bio-active foreign proteins have been expressed specifically in the mammary under the direction of particular milk regulatory sequences,such as:goatβ-casein,bovineαs1-casein,ovineβ-lactoglobulin (BLG) and rat whey acid protein(WAP).However,much of the evidences regarding the mammary as bioreactor indicated that only a small percentage of the mammary glands of transgenic animals expressed functional protein,and most of them were found at low levels in milk.
Several major regulatory elements are required in order to obtain satisfactory transgene expression,such as essential gene insulators,chromatin openers,enhancers,matrix attached regions,and introns.One of the most important elements may be the factor controlling the transgene's transcription and translation.In recent years,although lactoprotein gene promoters were widely used,it should be noted that their expression levels in milk were relatively low and unstable in many instances.
As an alternative to the widely used cytomegalovirus(Pcmv) enhancer for expressing transgene in eukaryon,there was no report for promoting transgenic expression in mammary gland,our aim was to build a series of promoters bearing at least one,or if possible,all of the following characteristics:promoters that would increase high levels of expressing transgene in milk,and expression specifically located in the mammary organs. We used the chimeric promoters/enhancers combining sequences of lactoprotein promoters from cow and goat,with the Pcmv or mono-milk-promoter without Pcmv or SV40ployA to construct vectors for expressing human lactoferrin(hLF) cDNA,their ability to drive high levels of specific recombinant human lactoferrin(rhLF) expression was determined in transgenic mice or goat mammary epithelial cells(GMECs) in vitro.Vectors of mono-promoters of lactoprotein elements were constructed as a control.Seven vectors (BnF95,pBnCL14,pBnLC2G,pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36) with different promoters/enhancers were constructed.Three vectors of BnF95,pBnCL14 and pBnLC2G were constructed by using milk regulatory elements of goat BLG and neomycin resistance marker gene(NEO~r) for transfecting of GMECs;pgCNCG/LF25,and pbCNCS/LF36 were constructed by using promoter enhance sequences of goat casein, bovineαs1-casein,chickenβ-globin insulators,Pcmv and SV40polyA for producing transgenic mice.pgCN/LF/g and pbCN/LF contained mono-promoters of casein as controls.
Primary GMEC lines were derived from biopsy tissue from artificial lactating goats. The cells were cultured in DMEM/F_(12) containing 10%fetal calf serum(FCS).GMECs could keep proliferate vigorously even 25 passages in vitro.The pure GMECs lines could be obtained within 2-3 passages by multi-alternation of digesting and adhering repeatedly. By electroporation,BnF95,pBnCL14 and pBnLC2G encoding neomycin resistance for selecting the positive cells were transfected into GMECs in vitro.After three-weeks-culture by G418(400ng·L~(-1)),the transfected cell colonies grew and proliferated in G418 medium. Single colonies were picked into another 24-well plate for continue culture,and 242 colonies were generated from 118 clonies of GMECs by neo-resistant culture.Three of cell clones from 242 colonies were respectively screened by PCR and each was confirmed to be transgenic colonies corresponded with NEO~r ones,the total 82,42 and 118 colonies were transfected transgenes of pBnCL14,BnF95 and pBnLC2G respectively.These colonies were induced with luteotropic hormone,and then the media were gleaned respectively at 48h or 72h.It was found that there were 30 colonies expressing recombinant human lactoferrin(rhLF) by ELISA at 72h.4 colonies of higher expression level of rhLF were about 50mg·L~(-1) from transgene of pBnCL14.These GMECs of expressing rhLF would be used as donor cells for hircine somatic cell nuclear transfer(SCNT) to produce the transgenic goats.
Four vectors(pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36) were used to generate transgenic mice.A total of 14 lines were selected out of 160 candidates.To verify transgenic integration,these PCR products were sequenced,and the sequences were analyzed using DNAStar software.Compared with correlative transgenes,the percentage of sequence identity was 100 except 99.75 for pgCNCG/LF25 founder line.Founder mice bearing the transgene were mated to C57 males or female and delivered 110 pups,of which 52 progeny were identified as being transgenic.
Using ELISA detection,results showed that the recombinant human lactoferrin rhLF expression was significantly increased in the milk of transgenic mice with chimeric promoter transgenes.The expression level was 2-8.1g·L~(-1) in the milk using chimeric promoter.In contrast,only 0～0.012g·L~(-1) was found in the milk using mono-casein promoter.In two of the highly expressing lines,pbCNCS/LF36 and pgCNCG/LF25,there was approximately a 10,000-fold increase in target protein in the milk of transgenic mice (P＜0.001).In addition,we have also shown that the expression of rhLF was strictly limited to the mammary organs in both founders and F1 offspring,because no rhLF was detected in the saliva or blood of transgenic mice expressing high levels rhLF in milk.This result suggested that the constructs with chimeric promoter/enhancer could increase the expression of heterogenous protein in the mammary gland of transgenic animals with high specificity.
Recently,continuing discoveries of new and surprising mechanism of gene regulation suggest many and perhaps all genes are regulated at multiple steps including transcription, post-transcriptional processing,nuclear export and localization,stability,and translation of mature mRNA molecules.Chimeric promoter/enhancer contains both mammary gland specific elements and Pcmv for mammary expression.Bio-informatics analysis shows that Pcmv sequence contains 10 uORF(upstream open reading frame) and 4 CCAAT/enhancer sites.Lodhi et al(2003) considered that the uORF could mediate constitutive effects on translation of mRNA from AUG codon.CCAAT sequence is an enhancer-binding site having been described in being linked to CCAAT-binding protein,some research show the increasing of the binding would mediate expression of functional gene,which might underlie observation that some cis-acting elements within the 5'-UTR including chimeric promoters of lactoprotein and CMV can activate initiation of translation.
From overall results,pgCNCG/LF25(composed of Pcmv chickenβ-globin and goatβ-casein promoter),pbCNCS/LF36(Pcmv,SV40polyA and bovineαs1-casein promoter),pBnCL14(Pcmv goatβ-BLG promoter AND SV40polyA) and pBnLC2G (Pcmv,2×chickenβ-globin and goatβ-BLG) were the most efficient under in vivo and in vitro conditions.The pBnCL14 was the most efficient in BnF95 in the expression of GMECs in three of pBnCL14,pBnCL14 and pBnLC2G,while the PgCNCG/LF25 was more efficient than the pbCNCS/LF36 in mammary gland of transgenic mice.It is worth mentioning that the binary promoter of Pcmv can improve mammary gland-specific expression,while ternary promoter of CMV,chickenβ-globin and lactoprotein may be more active than the binary one in mammary gland of transgenic mice.
Conclusively,although many long regulatory sequence can be useful for driving expression of targeted transgene in milk,their low efficiencies and uncertain expression,in most transgenic individuals,caused researchers to concentrate much time and effort on constructing over-expressing vectors for mammary specific-gland expression.Compared to constructing large size regulatory elements,the chimerc promoters/enhancers for conveniently constructing mammary-specific expression vectors,were shown to ascribe a high level of stability and efficiency of expression.We describe for the first time the synergistic effect achieved by the use of binary or ternary promoters/enhancers for mammary specific expression of rhLF in transfected GMECs and the milk of transgenic mice.
引文
[1]Houdebine L M.Production of pharmaceutical protein form transgenic animals[J].Biotech,1994,34:56-59.
[2]Niemann H,Wilfried A.Application of transgenesis in livestock for agriculture and biomedicine [J].Animal Reproduction Science,2003,79:291-317.
[3]Kevin R.Gene transfer in higher animals:theoretical considerations and key concepts[J].Journal of Biotechnology,2002,99:1-22.
[4]Palmiter R D,Brinster R L,Hammer R E.Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes[J].Nature,1982,300:611-615.
[5]Das R,roduction of therapeutic proteins from transgenic animals[J].Biobusiness,2001,30:60-64.
[6]Devinoy E,Thepot D,Stinnakre M G,et al.High level production of human growth hormone in the milk of transgenic mice:the upstream region of the rabbit whey acidic protein(WAP) gene targets transgene expression to the mammary gland[J].Transgenic Research,1994,3:79-89.
[7]Goldman IL,Kadulin SG,and Razin SV(2002) Transgenic goats in the world pharmaceutical industry of the 21st century.Russian Journal of Genetics.38:1-14
[8]Jaennisch R,Germ line integration and mendelian transmission of exogenous monloney leukemia virus[J].Pro.Natl.Acad.USA,1976,73:1260.
[9]Gordon JW,Scangos FH,Genetic transformation of mouse embryos by microinjection of purified DNA[J],Proc.Natl.Acad.Sci,USA,1980,77:7380-7384.
[10]Simons,JP,McClenaghan M,Clark AJ.Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice[J].Nature,1987,328:530-532.
[11]Gordon J W,Ruddle F H.Integration and stable germ line transmission of genes Injected into mouse pronuclei[J].Science,1981,241:1244-1246.
[12]Jones P B C,European Regulators Curdle Plans for Goat Milk Human Antithrombin,ISB News Report April 2006
[13]Paleyanda R k,Velander W H,Lee T K,Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Biotechnology,1990,8:443-446.
[14]Wright G,et al.High level expression of human alpha-antitrypsin in the milk of transgenic sheep[j].Biotechnology,1991,9:830-834.
[15]Close MJ,Howlett AR,Roskelley CD,Desprez PY.Lactoferrin expression in mammary epithelial cells is mediated by changes in cell shape and actin cytoskeleton[J].Journal of Cell science,1997,110:2861-2871.
[16]Wilmut I,Schnieke A E,McWhir J.Viable offspring derived from fetal and adult mammalian cells[J].Nature,1997,385:810-813.
[17]Ogden J E.Recombinant haemoglobin in the development of red-blood-cell substitutes [J].Trends Biotechnol,1992,10:91-96.
[18]Valander W H,Johnson J L,Page R L,et al.High-level expression of aheterogonous Protein in the milk of transgenic swine using of a cDNA encoding human protein[J].PNAS USA,1992,89:12003-12007.
[19]Gordon K,Lee E,Vitale J A.Production of human tissue plasminogen activator in transgenic mouse milk[J].Bio/Technoly,1987,5:1183-1187.
[20]黄英,张克忠,黄文英,等.乳腺表达人凝血因子Ⅸ的转基因小鼠的研究[J].科学通报,1998,43(1):732-735.
[21]Anon.Transgenic cows to produce human albumin by ton[J].Biotechnol News,1997,17(8):1
[22]Rudolph N S.Biopharmaleutical production in transgenic livestocks[J].Tibtech,1999,17:367-374.
[23]Toman P D,Chisholm G,McMullin H,et al.Production of Recombinant Human Type ⅠProcollagen Homotrimer in the Mammary Gland of Transgenic Mice[J].Transgertic Res,1999,8:415-427
[24]Wilmut I,Schnieke A E,McWhir J,Kind AJ Campbell H S.Viable offspring derived from fetal and adult mammalian cells[J].Nature,1997,385:810-813.
[25]McCreath K J,Howcroft J.Production of gene-targeted sheep by nuclear transfer from cultured somatic cells[J].Nature,2000,405:1066-1069.
[26]Krimpenfort P,Rademakers A,Eyestone W,et al.Generation of transgenic dairy cattle using in vitro embryo production[J].Biotechonlogy,1991,9:844-847.
[27]施履吉,郭礼和,倪祖梅等,家兔个体表达系统的建立。遗传学报,1993,12:13-16
[28]李宁,吴常信等.牛aS1酪蛋白基因启动区的克隆和序列分析.遗传,1997,19(1):4-8
[29]郑月茂,刘凤军,张涌.转人乳铁蛋白基因山羊乳腺上皮细胞的研究[J].畜牧兽医学报,2006,37(12):1282-1286.
[30]余珊珊,王毅彰,李宁.转基因小鼠乳腺表达人乳过氧化物酶的初步研究[J],科学导报,2005,206:31-34.
[31]张忠诚,李宁,陈永福.动物乳腺生物反应器的原理及研究进展[J],中国畜牧杂志,2000,2:51-53.
[32]汤家铭,成国祥.体细胞介导制备转基因动物的新途径[J].中国实验动物学报,2002,10(3):188-192.
[33]徐曼妮,厉曙光,成国祥.乳腺生物反应器表达载体的检测方法[J].生物技术通报,2003,5:23-26.
[34]舒建洪,刘津,张涌.动物乳腺生物反应器研究进展[J].西北农林科技大学学 报,2004,32(12):149-153.
[35]曾溢滔 主编.转基因动物与医药产业[M],上海教育出版社,2000.
[36]Liu Z.,Zhao C J,Fan B.et al.Variable expression of human lactoferrin gene in mice milk driven by its 90 Kb upstream flanking sequences[J].Animal Biotechnology,2004,15(1):21-310
[37]潘登科,张运海,孙秀柱,供体细胞对猪体细胞克隆胚胎早期发育的影响,畜牧兽医学报,2006,37(4):331-336
[38]李建许,陈杰,蔡亚飞,狼山鸡AA肉鸡嵌合体的制作及其转基因鸡研究,南京农业大学学报,2007,30(2):83-88
[39]Schild C,Claret FX,Wahli W.Nucleosome-dependent static loop potentiates estrogen-regulated transcription from the Xenopus vitellogenin B1 promoter in vitro[J].EMBO,1993,12(2):423-433
[40]Silva J,Mak W,Zvetkova I,et al.Establishment of histone h3 methylation on the inactive X chromosome requirest ransient recruitment of Eed-Enxl polycomb group complexes[J].Dev -elopement Cell,2003,4(4):481-495
[41]Park Y,Kuroda M I.Epigenetic aspects of X-chromosome dosage compensation[J].Science,2001,293(5532):1083-1085
[42]Reik W,Dean W,Walter J.Epigenetic reprogramming inmammalian development[J].Science,2001,293(5532):1089-1093
[43]朱静,王应雄.染色质的重塑与基因表达调控[M].外医学临床生物化学与检验学分册,2005,26(1):46-78
[44]劳为德主编.动物细胞与转基因动物制药[M].化学工业出版社,2003
[45]李震,陈永福.乳腺生物反应器细胞模型建立的问题探讨[J].农业生物技术学报,1997,5(2):148-152
[46]Paleyanda R k,velander W H,Lee T K,Scandella D H.Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Biotechnology,1990,8:443-446
[47]John Clark A.The Mammary Gland as a Bioreactor:Expression,Processing,and Production of Recombinant Proteins.Journal of Mammary Gland Biology and Neoplasia,2001,3(3):337-350
[48]Xul H L,Qian Y P,Nie W H.Construction,characterization and chromosomal mapping of bacterial artificial chromosome library of Yunnan snub-nosed monkey(Rhinopithecus bieti)[J].Chromosome Research,2004,12:251-262
[49]Ayares D.Gene targeting in livestocks[J].Transgenic Research Conference,1999,20:557-560.
[50]Kolb A F,Ansell R,Siddell S G.Insertion of a foreign gene into the b-casein locus by Cre-mediated site-specific recombination[J].Gene,1999,227:21-31
[51]田锦,阮晖,李志敏.乳蛋白基因及其应用的研究进展[J].中国奶牛,2003,1:33-36.
[52]Waston C,Gordon C E,Robertson M,et al.Interaction of DNA-binding proteins with milk protein gene promoter in vitro:indentification of a mammary gland-specific factor[J].Nucleic Acids Research,1991,19(23):6603-6610
[53]Whitelaw C,Grolli S,Broadhurst R J.Suppression of β-casein gene expression by inhibition of protein synthesis inmousemammary epithelial cells is associated with stimulation of NF-κB activity and blockage of prolactin-Stat5 signaling[J].Cell Tissue Res,2003,311:207-215
[54]Grosclaude F,Mahe M F,Juenet R A.Mendelian polymorphism underlying quantitative variation of goat s1-casein[J].Genet,1987,19:399-412
[55]Houdebine L M.Transgenic animal bioreactors[J].Transgenic Res,2000,9:305-320
[56]http://www.bcm.edu/rosenlab/publications.html
[57]Dai Chida,Hiroshi Wakao,Atsushi Miyajima.Transcriptional Regulation of the β-Casein Gene by ytokines:Cross-Talk between STAT5 and Other Signaling[J].Molecules Molecular Endocrinology,2006,12(11):1792-1806
[58]Adam C,Buser I,Wolfgang D.Progester one receptor repressing of Prolactin/Stat5-Mediated transcription of the β-Casein Gene in Mammary Epithelial Cells[J].Molecular Endocrinology,2006,10:210-297
[59]Subramanian A,Wang J M,Gil G.STAT 5 and NF-Y are involved in expression and growth hormone-mediated sexually dimorphic regulation of cytochrome P450 3A10/lithocholic acid 6[beta]-hydroxylase[J].Nucleic Acids Research,1998,26:2173-2178
[60]周明亮,吴登俊,曾云琦.β-酪蛋白基因的研究进展[M].内蒙古农业科技,2005,2:12-14.
[61]Pervaiz S,Brew K.Homology of beta-lactoglobulin serum retinol-binding protein and protein [J].Science,1985,228(4697):335-337
[62]Hayes H C,Petit E J.Mapping of the beta-lactoglobulin gene and of an immunoglobulin M heavy chain-like sequence to homoeologous cattle sheep and goat chromosomes[J].Mamm Genome,1993,4(4):207-210
[63]Folch J M,Coll A,Sanchez A.Rapid Communication:cloning and sequencing of the cDNA encoding goat beta2 lactoglobulin[J].Anim Sci,1993,71(10):2832
[64]http://www.watsonpharm.com/
[65]高建军,颜景斌,曾溢滔.山羊β乳球蛋白基因的克隆及其在转基因小鼠乳汁中的高效表达[J].遗传学杂志,2003,25(5):499-503
[66]Whitelaw C B A,Accomero P,et al.Matrix attachment region regulates basal β-lactogloblin transgene expression Gene[J],2000,244:73-80
[67]成军.转录因子C/EBP的生物学功能[J].世界华人消化杂志,2004,12(2):408-412
[68]Beaton A,Wilkins R J,Wheeler T T.Lactation-associated and prolactin-responsive changes in protein synthesis in mouse mammary cells[J].Tissue and Cell,1997,29:509-516
[69]Kadonaga J T.Regulation of RNA polymerase Ⅱ transcription by sequence-specific DNA binding factors[J].Cell,2004,116:247-257
[70]王付龙,徐祥,刘昕.转录因子圈套策略研究进展生物化学与生物物理进展[J].2001,28(6):802-804
[71]卡尔A.平克尔特(编),劳为德(译),转基因动物技术手册[M].2004,北京化学工业出版社
[72]Morris D R,Geballe A P.Upstream open reading frames as regulators of mRNA translation[J].Molecllar and Cellular Biology,2000,20:8635-8642
[73]Whitelaw CB,Archibald AL,Harris S,McClenaghan M,Simons JP,Clark AJ.Targeting expression to the mammary gland:Intronic sequences can enhance the efficiency of gene expression in transgenic mice.Transgenic Res.1991 Dec;1(1):3-13
[74]Lodhi K M,Ozdener M H,Shayiq R M.The upstream open reading frame mediates constitutive effects on translation of cytochrome P-450c27 from the seventh in-frame AUG codon in rat liver [J].Biol Chem,2003,278:40647-40657
[75]Wakao H,Gouilleux F,Groner B.Mammary gland factor(MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response[J].EMBO,1994,13:2182-2191
[76]Yang J,Kennelly J,Baracos V E.Physiological levels of Stat5 DNA binding activity and protein in bovine mammary gland[J].Anim Sci,2000,78:3126-3134
[77]潘玲,陈建军,陈常庆.山羊β-乳球蛋白基因5'-侧区的克隆、测序和序列分析生物工程学报,1997,13(2):132-137
[78]Grosveld F.Activation by locus control regions[J].Current Opinion in Genetics &Development,1999,9:152-157
[79]Lecilas-tagar F,Bell AC,Felsenfeld G.Position enhancer-blocking activity chicken β-globin 5'-HS4 insulators in transeintly transfected cells[J].Proc Natl.Acad Sci,1999,96:14354
[80]Pantano T,Jolivet G,Prince S,et al.,Effect of the Rabbit as1-Casein Gene Distal Enhancer onthe Expression of a Reporter Gene in Vitro and in Vivo.Biochemical and Biophysical Research Communications,2002,290,53-61
[81]于占洋,任蕴芳,沈倍奋.CMV启动子和RSV LTR启动子表达sIL-6R效率的比较[J].生物技术通讯,1999,10:95-99
[82]Morris D R,Geballe A P,Upstream open reading frames as regulators of mRNA translation[J].Molecllar and Cellular Biology,2000,20:8635-8642
[83]Hallauer P L,Hastings KEM.Human cytomegalovirus IEI promoter/enhancer drives variable gene expression in all fiber types in transgenic mouse skeletal muscle[J].BMC Genetics,2000,1:1-7
[84]Gehrke S,Jerome V,Muller R(2003) Chimeric transcriptional control units for improved liver-specific transgene expression.Gene 322:137-143
[85]Konopka W,Duniec K,Mioduszewska B,et al,Promoters for inducible gene expression in rat neurons in vitro and in vivo[J],2005,10:4-14
[86]Paleyanda RK,Velander WH,Lee TK,et al,Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Bio/technol,1990,8:443-446
[87]Wall R J.Transgenic livestock:progress and prospects for the future[J].Theriogenology,1996,45:57-68
[88]Houdebine LM(2000) Transgenic animal bioreactors.Transgenic Res,9:305-320
[89]Rudolph NS(1999) Biopharmaceutial production in transgenic livestock.Tibtech,17:367-374
[90]Wall RJ(1996) Transgenic livestock:progress and prospects for the future.Theriogenology 45:57-68
[91]Kim S J,Pak K W,Pak K W.Structure of the human lactoferrin gene and its chromosomal localization[J].Molecules and Cells.1998,8:663-668
[92]卢一凡,邓继先,肖成祖,等.内含子对提高转基因动物基因表达效率的影响[J],生物化学与生物物理进展,1995,22(4):322-326.
[93]Jenuwein T,Allis CD,Translating the histone code[J],Science,2001,293(5532):1074-1080.
[94]Fisher-Adams G,Grunstein M.Yeast histone H4 and H3 N-termini have different effects on the chromatin structure of the GAL1 promoter[J].EMBO J,1995,14(7):1468-1477.
[95]朱静,王应雄,染色质的重塑与基因表达调控,国外医学临床生物化学与检验学分册,2005,26(1):12-14
[1]Houdebine L M.Transgenic animal bioreactors[J].Transgenic Res,2000,9:305-320
[2]Wall R J.Transgenic livestock:progress and prospects for the future[J].Theriogenology,1996,45:57-68
[3]Maga E A,and Murray J D.Mammary gland expression of transgenes and the potential for altering the properties of milk[J].Bio/Technol,1995,13:1452-1457
[4]Paleyanda R K,Velander W H,Lee T K,et al.Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Bio/technol,1990,8:443-446
[5]Barash I,Faerman A,Richenstein M,Kari R,Damary G,Shani M and Bissell MJ(1999)In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with β-lactoglobulin and whey acidic protein promoters.Mol Repro.Dev.52:241-252
[6]Sφrensen P,Sheffield LG,Involvement of c-sic in b-Casein Expression by Mammary Epithelial Cells.Biochemical and Biophysical Research Communications,1997,241:710-713Ding Y,Wang P,Liu S,et al.Cloning of WAP promoter and construction of mammary-specific expression vector[J].2004,3:23-28
[7]Dale T C,Krnacik M J,Schmidhauser C,et al.High level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 30 untranslated region[J].Mol Cell Biol,1992,12:905-914
[8]Bonifer C.Long-distance chromatin mechanisms controlling tissue-specific gene locus activation[J].Gene,1999,238:277-289
[9]Giraldo P,Montoliu L.Size matters:use of YACs,BACs and PACs in transgenic animals[J].TransgenicRes,2001,10:83-103
[10]Jackson D A,Juranek S,Lipps H J,Designing Nonviral Vectors for Efficient Gene Transfer,2006,Molecular Therapy,14:613-627
[11]Boulos S,Meloni BP,Arthur PG,Bojarski C,Knuckey NW(2006) Assessment of CMV,RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures.Brain Research,12:1-8
[12]Arai Y,Ishiwata M,Baba S,et al.Neuron-specific activation of murine cytomegalovirus early gene e1 promoter in transgenic mice[J].American Journal of pathology,2003,163(2):643-652
[13] Gehrke S, Jerome V, Muller R. Chimeric transcriptional control units for improved liver-specific transgene expression [J] . Gene, 2003, 322:137-143
[14] F Raptis L, SV40 Protocols[M], Humana press, p275-286
[15] Orrester WC, Fernandez LA, Grosschedl R. Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer promoter interactions [J]. Gene Dev, 1999, 13:3003-3014
[16] Fedosyuk H, Peterson K R. Deletion of the human β -globin LCR 5' HS4 or 5' HS1 differentially affects β-like globin gene expression in β -YAC transgenic mice[J]. Blood Cells Mol Dis , 2007, 10:1016
[17] Cerdan M G, Young J I, Zino E, et al. Accurate spatial and temporal transgene expression driven by a 3.8-kilobase promoter of the bovine α -casein gene in the lactating mouse mammary gland [J] .Mol Reprod Dev, 1998, 49:236-245
[18] Van Berkel P H, Welling M M, Geerts M. Large scale production of recombinant human lactoferrin in the milk of transgenic cows [J] . Nat Biotechnol, 2002, 20(5):484-487
[19] Liu Z., Zhao, C.J., Fan B. et al. Variable expression of human lactoferrin gene in mice milk driven by its 90 Kb upstream flanking sequences [J] .Animal Biotechnology, 2004, 15(1): 21-31
[20] Sambrook, J, FritschEF, and Maniatis T. Molecular Cloning: A Laboratory Manual [M] . 2nd ed, Cold Spring Harbor Lab, New York, 1989
[21] Houdebine L M, Animal transgenesis and cloning [M] . John Wiley & Sons Ltd, 2003, pp. 14-15
[22] Nagy A, Gertsenstein M, Vintersten K, et al. Manipulating the mouse embryo: a laboratory manual [M] .Cold Spring Harbor Lab, New York, 1997, pp. 289-358
[23] Meade H, Gates L. Bovine a sl-casein gene sequence direct high level expression of active human urokinase in mouse milk [J]. Biotechnology, 1990, 8:443-448
[24] Oh K B, Cho i Y H, Kang Y K, et al. A hybrid bovine beta-casein and GH gene directs transgene expression to the lung and mammary gland of transgenic m ice[J ]. T ransgenic Res, 1999, 8 (4): 307-311
[25] Paleyanda RK, Velander WH, Lee TK, Scandella DH, Gwazdauskas FC, Meade H, Gates L, Lacy E and Lonberg N (1990) Bovine alpha sl-casein gene sequences direct high level expression of active human urokinase in mouse milk. Biotechnol 8:443-446
[26] Schumacher A, Koetsier PA, Hertz J, et al. Epigenetic and genotype-specific effects on the stability of de novo imposed methylation patterns in transgenic mice[ J]. J Biol Chem, 2000 , 275:37915-37921
[27] Lao WD, Liu W, Ceng GX, Xu SP, Cheng Y, Construction of transgenic mice producing CAT to milk by co-injection of two overlapping fragments of bovine α s1-casein-CAT gene Science in China(series C),1996,39:592-560
[28]Patick H C,Van Berkel,Mick M,et al.Large scale production of recombinant human lactoferrin in the milk of transgenic cows[J].Nature Biotechnology,2002,20:484-487
[29]陈红星,杨晓,黄培堂,乳腺生物反应器制备中表达载体的发展,生物工程学报18:136-140
[30]Rey M W,Woloshuk LW,deBoer H A,et al,Complete nucleotide sequence of human mammary gland lactoferrin,1990,Nucleic Acids Research,18:5288
[31]Israel DI.A PCR-based method for high stringency screening of DNA libraries.Nucleic[J].Acids Research.1993,21(11):2627-2631
[32]Gutierrez A,M eade H M,D itullio P,et al.Expression of a bovine kappa-CN cDNA in themammary gland of transgenicm ice utilizing a genomic milk protein gene as an exp ression cassette[J].T ransgenic Res,1996,5(4):271-279
[33]N inom iya T,H irabayash iM,Sagara J,et al.Functions of milk protein in gene 5'flanking region on human growth hormone gene[J].Mol Rep rod Dev,1994,37:276-283
[34]Benlhabib H,Herrera J E,Expression of the Op18 gene is maintained by the CCAAT-binding transcription factor NF-Y[J].Gene,2006,377:177-185
[35]Petitclerc D,Attal J,Theron MC,et al,The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice
[36]Xu ZL,Mizuguchi H,Ishii-Watabe A,Uchida E,Mayumi T,Hayakawa T(2001)Optimization of transcriptional regulatory elements for constructing plasmid vectors.Gene 272:149-156
[37]Kimet S J,Sohn B H,Sangkyun J,et al.High level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence[J].J Biol Chem,1999,126:320-325
[38]劳为德主编.动物细胞与转基因动物制药[M].化学工业出版社,2003.
[39]http://www.invitrogen.com/content.cfm?pageid=1
[40]萨姆布鲁克著,金冬雁等译分子克隆实验指南,第三版,科学出版社
[41]Chen LS,Tassone F,Sahota P,Hagerman PJ,(2003) The(CGG)n repeat element within the 5' untranslated region of the FMR1 message provides both positive and negative cis effects on in vivo translation of a downstream reporter.Human molecular genetics,12(23):3067-3074
[42]卡尔A.平克尔特编,劳为德译,转基因动物技术手册,2004,化学工业出版社,北京
[43]http://www.ncbi.nlm.nih.gov/sites/entrez
[44]https://www.invitrogen.com/content/sfs/manuals/pbc1_man.pdf
[45]Kim S J,Pak K W,Pak K W.Structure of the human lactoferrin gene and its chromosomal localization[J].Molecules and Cells.1998,8:663-668
[46]http://www.invitrogen.com/content/sfs/manuals/pcep4_man.pdf
[47]Wright G,Caryer A,Cottom D,et al.High level expression of active human alpha-1-anti trypsin in the milk of transgenic sheep Bio/Technology,1991,9(9):830-834.
[48]潘玲,陈建军,陈常庆,山羊β-乳球蛋白基因5′侧区的克隆、测序和序列分析,生物工程学报,1997,13(2):132-137.
[49]Folch J M,Coll A,Sanchez A.Rapid Communication:cloning and sequencing of the cDNA encoding goat beta2 lactoglobulin[J].Anim Sci,1993,71(10):28-32
[50]高建军,颜景斌,黄英,曾溢滔,山羊β乳球蛋白基因的克隆及其在转基因小鼠乳汁中的高效表达,遗传,2003,25(5):499-503
[51]Fedosyuk H,Peterson K R.Deletion of the human β-globin LCR 5' HS4 or 5' HS1differentially affects β-like globin gene expression in β-YAC transgenic mice[J].Blood Cells Mol Dis,2007,10:10-16
[52]Alami R,Greally JM,Tanimoto K,Hwang S,Feng YQ,Engel JD et al.(2000)Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice.Hum Mol Genet 9:631-636
[53]McKnight RA,Shamay A,Sankaran L,Wall RJ and Hennighausen L,(1992)Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice.Proc.Natl.Aacad Sci.,89:6943-6947
[54]Zhujing,Yangnana,Lihao,et al.A Research on the Transformation from λ Phage Library to Plasmid Fedosyuk H,Peterson K R.Deletion of the human β-globin LCR 5' HS4 or 5'HS1 differentially affects β-like globin gene expression in β-YAC transgenic mice[J].Blood Cells Mol Dis,2007,10:1016
[55]Boulos S,Meloni BP,Arthur PG,Bojarski C,Knuckey NW(2006)Assessment of CMV,RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures.Brain Research,12:1-8
[56]Gehrke S,Jerome V,Muller R.Chimeric transcriptional control units for improved liver-specific transgene expression[J].Gene,2003,322:137-143
[57]Smith RL,Traul D,Schaack L,Characterization of Promoter Function and Cell-Type-Specific Expression from Viral Vectors in the Nervous System,Journal of Virology,2000,74:11254-11261.
[58]Konopka W,Duniec K,Mioduszewska B,hCMV and Tet promoters for inducible gene expression in rat neurons in vitro and in vivo,Neurobiology of Disease,2005,xxx-xxx.
[1] Gordon JW SG, Plotkin DJ, Barbosa JA, et al. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A. 1980; 77:7380-7384.
[2] Sanes JR, Nicolas JF. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 1986; 5: 3133-3142.
[3] Zeng Y, Huang S, Ren Z, et al, Molecular Cloning and Sequence Analysis of Goat 8-Casein Gene [J] .Chinese Journal of Biochemistry and Molecular Biology, 2003, 15:5-8
[4] Li RY, TsKosugi I, utsui Y, Activation of murine cytomegalovirus immediate-early promoter in mouse brain after transplantation of the neural stem cells [J] . Acta Neuropathol, 2004, 107:406-412.
[5] Gossler ADT, Korn R, Serfling E, Kemler R. Transgenesis by means of blstocyst-derived embryonic stem cell line. Proc Natl Acad Sci USA. 1986, 83:9065-9069
[6] Arat S, Gibbons J, Rzucidlo S. et. al. In vitro development of bovine nuclear transfer embryos from transgenic clonal lines of adult and fetal fibroblast cells of the same genotype. Biol Reprod. 2002, 66:1768-1744
[7] Zakhartchenko V, Mueller S, Alberio R. et. al. Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts. Mol Reprod Dev. 2001, 60:362-369
[8] Groner B, Transcription factor regulation in mammary epithelial cells, Domestic Animal Endocrinology, 2002, 23:25-32
[9] Zou XG, Wang YG, Cheng Y, et al, Generation of cloned goats(Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle.Molecular reproduction and development , 2002, 61:164-172
[10] Barash I, Faerman A, Richenstein M, etal, In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with β -lactoglobulin and whey acidic protein promoters. Mol Repro. Dev. 1999, 52:241-252
[11] Pantschenko A.G, Woodcock-Mitchell J, Bushmich S.I, Yang T.J. Establishment and characterization of caprine mammary epithelial cell line (GMEC). In Vitro Cellular And Development Biology, 2000.6:26-37
[12] Stelwagen K, McFadden HA, Demmer J, Prolactin, alone or in combination with glucocorticoids, enhances tight junction formation and expression of the tight junction protein occludin in mammary cells [J]. Molecular and Cellular Endocrinology, 1999, 156:55-61
[13] Lopez-Soriano J, Lopez-Soriano FJ, Carbo N, et al, Leptin administration to lactating rats is unable to induce changes in lipid metabolism in white adipose tissue or mammary gland [J]. European Journal of Obstetrics & Gynecology and Reproductive Biology,1999,84:93-97
[14]Zheng YM,An ZX,Peng XR,.Establishment of Goat Mammary Epithelial Cell Line and Expression of EGFP Gene[J].Journal of Agricultural Biotechnology.2005,03
[15]Gehrke S,Jerome V,Muller R.Chimeric transcriptional control units for improved liver-specific transgene expression[J].Gene,2003,322:137-143
[16]欧阳五庆,钱菊汾.山羊乳腺上皮细胞培养体系的建立[J].西北农林科技大学学报(自然科学版),2003,31:30-34
[17]任芳丽.山羊乳腺上皮细胞的分离与体外培养[D].西北农林科技大学2002硕士论文,陕西杨凌,p81-82.
[18]Xun Xu,Jun Han,Jun Han.Developmental expression of Smad1-7 suggests critical function of TGF-β/BMP signaling in regulating epithelial mesenchymal interaction during tooth morphogenesis[J].International Journal of Developmental Biology.2003,47:31-39
[19]Houdebine L M.Transgenic animal bioreactors[J].Transgenic Res,2000,9:305-320
[20]Wall R J.Transgenic livestock:progress and prospects for the future[J].Theriogenology,1996,45:57-68
[21]Baveye S Mazurier J Mazurier J.Lactoferrin:A multifunctional glycoprotein involved in the modulation of the inflammatory process[J].Clinical Chemistry and Laboratory Medicine.1999,37:281-286
[22]李向臣,任芳丽,张涌.山羊乳腺上皮细胞的分离、培养与超微结构[J].畜牧兽医学报,2005,36(2):122～126.Israel DI.
[23]李扬,吴凯峰,郭旭东等,脂质体介导外源基因体外转染,遗传,2002,24:653-655
[24]Tomov TCH.Quantitative dependence of electroporation on the pulse parameters.Bioelectrochemisty and Bioenergetics.1995,37:101-107
[25]萨姆布鲁克著,金冬雁等译分子克隆实验指南,第三版,科学出版社
[26]Transfection protocol(epphendorf company),L929,Protocol.No.4308 915.025-11/1999
[27]A PCR-based method for high stringency screening of DNA libraries.Nucleic[J].Acids Research.1993,21(11):2627-2631
[28]Maga E A,and Murray J D.Mammary gland expression of transgenes and the potential for altering the properties of milk[J].Bio Technol,1995,13:1452-1457
[29]徐宜为编著,免疫检测技术 第二版,科学出版社,1991,北京Wang CD,Lee YK,Lee YK.A functional composite cis-element for NF-kappaB and RBJKappa in the rat pregnancy-specific glycoprotein gene[J].Biology of Reproduction.2003,65:1437-1443
[30]http://baike.baidu.com/view/676752.htm
[31]Sun YL,Lin CS,Chou YC,Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows[J].Cell Biology International,2005,29:576-582
[32]汪和睦,谢廷栋.细胞电穿孔电融合电刺激原理及应用 天津科学技术出版社 2000.8
[33]Lu jian,Chen yu xia.Effect of glucocorticoid receptor beta on the transactivation function of glucocorticoid receptor alpha[J].Chinese Journal of Pathophysiology 2002,10
[34]Jiang xiang qing,Zhu li ping,Studies on Three-Dimensional Quantitative Structure-Activity Relationships of Nonsteroidal Progesterone Receptor Agonists 5-Benzylidene 1,2-dihydro 5H-chromeno quinolines式会社[J].Journal of Fudan University(Medical Sciences) 2005,01
[35]Email:epphemdorf@epphemdorf.com;internet:www.epphendorf.com
[36]Baum C,Forster P,Hegewisch-Becker S,Harbers K,An optimized electroporation protocol applicable to a wide range of cell lines[J].Biotechniques,1994,17:10:58-62.
[37]Baisong L,peitang H.The efferts of DNA on efficiency of electroporation[J]Prog.Biochem.Biophys,1995:256-260
[38]钱锋;肖成组,电击缓冲液对哺乳动物细胞电穿孔效率的影响[J],军事医学科学院院刊,1999,02:22-24
[39]钱锋,肖成组,影响细胞电穿孔效率的因素[J]生物技术 1998,8(5):27-31
[40]Arai Y,Ishiwata M,Baba S,et al.Neuron-specific activation of murine cytomegalovirus early gene e1 promoter in transgenic mice[J].American Journal of pathology,2003,163(2):643-652
[41]Hallauer P L,Hastings KEM,Human cytomegalovirus IEI promoter/enhancer drives variable gene expression in all fiber types in transgenic mouse skeletal muscle[J].BMC Genetics,2000,1:1-7
[42]Cha-Molstad H,Young DP,Kushner I,et al.The interaction of C-Rel with C/EBPbeta enhances C/EBPbeta binding to the C-reactive protein gene promoter[J].Molecular Immunology,2007,44:2933-2942
[43]Dobie K W,Lee M,Fantes J A,et al.Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus[J].Proc Natl Acad Sci,1996,93:6659-6664
[44]Lao WD,Liu W,Ceng GX,Xu SP,Cheng Y,Construction of transgenic mice producing CAT to milk by co-injection of two overlapping fragments of bovine α s1-casein-CAT gene Science in China(series C),1996,39:592-560.
[45]Fedosyuk H,Peterson K R.Deletion of the human β-globin LCR 5' HS4 or 5' HS1differentially affects β-like globin gene expression in β-YAC transgenic mice[J].Blood Cells Mol Dis,2007,10:1016
[46]Giraldo P,Montoliu L.Size matters:use of YACs,BACs and PACs in transgenic animals[J].Transgenic Res,2001,10:83-103
[47]Bonifer C.Long-distance chromatin mechanisms controlling tissue-specific gene locus activation[J].Gene,1999,238:277-289
[48]Sφrensen P,Sheffield LG,Involvement of c-sic in b-Casein Expression by Mammary Epithelial Cells.Biochemical and Biophysical Research Communications,1997,241:710-713.
[49]Kim S J,Pak K W,Pak K W.Structure of the human lactoferrin gene and its chromosomal localization[J].Molecules and Cells.1998,8:663-668
[50]Chen CM,Wu SC,Wu SCT.Temporal and spatial expression of biologically active human factor Ⅷ in the milk of transgenic mice driven by mammary-specific bovine alpha lactalbumin Ⅲregulator sequences[J]Transgenic Research,2002,11:257
[51]Nagy A,Gertsenstein M,Vintersten K,et al,Manipulating the mouse embryo:a laboratory manual[M].Cold Spring Harbor Lab,New York,1997,pp.289-358
[52]田锦,阮晖,牛冬,等.乳蛋白基因及其应用的研究进展[J].中国奶牛,2003,1:33-36
[53]Chen gang,Shi wei qin,Wu mei juan,et al.Cloning and sequencing of β-lactoglobulin gene of dairy goats[J],Journal of Yangzhou University(Agricultural and Life Science Edition),2001,2
[1]Nagy A,Gertsenstein M,Vintersten K,Behringer R,(1997) Manipulating the mouse embryo:a laboratory manual.Cold Spring Harbor Lab,New York,(pp.289-358)
[2]Houdebine LM,(2003) Animal transgenesis and cloning.John Wiley & Sons,Ltd.USA.
[3]Clark AJ,(1998) The mammary gland as a bioreactor:expression,processing,and production of recombinant proteins.Journal of Mammary Gland Biology and Neoplasia,3(3):337-350
[4]Maga EA,and Murray JD(1995) Mammary gland expression of transgenes and the potential for altering the properties of milk.Bio/Technol,13:1452-1457
[5]Clarke AR,(2002) Transgenesis Techniques.2nd ed,School of Biosciences,Cardiff University Cardiff,UK(pp.305-330)
[6]A.西德-阿里吉尔,A.加西亚-卡冉卡编著,张玉静等译,微注射和转基因实验指南,科学出版社,北京,2002,第一版
[7]Israel DI.A PCR-based method for high stringency screening of DNA libraries.Nucleic[J].Acids Research.1993,21(11):2627-2631
[8]张阳德编著,生物信息学,科学出版社,北京,2004,第一版
[9]Paleyanda RK,Velander WH,Lee TK,Scandella DH,Gwazdauskas FC,Meade H,Gates L,Lacy E and Lonberg N(1990) Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk.Biotechnol 8:443-446
[10]Liu Z.,Zhao,C.J.,Fan B.et al.Variable expression of human lactoferrin gene in mice milk driven by its 90 Kb upstream-flanking sequences[J].Animal Biotechnology,2004,15(1):21-31
[11]Paleyanda R K,Velander W H,Lee T K,et al.Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Bio/technol,1990,8:443-446
[12]Houdebine L M.Transgenic animal bioreactors[J].Transgenic Res,2000,9:305-320
[13]徐宜为编著,免疫检测技术 第二版,科学出版社,1991,北京
[14]郭尧君编著,蛋白质电泳实验技术,科学出版社,1999,北京
[15]汪家政,范明主编,蛋白质技术手册,科学出版社,2000,北京
[16]Forrester WC,Fernandez LA,Grosschedl R(1999) Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer promoter interactions. Gene Dev. 13: 3003-3014.
[17] Whitelaw C B A, Accomero P, et al. Matrix attachment region regulates basal β-lactogloblin transgene expression Gene [J], 2000, 244:73-80.
[18] Alami R, Greally JM, Tanimoto K, Hwang S, Feng YQ, Engel JD et al. (2000) Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice. Hum Mol Genet 9: 631-636
[19] McKnight RA, Shamay A, Sankaran L, Wall RJ and Hennighausen L, (1992) Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice. Proc. Natl. Aacad Sci., 89:6943-6947
[20] Fedosyuk H, Peterson K R. Deletion of the human β -globin LCR 5' HS4 or 5' HS1 differentially affects β -like globin gene expression in β -YAC transgenic mice[J]. Blood Cells Mol Dis , 2007, 10:1016
[21] Orrester WC, Fernandez LA, Grosschedl R. Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer promoter interactions [J]. Gene Dev, 1999, 13:3003-3014
[22] Fedosyuk H, Peterson K R. Deletion of the human β -globin LCR 5' HS4 or 5' HS1 differentially affects β-like globin gene expression in β -YAC transgenic mice[J]. Blood Cells Mol Dis , 2007, 10:1016
[23] Bonifer C. Long-distance chromatin mechanisms controlling tissue-specific gene locus activation [J] .Gene, 1999, 238:277-289
[24] Arai Y, Ishiwata M, Baba S, et al. Neuron-specific activation of murine cytomegalovirus early gene el promoter in transgenic mice [J] . American Journal of pathology, 2003, 163(2):643-652
[25] Hallauer PL, Hastings KEM, (2000) Human cytomegalovirus IEI promoter/enhancer drives variable gene expression in all fiber types in transgenic mouse skeletal muscle. BMC Genetics, 1:1-7
[26] Boulos S, Meloni BP, Arthur PG, Bojarski C, Knuckey NW (2006) Assessment of CMV, RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures. Brain Research, 12: 1-8
[27] Gehrke S, Jerome V, Muller R. Chimeric transcriptional control units for improved liver-specific transgene expression [J] . Gene, 2003, 322:137-143
[28] Smith RL, Traul D , Schaack L, Characterization of Promoter Function and Cell-Type-Specific Expression from Viral Vectors in the Nervous System, Journal of Virology, 2000, 74:11254-11261.
[29]Konopka W,Duniec K,Mioduszewska B,hCMV and Tet promoters for inducible gene expression in rat neurons in vitro and in vivo,Neurobiology of Disease,2005,xxx-xxx.
[30]Everett RS,Evans HK,Hodges BL,Strain-specific rate of shutdown of CMVenhancer activity in murine liver confirmed by use of persistent[E1_,E2b_]adenoviral vectors,Virology,2004,325:96-105
[31]Schumacher A,Koetsier PA,Hertz J,et al.Epigenetic and genotype-specific effects on the stability of de novo imposed methylation patterns in transgenic mice[J].J Biol Chem,2000,275:37915-37921.
[32]Palmer CA,Lubon H,McManaman JL,Transgenic mice expressing recombinant human protein C exhibit defects in lactation and impaired mammary gland development[J].Transgenic Research,2003,12:283-292
[33]Kimet S J,Sohn B H,Sangkyun J,et al.High level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence[J].J Biol Chem,1999,126:320-325
[34]Ono E,Amagai K,Taharaguchi S,Tomioka Y,Yoshino S,Watanabe Y,Houdebine LM,Adam M,Eloit M,Inobe M and Uede T(2004) Transgenic mice expressing a soluble form of porcine nectin-1 herpesvirus entry mediator C as a model for seudorabies-resistant livestock[J].Proc.Natl.Aacad Sci.101(46):16150-16155
[35]De Groot N,Van Kuik-Romeijn P,Lee SH and De Boer H(1999) Over-expression of the murine polymeric immunoglobulin receptor gene in the mammary gland of transgenic mice[J].Transgenic Res.8:125-135
[36]Cerdan MG,Young JI,Zino E,Falzone TL,Otero V,Torres HN and Rubinstein M(1998)Accurate spatial and temporal transgene expression driven by a 3.8-kilobase promoter of the bovine α-casein gene in the lactating mouse mammary gland[J].Mol.Reprod.Dev.49:236-245
[37]Bonifer C(1999) Long-distance chromatin mechanisms controlling tissue-specific gene locus activation[J].Gene 238:277-289
[38]成勇,成国祥,王杏龙等,乳腺特异性基因转基因山羊表达特性的研究[J],畜牧兽医学报,1999,30(3):193-199
[39]Han YM,Kim SJ,Yu DY,et al.(1996) Studies on the generation of transgenic cow producing human lactoferrin in the milk[J].Korean J.Anim.Reprod.20:371-378
[40]Giraldo,P.and Montoliu L(2001) Size matters:use of YACs,BACs and PACs in transgenic animals[J].Transgenic Res.10:83-103
[41]Barash I,Faerman A,Richenstein M,Kari R,Damary G,Shani M and Bissell MJ(1999)In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with β -lactoglobulin and whey acidic protein promoters [J] . Mol Repro. Dev. 52: 241-252
[42] Bell AC and Felsenfeld G (1999) Stopped at the border: boundaries and insulators[J]. Cur. Opin. Gene Dev. 9:191-198
[43] Gehrke S, Jerome V, Muller R (2003) Chimeric transcriptional control units for improved liver-specific transgene expression [J] . Gene 322:137-143
[44] Whitelaw CB, Archibald AL, Harris S, McClenaghan M, Simons JP, Clark AJ. Targeting expression to the mammary gland: Intronic sequences can enhance the efficiency of gene expression in transgenic mice [J] . Transgenic Res. 1991 Dec;1(1):3-13
[45] Reid LH, Gregg RG, Smithes O, et al, Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cell [J] . Proc Natl Acad Sci USA, 1990, 87:4299-4303
[46] Dorsett D (1999) Distant liaisons: long-range enhancer-promoter interactions in Drosophila [ J] . Curr. Opin. Gene Dev. 9: 505-514
[47] Van Berkel PH, Welling MM, Geerts M, van Veen HA, Ravensbergen B, Salaheddine M, Pauwels EK, Pieper F, Nuijens JH, Nibbering PH. (2002)Large scale production of recombinant human lactoferrin in the milk of transgenic cows [ J ] . Nat Biotechnol. 20(5):484-487
[48] Sambrook, J., Fritsch, E.F., and Maniatis, T, (1989) Molecular Cloning: A Laboratory Manual. 2nd ed [M], Cold Spring Harbor Lab, New York
[49] Xu ZL, Mizuguchi H, Ishii-Watabe A, Uchida E, Mayumi T, Hayakawa T (2001) Optimization of transcriptional regulatory elements for constructing plasmid vectors [ J] . Gene 272:149-156
[50] Chen LS, Tassone F, Sahota P, Hagerman PJ (2003) The (CGG)n repeat element within the 5' untranslated region of the FMR1 message provides both positive and negative cis effects on in vivo translation of a downstream reporter [ J ] . Human molecular genetics, 12(23):3067-3074
[51] Giraldo P, Rival-Gervier S, Houdebine LM and Montoliu L (2003) The potential benefits of insulators on heterologous constructs in transgenic animals [J] . Transgenic Res., 12:751-755
[52] Lodhi K M, Ozdener M H, Shayiq R M, The upstream open reading frame mediates constitutive effects on translation of cytochrome P-450c27 from the seventh in-frame AUG codon in rat liver [J] J Biol Chem, 2003, 278:40647-40657
[53] Morris D R, Geballe A P, Upstream open reading frames as regulators of mRNA translation [J].Molecllar and Cellular Biology,2000,20:8635-8642
[54]Maga E A,and Murray J D.Mammary gland expression of transgenes and the potential for altering the properties of milk[J].Bio Technol,1995,13:1452-1457
[55]Wang CD,Lee YK,Lee YK.A functional composite cis-element for NF-kappaB and RBJKappa in the rat pregnancy-specific glycoprotein gene[J].Biology of Reproduction.2003,65:1437-1443
[56]Lu jian,Chen yu xia.Effect of glucocorticoid receptor beta on the transactivation function of glucocorticoid receptor alpha[J].Chinese Journal of Pathophysiology 2002,10
[57]Dobie K W,Lee M,Fantes J A,et al.Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus[J].Proc Natl Acad Sci,1996,93:6659-6664
[58]Lao WD,Liu W,Ceng GX,Xu SP,Cheng Y,Construction of transgenic mice producing CAT to milk by co-injection of two overlapping fragments of bovine αs1-casein-CAT gene Science in China[J](series C),1996,39:592-560
[59]Giraldo P,Montoliu L.Size matters:use of YACs,BACs and PACs in transgenic animals[J].Transgenic Res,2001,10:83-103
[60]Bonifer C.Long-distance chromatin mechanisms controlling tissue-specific gene locus activation[J].Gene,1999,238:277-289
[61]Paleyanda RK,Velander WH,Lee TK,et al,Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Bio/technol,1990,8:443-446
[62]Wall R J.Transgenic livestock:progress and prospects for the future[J].Theriogenology,1996,45:57-68
[63]Houdebine LM(2000) Transgenic animal bioreactors[J].Transgenic Res,9:305-320
[64]Rudolph NS(1999) Biopharmaceutial production in transgenic livestock.Tibtech,17:367-374
[65]Wall RJ(1996) Transgenic livestock:progress and prospects for the future[J].Theriogenology 45:57-68
[66]Goldman IL,Kadulin SG,and Razin SV(2002) Transgenic goats in the world pharmaceutical industry of the 21st century[J].Russian Journal of Genetics.38:1-14
[67]徐青,扈荣良,张守峰,梁冠生,山羊β-酪蛋白启动子指导的通用表达载体及乳腺暂时表达系统[J].中国兽医学报,2003,23:576-581
[68]成勇,徐少甫,劳为德等,牛α s1酪蛋白/HBsAg基因在山羊乳腺中暂态表达[J].扬州大学学报,1998,1(3):27-31.
[69]Ebert KM,DiTullio P,Barry CA et al.,Induction of human tissue plasminogen activator in the mammary gland of transgenic goats[J].1994,Biotechnology(N Y).12(7):699-702.
[70]Denman J,Hayes M,O'Day C et al.,Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk:purification and characterization of the recombinant enzyme [J]..Biotechnology,(N Y).1991
[1]鲍文奎,机会与风险——40年育种研究的思考[J].植物杂志,1990,4:4-5
[2]D'haeseleer P,Liang S,Somogyi R.Genetic network inference:from co-expression clustering to reverse engineering[J].Bioinformatics,2000,16:707-726.
[3]胡建广,杨金水,陈金婷,作物杂种优势的遗传学基础[J].遗传,1999,2:34-36
[4]Morris D R,Geballe A P.Upstream open reading frames as regulators of mRNA translation[J].Molecllar and Cellular Biology,2000,20:8635-8642.
[5]黄峙,周天鸿,郭宝江,NMD作用的顺式调控元件[J].遗传学报,2004,31:1321-1326
[6]Kimet S J,Sohn B H,Sangkyun J,et al.High level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence[J].J Biol Chem,1999,126:320-325
[7]Liu Z.,Zhao,C.J.,Fan B.et al.Variable expression of human lactoferrin gene in mice milk driven by its 90 Kb upstream flanking sequences[J].Animal Biotechnology,2004,15(1):21-31
[8]Lodhi K M,Ozdener M H,Shayiq R M.The upstream open reading frame mediates constitutive effects on translation of cytochrome P-450c27 from the seventh in-frame AUG codon in rat liver[J].Biol Chem,2003,278:40647-40657.
[9]Schumacher A,Koetsier PA,Hertz J,et al.Epigenetic and genotype-specific effects on the stability of de novo imposed methylation patterns in transgenic mice[J].J Biol Chem,2000,275:37915-37921.
[10]Gillio-Meina C,Hui Y Y,LaVoie HA,Expression of CCAAT/Enhancer Binding Proteins Alpha and Beta in the Porcine Ovary and Regulation in Primary Cultures of Granulosa Cells,Biology of Reproduction[J],2005,72:1194-1204
[11]Hallauer P L,Hastings KEM,Human cytomegalovirus IEI promoter/enhancer drives variable gene expression in all fiber types in transgenic mouse skeletal muscle[J].BMC Genetics,2000,1:1-7
[12]Cha-Molstad H,Young DP,Kushner I,et al.The interaction of C-Rel with C/EBPbeta enhances C/EBPbeta binding to the C-reactive protein gene promoter[J].Molecular Immunology,2007,44:2933-2942
[13]Sun YL,Lin CS,Chou YC,Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows[J].Cell Biology International,2005,29:576-582
[14]Barash I,Faerman A,Richenstein M,etal,In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with β-lactoglobulin and whey acidic protein promoters.Mol Repro.Dev.1999,52:241-252
[15]徐肖江,王连水,丁达夫,从酵母表达时间序列估计基因调控网络[J].生物化学和生物物理学报,2003,35:707-716
[2]Niemann H,Wilfried A.Application of transgenesis in livestock for agriculture and biomedicine [J].Animal Reproduction Science,2003,79:291-317.
[3]Kevin R.Gene transfer in higher animals:theoretical considerations and key concepts[J].Journal of Biotechnology,2002,99:1-22.
[4]Palmiter R D,Brinster R L,Hammer R E.Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes[J].Nature,1982,300:611-615.
[5]Das R,roduction of therapeutic proteins from transgenic animals[J].Biobusiness,2001,30:60-64.
[6]Devinoy E,Thepot D,Stinnakre M G,et al.High level production of human growth hormone in the milk of transgenic mice:the upstream region of the rabbit whey acidic protein(WAP) gene targets transgene expression to the mammary gland[J].Transgenic Research,1994,3:79-89.
[7]Goldman IL,Kadulin SG,and Razin SV(2002) Transgenic goats in the world pharmaceutical industry of the 21st century.Russian Journal of Genetics.38:1-14
[8]Jaennisch R,Germ line integration and mendelian transmission of exogenous monloney leukemia virus[J].Pro.Natl.Acad.USA,1976,73:1260.
[9]Gordon JW,Scangos FH,Genetic transformation of mouse embryos by microinjection of purified DNA[J],Proc.Natl.Acad.Sci,USA,1980,77:7380-7384.
[10]Simons,JP,McClenaghan M,Clark AJ.Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice[J].Nature,1987,328:530-532.
[11]Gordon J W,Ruddle F H.Integration and stable germ line transmission of genes Injected into mouse pronuclei[J].Science,1981,241:1244-1246.
[12]Jones P B C,European Regulators Curdle Plans for Goat Milk Human Antithrombin,ISB News Report April 2006
[13]Paleyanda R k,Velander W H,Lee T K,Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Biotechnology,1990,8:443-446.
[14]Wright G,et al.High level expression of human alpha-antitrypsin in the milk of transgenic sheep[j].Biotechnology,1991,9:830-834.
[15]Close MJ,Howlett AR,Roskelley CD,Desprez PY.Lactoferrin expression in mammary epithelial cells is mediated by changes in cell shape and actin cytoskeleton[J].Journal of Cell science,1997,110:2861-2871.
[16]Wilmut I,Schnieke A E,McWhir J.Viable offspring derived from fetal and adult mammalian cells[J].Nature,1997,385:810-813.
[17]Ogden J E.Recombinant haemoglobin in the development of red-blood-cell substitutes [J].Trends Biotechnol,1992,10:91-96.
[18]Valander W H,Johnson J L,Page R L,et al.High-level expression of aheterogonous Protein in the milk of transgenic swine using of a cDNA encoding human protein[J].PNAS USA,1992,89:12003-12007.
[19]Gordon K,Lee E,Vitale J A.Production of human tissue plasminogen activator in transgenic mouse milk[J].Bio/Technoly,1987,5:1183-1187.
[20]黄英,张克忠,黄文英,等.乳腺表达人凝血因子Ⅸ的转基因小鼠的研究[J].科学通报,1998,43(1):732-735.
[21]Anon.Transgenic cows to produce human albumin by ton[J].Biotechnol News,1997,17(8):1
[22]Rudolph N S.Biopharmaleutical production in transgenic livestocks[J].Tibtech,1999,17:367-374.
[23]Toman P D,Chisholm G,McMullin H,et al.Production of Recombinant Human Type ⅠProcollagen Homotrimer in the Mammary Gland of Transgenic Mice[J].Transgertic Res,1999,8:415-427
[24]Wilmut I,Schnieke A E,McWhir J,Kind AJ Campbell H S.Viable offspring derived from fetal and adult mammalian cells[J].Nature,1997,385:810-813.
[25]McCreath K J,Howcroft J.Production of gene-targeted sheep by nuclear transfer from cultured somatic cells[J].Nature,2000,405:1066-1069.
[26]Krimpenfort P,Rademakers A,Eyestone W,et al.Generation of transgenic dairy cattle using in vitro embryo production[J].Biotechonlogy,1991,9:844-847.
[27]施履吉,郭礼和,倪祖梅等,家兔个体表达系统的建立。遗传学报,1993,12:13-16
[28]李宁,吴常信等.牛aS1酪蛋白基因启动区的克隆和序列分析.遗传,1997,19(1):4-8
[29]郑月茂,刘凤军,张涌.转人乳铁蛋白基因山羊乳腺上皮细胞的研究[J].畜牧兽医学报,2006,37(12):1282-1286.
[30]余珊珊,王毅彰,李宁.转基因小鼠乳腺表达人乳过氧化物酶的初步研究[J],科学导报,2005,206:31-34.
[31]张忠诚,李宁,陈永福.动物乳腺生物反应器的原理及研究进展[J],中国畜牧杂志,2000,2:51-53.
[32]汤家铭,成国祥.体细胞介导制备转基因动物的新途径[J].中国实验动物学报,2002,10(3):188-192.
[33]徐曼妮,厉曙光,成国祥.乳腺生物反应器表达载体的检测方法[J].生物技术通报,2003,5:23-26.
[34]舒建洪,刘津,张涌.动物乳腺生物反应器研究进展[J].西北农林科技大学学 报,2004,32(12):149-153.
[35]曾溢滔 主编.转基因动物与医药产业[M],上海教育出版社,2000.
[36]Liu Z.,Zhao C J,Fan B.et al.Variable expression of human lactoferrin gene in mice milk driven by its 90 Kb upstream flanking sequences[J].Animal Biotechnology,2004,15(1):21-310
[37]潘登科,张运海,孙秀柱,供体细胞对猪体细胞克隆胚胎早期发育的影响,畜牧兽医学报,2006,37(4):331-336
[38]李建许,陈杰,蔡亚飞,狼山鸡AA肉鸡嵌合体的制作及其转基因鸡研究,南京农业大学学报,2007,30(2):83-88
[39]Schild C,Claret FX,Wahli W.Nucleosome-dependent static loop potentiates estrogen-regulated transcription from the Xenopus vitellogenin B1 promoter in vitro[J].EMBO,1993,12(2):423-433
[40]Silva J,Mak W,Zvetkova I,et al.Establishment of histone h3 methylation on the inactive X chromosome requirest ransient recruitment of Eed-Enxl polycomb group complexes[J].Dev -elopement Cell,2003,4(4):481-495
[41]Park Y,Kuroda M I.Epigenetic aspects of X-chromosome dosage compensation[J].Science,2001,293(5532):1083-1085
[42]Reik W,Dean W,Walter J.Epigenetic reprogramming inmammalian development[J].Science,2001,293(5532):1089-1093
[43]朱静,王应雄.染色质的重塑与基因表达调控[M].外医学临床生物化学与检验学分册,2005,26(1):46-78
[44]劳为德主编.动物细胞与转基因动物制药[M].化学工业出版社,2003
[45]李震,陈永福.乳腺生物反应器细胞模型建立的问题探讨[J].农业生物技术学报,1997,5(2):148-152
[46]Paleyanda R k,velander W H,Lee T K,Scandella D H.Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Biotechnology,1990,8:443-446
[47]John Clark A.The Mammary Gland as a Bioreactor:Expression,Processing,and Production of Recombinant Proteins.Journal of Mammary Gland Biology and Neoplasia,2001,3(3):337-350
[48]Xul H L,Qian Y P,Nie W H.Construction,characterization and chromosomal mapping of bacterial artificial chromosome library of Yunnan snub-nosed monkey(Rhinopithecus bieti)[J].Chromosome Research,2004,12:251-262
[49]Ayares D.Gene targeting in livestocks[J].Transgenic Research Conference,1999,20:557-560.
[50]Kolb A F,Ansell R,Siddell S G.Insertion of a foreign gene into the b-casein locus by Cre-mediated site-specific recombination[J].Gene,1999,227:21-31
[51]田锦,阮晖,李志敏.乳蛋白基因及其应用的研究进展[J].中国奶牛,2003,1:33-36.
[52]Waston C,Gordon C E,Robertson M,et al.Interaction of DNA-binding proteins with milk protein gene promoter in vitro:indentification of a mammary gland-specific factor[J].Nucleic Acids Research,1991,19(23):6603-6610
[53]Whitelaw C,Grolli S,Broadhurst R J.Suppression of β-casein gene expression by inhibition of protein synthesis inmousemammary epithelial cells is associated with stimulation of NF-κB activity and blockage of prolactin-Stat5 signaling[J].Cell Tissue Res,2003,311:207-215
[54]Grosclaude F,Mahe M F,Juenet R A.Mendelian polymorphism underlying quantitative variation of goat s1-casein[J].Genet,1987,19:399-412
[55]Houdebine L M.Transgenic animal bioreactors[J].Transgenic Res,2000,9:305-320
[56]http://www.bcm.edu/rosenlab/publications.html
[57]Dai Chida,Hiroshi Wakao,Atsushi Miyajima.Transcriptional Regulation of the β-Casein Gene by ytokines:Cross-Talk between STAT5 and Other Signaling[J].Molecules Molecular Endocrinology,2006,12(11):1792-1806
[58]Adam C,Buser I,Wolfgang D.Progester one receptor repressing of Prolactin/Stat5-Mediated transcription of the β-Casein Gene in Mammary Epithelial Cells[J].Molecular Endocrinology,2006,10:210-297
[59]Subramanian A,Wang J M,Gil G.STAT 5 and NF-Y are involved in expression and growth hormone-mediated sexually dimorphic regulation of cytochrome P450 3A10/lithocholic acid 6[beta]-hydroxylase[J].Nucleic Acids Research,1998,26:2173-2178
[60]周明亮,吴登俊,曾云琦.β-酪蛋白基因的研究进展[M].内蒙古农业科技,2005,2:12-14.
[61]Pervaiz S,Brew K.Homology of beta-lactoglobulin serum retinol-binding protein and protein [J].Science,1985,228(4697):335-337
[62]Hayes H C,Petit E J.Mapping of the beta-lactoglobulin gene and of an immunoglobulin M heavy chain-like sequence to homoeologous cattle sheep and goat chromosomes[J].Mamm Genome,1993,4(4):207-210
[63]Folch J M,Coll A,Sanchez A.Rapid Communication:cloning and sequencing of the cDNA encoding goat beta2 lactoglobulin[J].Anim Sci,1993,71(10):2832
[64]http://www.watsonpharm.com/
[65]高建军,颜景斌,曾溢滔.山羊β乳球蛋白基因的克隆及其在转基因小鼠乳汁中的高效表达[J].遗传学杂志,2003,25(5):499-503
[66]Whitelaw C B A,Accomero P,et al.Matrix attachment region regulates basal β-lactogloblin transgene expression Gene[J],2000,244:73-80
[67]成军.转录因子C/EBP的生物学功能[J].世界华人消化杂志,2004,12(2):408-412
[68]Beaton A,Wilkins R J,Wheeler T T.Lactation-associated and prolactin-responsive changes in protein synthesis in mouse mammary cells[J].Tissue and Cell,1997,29:509-516
[69]Kadonaga J T.Regulation of RNA polymerase Ⅱ transcription by sequence-specific DNA binding factors[J].Cell,2004,116:247-257
[70]王付龙,徐祥,刘昕.转录因子圈套策略研究进展生物化学与生物物理进展[J].2001,28(6):802-804
[71]卡尔A.平克尔特(编),劳为德(译),转基因动物技术手册[M].2004,北京化学工业出版社
[72]Morris D R,Geballe A P.Upstream open reading frames as regulators of mRNA translation[J].Molecllar and Cellular Biology,2000,20:8635-8642
[73]Whitelaw CB,Archibald AL,Harris S,McClenaghan M,Simons JP,Clark AJ.Targeting expression to the mammary gland:Intronic sequences can enhance the efficiency of gene expression in transgenic mice.Transgenic Res.1991 Dec;1(1):3-13
[74]Lodhi K M,Ozdener M H,Shayiq R M.The upstream open reading frame mediates constitutive effects on translation of cytochrome P-450c27 from the seventh in-frame AUG codon in rat liver [J].Biol Chem,2003,278:40647-40657
[75]Wakao H,Gouilleux F,Groner B.Mammary gland factor(MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response[J].EMBO,1994,13:2182-2191
[76]Yang J,Kennelly J,Baracos V E.Physiological levels of Stat5 DNA binding activity and protein in bovine mammary gland[J].Anim Sci,2000,78:3126-3134
[77]潘玲,陈建军,陈常庆.山羊β-乳球蛋白基因5'-侧区的克隆、测序和序列分析生物工程学报,1997,13(2):132-137
[78]Grosveld F.Activation by locus control regions[J].Current Opinion in Genetics &Development,1999,9:152-157
[79]Lecilas-tagar F,Bell AC,Felsenfeld G.Position enhancer-blocking activity chicken β-globin 5'-HS4 insulators in transeintly transfected cells[J].Proc Natl.Acad Sci,1999,96:14354
[80]Pantano T,Jolivet G,Prince S,et al.,Effect of the Rabbit as1-Casein Gene Distal Enhancer onthe Expression of a Reporter Gene in Vitro and in Vivo.Biochemical and Biophysical Research Communications,2002,290,53-61
[81]于占洋,任蕴芳,沈倍奋.CMV启动子和RSV LTR启动子表达sIL-6R效率的比较[J].生物技术通讯,1999,10:95-99
[82]Morris D R,Geballe A P,Upstream open reading frames as regulators of mRNA translation[J].Molecllar and Cellular Biology,2000,20:8635-8642
[83]Hallauer P L,Hastings KEM.Human cytomegalovirus IEI promoter/enhancer drives variable gene expression in all fiber types in transgenic mouse skeletal muscle[J].BMC Genetics,2000,1:1-7
[84]Gehrke S,Jerome V,Muller R(2003) Chimeric transcriptional control units for improved liver-specific transgene expression.Gene 322:137-143
[85]Konopka W,Duniec K,Mioduszewska B,et al,Promoters for inducible gene expression in rat neurons in vitro and in vivo[J],2005,10:4-14
[86]Paleyanda RK,Velander WH,Lee TK,et al,Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Bio/technol,1990,8:443-446
[87]Wall R J.Transgenic livestock:progress and prospects for the future[J].Theriogenology,1996,45:57-68
[88]Houdebine LM(2000) Transgenic animal bioreactors.Transgenic Res,9:305-320
[89]Rudolph NS(1999) Biopharmaceutial production in transgenic livestock.Tibtech,17:367-374
[90]Wall RJ(1996) Transgenic livestock:progress and prospects for the future.Theriogenology 45:57-68
[91]Kim S J,Pak K W,Pak K W.Structure of the human lactoferrin gene and its chromosomal localization[J].Molecules and Cells.1998,8:663-668
[92]卢一凡,邓继先,肖成祖,等.内含子对提高转基因动物基因表达效率的影响[J],生物化学与生物物理进展,1995,22(4):322-326.
[93]Jenuwein T,Allis CD,Translating the histone code[J],Science,2001,293(5532):1074-1080.
[94]Fisher-Adams G,Grunstein M.Yeast histone H4 and H3 N-termini have different effects on the chromatin structure of the GAL1 promoter[J].EMBO J,1995,14(7):1468-1477.
[95]朱静,王应雄,染色质的重塑与基因表达调控,国外医学临床生物化学与检验学分册,2005,26(1):12-14
[1]Houdebine L M.Transgenic animal bioreactors[J].Transgenic Res,2000,9:305-320
[2]Wall R J.Transgenic livestock:progress and prospects for the future[J].Theriogenology,1996,45:57-68
[3]Maga E A,and Murray J D.Mammary gland expression of transgenes and the potential for altering the properties of milk[J].Bio/Technol,1995,13:1452-1457
[4]Paleyanda R K,Velander W H,Lee T K,et al.Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Bio/technol,1990,8:443-446
[5]Barash I,Faerman A,Richenstein M,Kari R,Damary G,Shani M and Bissell MJ(1999)In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with β-lactoglobulin and whey acidic protein promoters.Mol Repro.Dev.52:241-252
[6]Sφrensen P,Sheffield LG,Involvement of c-sic in b-Casein Expression by Mammary Epithelial Cells.Biochemical and Biophysical Research Communications,1997,241:710-713Ding Y,Wang P,Liu S,et al.Cloning of WAP promoter and construction of mammary-specific expression vector[J].2004,3:23-28
[7]Dale T C,Krnacik M J,Schmidhauser C,et al.High level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 30 untranslated region[J].Mol Cell Biol,1992,12:905-914
[8]Bonifer C.Long-distance chromatin mechanisms controlling tissue-specific gene locus activation[J].Gene,1999,238:277-289
[9]Giraldo P,Montoliu L.Size matters:use of YACs,BACs and PACs in transgenic animals[J].TransgenicRes,2001,10:83-103
[10]Jackson D A,Juranek S,Lipps H J,Designing Nonviral Vectors for Efficient Gene Transfer,2006,Molecular Therapy,14:613-627
[11]Boulos S,Meloni BP,Arthur PG,Bojarski C,Knuckey NW(2006) Assessment of CMV,RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures.Brain Research,12:1-8
[12]Arai Y,Ishiwata M,Baba S,et al.Neuron-specific activation of murine cytomegalovirus early gene e1 promoter in transgenic mice[J].American Journal of pathology,2003,163(2):643-652
[13] Gehrke S, Jerome V, Muller R. Chimeric transcriptional control units for improved liver-specific transgene expression [J] . Gene, 2003, 322:137-143
[14] F Raptis L, SV40 Protocols[M], Humana press, p275-286
[15] Orrester WC, Fernandez LA, Grosschedl R. Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer promoter interactions [J]. Gene Dev, 1999, 13:3003-3014
[16] Fedosyuk H, Peterson K R. Deletion of the human β -globin LCR 5' HS4 or 5' HS1 differentially affects β-like globin gene expression in β -YAC transgenic mice[J]. Blood Cells Mol Dis , 2007, 10:1016
[17] Cerdan M G, Young J I, Zino E, et al. Accurate spatial and temporal transgene expression driven by a 3.8-kilobase promoter of the bovine α -casein gene in the lactating mouse mammary gland [J] .Mol Reprod Dev, 1998, 49:236-245
[18] Van Berkel P H, Welling M M, Geerts M. Large scale production of recombinant human lactoferrin in the milk of transgenic cows [J] . Nat Biotechnol, 2002, 20(5):484-487
[19] Liu Z., Zhao, C.J., Fan B. et al. Variable expression of human lactoferrin gene in mice milk driven by its 90 Kb upstream flanking sequences [J] .Animal Biotechnology, 2004, 15(1): 21-31
[20] Sambrook, J, FritschEF, and Maniatis T. Molecular Cloning: A Laboratory Manual [M] . 2nd ed, Cold Spring Harbor Lab, New York, 1989
[21] Houdebine L M, Animal transgenesis and cloning [M] . John Wiley & Sons Ltd, 2003, pp. 14-15
[22] Nagy A, Gertsenstein M, Vintersten K, et al. Manipulating the mouse embryo: a laboratory manual [M] .Cold Spring Harbor Lab, New York, 1997, pp. 289-358
[23] Meade H, Gates L. Bovine a sl-casein gene sequence direct high level expression of active human urokinase in mouse milk [J]. Biotechnology, 1990, 8:443-448
[24] Oh K B, Cho i Y H, Kang Y K, et al. A hybrid bovine beta-casein and GH gene directs transgene expression to the lung and mammary gland of transgenic m ice[J ]. T ransgenic Res, 1999, 8 (4): 307-311
[25] Paleyanda RK, Velander WH, Lee TK, Scandella DH, Gwazdauskas FC, Meade H, Gates L, Lacy E and Lonberg N (1990) Bovine alpha sl-casein gene sequences direct high level expression of active human urokinase in mouse milk. Biotechnol 8:443-446
[26] Schumacher A, Koetsier PA, Hertz J, et al. Epigenetic and genotype-specific effects on the stability of de novo imposed methylation patterns in transgenic mice[ J]. J Biol Chem, 2000 , 275:37915-37921
[27] Lao WD, Liu W, Ceng GX, Xu SP, Cheng Y, Construction of transgenic mice producing CAT to milk by co-injection of two overlapping fragments of bovine α s1-casein-CAT gene Science in China(series C),1996,39:592-560
[28]Patick H C,Van Berkel,Mick M,et al.Large scale production of recombinant human lactoferrin in the milk of transgenic cows[J].Nature Biotechnology,2002,20:484-487
[29]陈红星,杨晓,黄培堂,乳腺生物反应器制备中表达载体的发展,生物工程学报18:136-140
[30]Rey M W,Woloshuk LW,deBoer H A,et al,Complete nucleotide sequence of human mammary gland lactoferrin,1990,Nucleic Acids Research,18:5288
[31]Israel DI.A PCR-based method for high stringency screening of DNA libraries.Nucleic[J].Acids Research.1993,21(11):2627-2631
[32]Gutierrez A,M eade H M,D itullio P,et al.Expression of a bovine kappa-CN cDNA in themammary gland of transgenicm ice utilizing a genomic milk protein gene as an exp ression cassette[J].T ransgenic Res,1996,5(4):271-279
[33]N inom iya T,H irabayash iM,Sagara J,et al.Functions of milk protein in gene 5'flanking region on human growth hormone gene[J].Mol Rep rod Dev,1994,37:276-283
[34]Benlhabib H,Herrera J E,Expression of the Op18 gene is maintained by the CCAAT-binding transcription factor NF-Y[J].Gene,2006,377:177-185
[35]Petitclerc D,Attal J,Theron MC,et al,The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice
[36]Xu ZL,Mizuguchi H,Ishii-Watabe A,Uchida E,Mayumi T,Hayakawa T(2001)Optimization of transcriptional regulatory elements for constructing plasmid vectors.Gene 272:149-156
[37]Kimet S J,Sohn B H,Sangkyun J,et al.High level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence[J].J Biol Chem,1999,126:320-325
[38]劳为德主编.动物细胞与转基因动物制药[M].化学工业出版社,2003.
[39]http://www.invitrogen.com/content.cfm?pageid=1
[40]萨姆布鲁克著,金冬雁等译分子克隆实验指南,第三版,科学出版社
[41]Chen LS,Tassone F,Sahota P,Hagerman PJ,(2003) The(CGG)n repeat element within the 5' untranslated region of the FMR1 message provides both positive and negative cis effects on in vivo translation of a downstream reporter.Human molecular genetics,12(23):3067-3074
[42]卡尔A.平克尔特编,劳为德译,转基因动物技术手册,2004,化学工业出版社,北京
[43]http://www.ncbi.nlm.nih.gov/sites/entrez
[44]https://www.invitrogen.com/content/sfs/manuals/pbc1_man.pdf
[45]Kim S J,Pak K W,Pak K W.Structure of the human lactoferrin gene and its chromosomal localization[J].Molecules and Cells.1998,8:663-668
[46]http://www.invitrogen.com/content/sfs/manuals/pcep4_man.pdf
[47]Wright G,Caryer A,Cottom D,et al.High level expression of active human alpha-1-anti trypsin in the milk of transgenic sheep Bio/Technology,1991,9(9):830-834.
[48]潘玲,陈建军,陈常庆,山羊β-乳球蛋白基因5′侧区的克隆、测序和序列分析,生物工程学报,1997,13(2):132-137.
[49]Folch J M,Coll A,Sanchez A.Rapid Communication:cloning and sequencing of the cDNA encoding goat beta2 lactoglobulin[J].Anim Sci,1993,71(10):28-32
[50]高建军,颜景斌,黄英,曾溢滔,山羊β乳球蛋白基因的克隆及其在转基因小鼠乳汁中的高效表达,遗传,2003,25(5):499-503
[51]Fedosyuk H,Peterson K R.Deletion of the human β-globin LCR 5' HS4 or 5' HS1differentially affects β-like globin gene expression in β-YAC transgenic mice[J].Blood Cells Mol Dis,2007,10:10-16
[52]Alami R,Greally JM,Tanimoto K,Hwang S,Feng YQ,Engel JD et al.(2000)Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice.Hum Mol Genet 9:631-636
[53]McKnight RA,Shamay A,Sankaran L,Wall RJ and Hennighausen L,(1992)Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice.Proc.Natl.Aacad Sci.,89:6943-6947
[54]Zhujing,Yangnana,Lihao,et al.A Research on the Transformation from λ Phage Library to Plasmid Fedosyuk H,Peterson K R.Deletion of the human β-globin LCR 5' HS4 or 5'HS1 differentially affects β-like globin gene expression in β-YAC transgenic mice[J].Blood Cells Mol Dis,2007,10:1016
[55]Boulos S,Meloni BP,Arthur PG,Bojarski C,Knuckey NW(2006)Assessment of CMV,RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures.Brain Research,12:1-8
[56]Gehrke S,Jerome V,Muller R.Chimeric transcriptional control units for improved liver-specific transgene expression[J].Gene,2003,322:137-143
[57]Smith RL,Traul D,Schaack L,Characterization of Promoter Function and Cell-Type-Specific Expression from Viral Vectors in the Nervous System,Journal of Virology,2000,74:11254-11261.
[58]Konopka W,Duniec K,Mioduszewska B,hCMV and Tet promoters for inducible gene expression in rat neurons in vitro and in vivo,Neurobiology of Disease,2005,xxx-xxx.
[1] Gordon JW SG, Plotkin DJ, Barbosa JA, et al. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A. 1980; 77:7380-7384.
[2] Sanes JR, Nicolas JF. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 1986; 5: 3133-3142.
[3] Zeng Y, Huang S, Ren Z, et al, Molecular Cloning and Sequence Analysis of Goat 8-Casein Gene [J] .Chinese Journal of Biochemistry and Molecular Biology, 2003, 15:5-8
[4] Li RY, TsKosugi I, utsui Y, Activation of murine cytomegalovirus immediate-early promoter in mouse brain after transplantation of the neural stem cells [J] . Acta Neuropathol, 2004, 107:406-412.
[5] Gossler ADT, Korn R, Serfling E, Kemler R. Transgenesis by means of blstocyst-derived embryonic stem cell line. Proc Natl Acad Sci USA. 1986, 83:9065-9069
[6] Arat S, Gibbons J, Rzucidlo S. et. al. In vitro development of bovine nuclear transfer embryos from transgenic clonal lines of adult and fetal fibroblast cells of the same genotype. Biol Reprod. 2002, 66:1768-1744
[7] Zakhartchenko V, Mueller S, Alberio R. et. al. Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts. Mol Reprod Dev. 2001, 60:362-369
[8] Groner B, Transcription factor regulation in mammary epithelial cells, Domestic Animal Endocrinology, 2002, 23:25-32
[9] Zou XG, Wang YG, Cheng Y, et al, Generation of cloned goats(Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle.Molecular reproduction and development , 2002, 61:164-172
[10] Barash I, Faerman A, Richenstein M, etal, In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with β -lactoglobulin and whey acidic protein promoters. Mol Repro. Dev. 1999, 52:241-252
[11] Pantschenko A.G, Woodcock-Mitchell J, Bushmich S.I, Yang T.J. Establishment and characterization of caprine mammary epithelial cell line (GMEC). In Vitro Cellular And Development Biology, 2000.6:26-37
[12] Stelwagen K, McFadden HA, Demmer J, Prolactin, alone or in combination with glucocorticoids, enhances tight junction formation and expression of the tight junction protein occludin in mammary cells [J]. Molecular and Cellular Endocrinology, 1999, 156:55-61
[13] Lopez-Soriano J, Lopez-Soriano FJ, Carbo N, et al, Leptin administration to lactating rats is unable to induce changes in lipid metabolism in white adipose tissue or mammary gland [J]. European Journal of Obstetrics & Gynecology and Reproductive Biology,1999,84:93-97
[14]Zheng YM,An ZX,Peng XR,.Establishment of Goat Mammary Epithelial Cell Line and Expression of EGFP Gene[J].Journal of Agricultural Biotechnology.2005,03
[15]Gehrke S,Jerome V,Muller R.Chimeric transcriptional control units for improved liver-specific transgene expression[J].Gene,2003,322:137-143
[16]欧阳五庆,钱菊汾.山羊乳腺上皮细胞培养体系的建立[J].西北农林科技大学学报(自然科学版),2003,31:30-34
[17]任芳丽.山羊乳腺上皮细胞的分离与体外培养[D].西北农林科技大学2002硕士论文,陕西杨凌,p81-82.
[18]Xun Xu,Jun Han,Jun Han.Developmental expression of Smad1-7 suggests critical function of TGF-β/BMP signaling in regulating epithelial mesenchymal interaction during tooth morphogenesis[J].International Journal of Developmental Biology.2003,47:31-39
[19]Houdebine L M.Transgenic animal bioreactors[J].Transgenic Res,2000,9:305-320
[20]Wall R J.Transgenic livestock:progress and prospects for the future[J].Theriogenology,1996,45:57-68
[21]Baveye S Mazurier J Mazurier J.Lactoferrin:A multifunctional glycoprotein involved in the modulation of the inflammatory process[J].Clinical Chemistry and Laboratory Medicine.1999,37:281-286
[22]李向臣,任芳丽,张涌.山羊乳腺上皮细胞的分离、培养与超微结构[J].畜牧兽医学报,2005,36(2):122～126.Israel DI.
[23]李扬,吴凯峰,郭旭东等,脂质体介导外源基因体外转染,遗传,2002,24:653-655
[24]Tomov TCH.Quantitative dependence of electroporation on the pulse parameters.Bioelectrochemisty and Bioenergetics.1995,37:101-107
[25]萨姆布鲁克著,金冬雁等译分子克隆实验指南,第三版,科学出版社
[26]Transfection protocol(epphendorf company),L929,Protocol.No.4308 915.025-11/1999
[27]A PCR-based method for high stringency screening of DNA libraries.Nucleic[J].Acids Research.1993,21(11):2627-2631
[28]Maga E A,and Murray J D.Mammary gland expression of transgenes and the potential for altering the properties of milk[J].Bio Technol,1995,13:1452-1457
[29]徐宜为编著,免疫检测技术 第二版,科学出版社,1991,北京Wang CD,Lee YK,Lee YK.A functional composite cis-element for NF-kappaB and RBJKappa in the rat pregnancy-specific glycoprotein gene[J].Biology of Reproduction.2003,65:1437-1443
[30]http://baike.baidu.com/view/676752.htm
[31]Sun YL,Lin CS,Chou YC,Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows[J].Cell Biology International,2005,29:576-582
[32]汪和睦,谢廷栋.细胞电穿孔电融合电刺激原理及应用 天津科学技术出版社 2000.8
[33]Lu jian,Chen yu xia.Effect of glucocorticoid receptor beta on the transactivation function of glucocorticoid receptor alpha[J].Chinese Journal of Pathophysiology 2002,10
[34]Jiang xiang qing,Zhu li ping,Studies on Three-Dimensional Quantitative Structure-Activity Relationships of Nonsteroidal Progesterone Receptor Agonists 5-Benzylidene 1,2-dihydro 5H-chromeno quinolines式会社[J].Journal of Fudan University(Medical Sciences) 2005,01
[35]Email:epphemdorf@epphemdorf.com;internet:www.epphendorf.com
[36]Baum C,Forster P,Hegewisch-Becker S,Harbers K,An optimized electroporation protocol applicable to a wide range of cell lines[J].Biotechniques,1994,17:10:58-62.
[37]Baisong L,peitang H.The efferts of DNA on efficiency of electroporation[J]Prog.Biochem.Biophys,1995:256-260
[38]钱锋;肖成组,电击缓冲液对哺乳动物细胞电穿孔效率的影响[J],军事医学科学院院刊,1999,02:22-24
[39]钱锋,肖成组,影响细胞电穿孔效率的因素[J]生物技术 1998,8(5):27-31
[40]Arai Y,Ishiwata M,Baba S,et al.Neuron-specific activation of murine cytomegalovirus early gene e1 promoter in transgenic mice[J].American Journal of pathology,2003,163(2):643-652
[41]Hallauer P L,Hastings KEM,Human cytomegalovirus IEI promoter/enhancer drives variable gene expression in all fiber types in transgenic mouse skeletal muscle[J].BMC Genetics,2000,1:1-7
[42]Cha-Molstad H,Young DP,Kushner I,et al.The interaction of C-Rel with C/EBPbeta enhances C/EBPbeta binding to the C-reactive protein gene promoter[J].Molecular Immunology,2007,44:2933-2942
[43]Dobie K W,Lee M,Fantes J A,et al.Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus[J].Proc Natl Acad Sci,1996,93:6659-6664
[44]Lao WD,Liu W,Ceng GX,Xu SP,Cheng Y,Construction of transgenic mice producing CAT to milk by co-injection of two overlapping fragments of bovine α s1-casein-CAT gene Science in China(series C),1996,39:592-560.
[45]Fedosyuk H,Peterson K R.Deletion of the human β-globin LCR 5' HS4 or 5' HS1differentially affects β-like globin gene expression in β-YAC transgenic mice[J].Blood Cells Mol Dis,2007,10:1016
[46]Giraldo P,Montoliu L.Size matters:use of YACs,BACs and PACs in transgenic animals[J].Transgenic Res,2001,10:83-103
[47]Bonifer C.Long-distance chromatin mechanisms controlling tissue-specific gene locus activation[J].Gene,1999,238:277-289
[48]Sφrensen P,Sheffield LG,Involvement of c-sic in b-Casein Expression by Mammary Epithelial Cells.Biochemical and Biophysical Research Communications,1997,241:710-713.
[49]Kim S J,Pak K W,Pak K W.Structure of the human lactoferrin gene and its chromosomal localization[J].Molecules and Cells.1998,8:663-668
[50]Chen CM,Wu SC,Wu SCT.Temporal and spatial expression of biologically active human factor Ⅷ in the milk of transgenic mice driven by mammary-specific bovine alpha lactalbumin Ⅲregulator sequences[J]Transgenic Research,2002,11:257
[51]Nagy A,Gertsenstein M,Vintersten K,et al,Manipulating the mouse embryo:a laboratory manual[M].Cold Spring Harbor Lab,New York,1997,pp.289-358
[52]田锦,阮晖,牛冬,等.乳蛋白基因及其应用的研究进展[J].中国奶牛,2003,1:33-36
[53]Chen gang,Shi wei qin,Wu mei juan,et al.Cloning and sequencing of β-lactoglobulin gene of dairy goats[J],Journal of Yangzhou University(Agricultural and Life Science Edition),2001,2
[1]Nagy A,Gertsenstein M,Vintersten K,Behringer R,(1997) Manipulating the mouse embryo:a laboratory manual.Cold Spring Harbor Lab,New York,(pp.289-358)
[2]Houdebine LM,(2003) Animal transgenesis and cloning.John Wiley & Sons,Ltd.USA.
[3]Clark AJ,(1998) The mammary gland as a bioreactor:expression,processing,and production of recombinant proteins.Journal of Mammary Gland Biology and Neoplasia,3(3):337-350
[4]Maga EA,and Murray JD(1995) Mammary gland expression of transgenes and the potential for altering the properties of milk.Bio/Technol,13:1452-1457
[5]Clarke AR,(2002) Transgenesis Techniques.2nd ed,School of Biosciences,Cardiff University Cardiff,UK(pp.305-330)
[6]A.西德-阿里吉尔,A.加西亚-卡冉卡编著,张玉静等译,微注射和转基因实验指南,科学出版社,北京,2002,第一版
[7]Israel DI.A PCR-based method for high stringency screening of DNA libraries.Nucleic[J].Acids Research.1993,21(11):2627-2631
[8]张阳德编著,生物信息学,科学出版社,北京,2004,第一版
[9]Paleyanda RK,Velander WH,Lee TK,Scandella DH,Gwazdauskas FC,Meade H,Gates L,Lacy E and Lonberg N(1990) Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk.Biotechnol 8:443-446
[10]Liu Z.,Zhao,C.J.,Fan B.et al.Variable expression of human lactoferrin gene in mice milk driven by its 90 Kb upstream-flanking sequences[J].Animal Biotechnology,2004,15(1):21-31
[11]Paleyanda R K,Velander W H,Lee T K,et al.Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Bio/technol,1990,8:443-446
[12]Houdebine L M.Transgenic animal bioreactors[J].Transgenic Res,2000,9:305-320
[13]徐宜为编著,免疫检测技术 第二版,科学出版社,1991,北京
[14]郭尧君编著,蛋白质电泳实验技术,科学出版社,1999,北京
[15]汪家政,范明主编,蛋白质技术手册,科学出版社,2000,北京
[16]Forrester WC,Fernandez LA,Grosschedl R(1999) Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer promoter interactions. Gene Dev. 13: 3003-3014.
[17] Whitelaw C B A, Accomero P, et al. Matrix attachment region regulates basal β-lactogloblin transgene expression Gene [J], 2000, 244:73-80.
[18] Alami R, Greally JM, Tanimoto K, Hwang S, Feng YQ, Engel JD et al. (2000) Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice. Hum Mol Genet 9: 631-636
[19] McKnight RA, Shamay A, Sankaran L, Wall RJ and Hennighausen L, (1992) Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice. Proc. Natl. Aacad Sci., 89:6943-6947
[20] Fedosyuk H, Peterson K R. Deletion of the human β -globin LCR 5' HS4 or 5' HS1 differentially affects β -like globin gene expression in β -YAC transgenic mice[J]. Blood Cells Mol Dis , 2007, 10:1016
[21] Orrester WC, Fernandez LA, Grosschedl R. Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer promoter interactions [J]. Gene Dev, 1999, 13:3003-3014
[22] Fedosyuk H, Peterson K R. Deletion of the human β -globin LCR 5' HS4 or 5' HS1 differentially affects β-like globin gene expression in β -YAC transgenic mice[J]. Blood Cells Mol Dis , 2007, 10:1016
[23] Bonifer C. Long-distance chromatin mechanisms controlling tissue-specific gene locus activation [J] .Gene, 1999, 238:277-289
[24] Arai Y, Ishiwata M, Baba S, et al. Neuron-specific activation of murine cytomegalovirus early gene el promoter in transgenic mice [J] . American Journal of pathology, 2003, 163(2):643-652
[25] Hallauer PL, Hastings KEM, (2000) Human cytomegalovirus IEI promoter/enhancer drives variable gene expression in all fiber types in transgenic mouse skeletal muscle. BMC Genetics, 1:1-7
[26] Boulos S, Meloni BP, Arthur PG, Bojarski C, Knuckey NW (2006) Assessment of CMV, RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures. Brain Research, 12: 1-8
[27] Gehrke S, Jerome V, Muller R. Chimeric transcriptional control units for improved liver-specific transgene expression [J] . Gene, 2003, 322:137-143
[28] Smith RL, Traul D , Schaack L, Characterization of Promoter Function and Cell-Type-Specific Expression from Viral Vectors in the Nervous System, Journal of Virology, 2000, 74:11254-11261.
[29]Konopka W,Duniec K,Mioduszewska B,hCMV and Tet promoters for inducible gene expression in rat neurons in vitro and in vivo,Neurobiology of Disease,2005,xxx-xxx.
[30]Everett RS,Evans HK,Hodges BL,Strain-specific rate of shutdown of CMVenhancer activity in murine liver confirmed by use of persistent[E1_,E2b_]adenoviral vectors,Virology,2004,325:96-105
[31]Schumacher A,Koetsier PA,Hertz J,et al.Epigenetic and genotype-specific effects on the stability of de novo imposed methylation patterns in transgenic mice[J].J Biol Chem,2000,275:37915-37921.
[32]Palmer CA,Lubon H,McManaman JL,Transgenic mice expressing recombinant human protein C exhibit defects in lactation and impaired mammary gland development[J].Transgenic Research,2003,12:283-292
[33]Kimet S J,Sohn B H,Sangkyun J,et al.High level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence[J].J Biol Chem,1999,126:320-325
[34]Ono E,Amagai K,Taharaguchi S,Tomioka Y,Yoshino S,Watanabe Y,Houdebine LM,Adam M,Eloit M,Inobe M and Uede T(2004) Transgenic mice expressing a soluble form of porcine nectin-1 herpesvirus entry mediator C as a model for seudorabies-resistant livestock[J].Proc.Natl.Aacad Sci.101(46):16150-16155
[35]De Groot N,Van Kuik-Romeijn P,Lee SH and De Boer H(1999) Over-expression of the murine polymeric immunoglobulin receptor gene in the mammary gland of transgenic mice[J].Transgenic Res.8:125-135
[36]Cerdan MG,Young JI,Zino E,Falzone TL,Otero V,Torres HN and Rubinstein M(1998)Accurate spatial and temporal transgene expression driven by a 3.8-kilobase promoter of the bovine α-casein gene in the lactating mouse mammary gland[J].Mol.Reprod.Dev.49:236-245
[37]Bonifer C(1999) Long-distance chromatin mechanisms controlling tissue-specific gene locus activation[J].Gene 238:277-289
[38]成勇,成国祥,王杏龙等,乳腺特异性基因转基因山羊表达特性的研究[J],畜牧兽医学报,1999,30(3):193-199
[39]Han YM,Kim SJ,Yu DY,et al.(1996) Studies on the generation of transgenic cow producing human lactoferrin in the milk[J].Korean J.Anim.Reprod.20:371-378
[40]Giraldo,P.and Montoliu L(2001) Size matters:use of YACs,BACs and PACs in transgenic animals[J].Transgenic Res.10:83-103
[41]Barash I,Faerman A,Richenstein M,Kari R,Damary G,Shani M and Bissell MJ(1999)In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with β -lactoglobulin and whey acidic protein promoters [J] . Mol Repro. Dev. 52: 241-252
[42] Bell AC and Felsenfeld G (1999) Stopped at the border: boundaries and insulators[J]. Cur. Opin. Gene Dev. 9:191-198
[43] Gehrke S, Jerome V, Muller R (2003) Chimeric transcriptional control units for improved liver-specific transgene expression [J] . Gene 322:137-143
[44] Whitelaw CB, Archibald AL, Harris S, McClenaghan M, Simons JP, Clark AJ. Targeting expression to the mammary gland: Intronic sequences can enhance the efficiency of gene expression in transgenic mice [J] . Transgenic Res. 1991 Dec;1(1):3-13
[45] Reid LH, Gregg RG, Smithes O, et al, Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cell [J] . Proc Natl Acad Sci USA, 1990, 87:4299-4303
[46] Dorsett D (1999) Distant liaisons: long-range enhancer-promoter interactions in Drosophila [ J] . Curr. Opin. Gene Dev. 9: 505-514
[47] Van Berkel PH, Welling MM, Geerts M, van Veen HA, Ravensbergen B, Salaheddine M, Pauwels EK, Pieper F, Nuijens JH, Nibbering PH. (2002)Large scale production of recombinant human lactoferrin in the milk of transgenic cows [ J ] . Nat Biotechnol. 20(5):484-487
[48] Sambrook, J., Fritsch, E.F., and Maniatis, T, (1989) Molecular Cloning: A Laboratory Manual. 2nd ed [M], Cold Spring Harbor Lab, New York
[49] Xu ZL, Mizuguchi H, Ishii-Watabe A, Uchida E, Mayumi T, Hayakawa T (2001) Optimization of transcriptional regulatory elements for constructing plasmid vectors [ J] . Gene 272:149-156
[50] Chen LS, Tassone F, Sahota P, Hagerman PJ (2003) The (CGG)n repeat element within the 5' untranslated region of the FMR1 message provides both positive and negative cis effects on in vivo translation of a downstream reporter [ J ] . Human molecular genetics, 12(23):3067-3074
[51] Giraldo P, Rival-Gervier S, Houdebine LM and Montoliu L (2003) The potential benefits of insulators on heterologous constructs in transgenic animals [J] . Transgenic Res., 12:751-755
[52] Lodhi K M, Ozdener M H, Shayiq R M, The upstream open reading frame mediates constitutive effects on translation of cytochrome P-450c27 from the seventh in-frame AUG codon in rat liver [J] J Biol Chem, 2003, 278:40647-40657
[53] Morris D R, Geballe A P, Upstream open reading frames as regulators of mRNA translation [J].Molecllar and Cellular Biology,2000,20:8635-8642
[54]Maga E A,and Murray J D.Mammary gland expression of transgenes and the potential for altering the properties of milk[J].Bio Technol,1995,13:1452-1457
[55]Wang CD,Lee YK,Lee YK.A functional composite cis-element for NF-kappaB and RBJKappa in the rat pregnancy-specific glycoprotein gene[J].Biology of Reproduction.2003,65:1437-1443
[56]Lu jian,Chen yu xia.Effect of glucocorticoid receptor beta on the transactivation function of glucocorticoid receptor alpha[J].Chinese Journal of Pathophysiology 2002,10
[57]Dobie K W,Lee M,Fantes J A,et al.Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus[J].Proc Natl Acad Sci,1996,93:6659-6664
[58]Lao WD,Liu W,Ceng GX,Xu SP,Cheng Y,Construction of transgenic mice producing CAT to milk by co-injection of two overlapping fragments of bovine αs1-casein-CAT gene Science in China[J](series C),1996,39:592-560
[59]Giraldo P,Montoliu L.Size matters:use of YACs,BACs and PACs in transgenic animals[J].Transgenic Res,2001,10:83-103
[60]Bonifer C.Long-distance chromatin mechanisms controlling tissue-specific gene locus activation[J].Gene,1999,238:277-289
[61]Paleyanda RK,Velander WH,Lee TK,et al,Bovine alpha s1-casein gene sequences direct high level expression of active human urokinase in mouse milk[J].Bio/technol,1990,8:443-446
[62]Wall R J.Transgenic livestock:progress and prospects for the future[J].Theriogenology,1996,45:57-68
[63]Houdebine LM(2000) Transgenic animal bioreactors[J].Transgenic Res,9:305-320
[64]Rudolph NS(1999) Biopharmaceutial production in transgenic livestock.Tibtech,17:367-374
[65]Wall RJ(1996) Transgenic livestock:progress and prospects for the future[J].Theriogenology 45:57-68
[66]Goldman IL,Kadulin SG,and Razin SV(2002) Transgenic goats in the world pharmaceutical industry of the 21st century[J].Russian Journal of Genetics.38:1-14
[67]徐青,扈荣良,张守峰,梁冠生,山羊β-酪蛋白启动子指导的通用表达载体及乳腺暂时表达系统[J].中国兽医学报,2003,23:576-581
[68]成勇,徐少甫,劳为德等,牛α s1酪蛋白/HBsAg基因在山羊乳腺中暂态表达[J].扬州大学学报,1998,1(3):27-31.
[69]Ebert KM,DiTullio P,Barry CA et al.,Induction of human tissue plasminogen activator in the mammary gland of transgenic goats[J].1994,Biotechnology(N Y).12(7):699-702.
[70]Denman J,Hayes M,O'Day C et al.,Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk:purification and characterization of the recombinant enzyme [J]..Biotechnology,(N Y).1991
[1]鲍文奎,机会与风险——40年育种研究的思考[J].植物杂志,1990,4:4-5
[2]D'haeseleer P,Liang S,Somogyi R.Genetic network inference:from co-expression clustering to reverse engineering[J].Bioinformatics,2000,16:707-726.
[3]胡建广,杨金水,陈金婷,作物杂种优势的遗传学基础[J].遗传,1999,2:34-36
[4]Morris D R,Geballe A P.Upstream open reading frames as regulators of mRNA translation[J].Molecllar and Cellular Biology,2000,20:8635-8642.
[5]黄峙,周天鸿,郭宝江,NMD作用的顺式调控元件[J].遗传学报,2004,31:1321-1326
[6]Kimet S J,Sohn B H,Sangkyun J,et al.High level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence[J].J Biol Chem,1999,126:320-325
[7]Liu Z.,Zhao,C.J.,Fan B.et al.Variable expression of human lactoferrin gene in mice milk driven by its 90 Kb upstream flanking sequences[J].Animal Biotechnology,2004,15(1):21-31
[8]Lodhi K M,Ozdener M H,Shayiq R M.The upstream open reading frame mediates constitutive effects on translation of cytochrome P-450c27 from the seventh in-frame AUG codon in rat liver[J].Biol Chem,2003,278:40647-40657.
[9]Schumacher A,Koetsier PA,Hertz J,et al.Epigenetic and genotype-specific effects on the stability of de novo imposed methylation patterns in transgenic mice[J].J Biol Chem,2000,275:37915-37921.
[10]Gillio-Meina C,Hui Y Y,LaVoie HA,Expression of CCAAT/Enhancer Binding Proteins Alpha and Beta in the Porcine Ovary and Regulation in Primary Cultures of Granulosa Cells,Biology of Reproduction[J],2005,72:1194-1204
[11]Hallauer P L,Hastings KEM,Human cytomegalovirus IEI promoter/enhancer drives variable gene expression in all fiber types in transgenic mouse skeletal muscle[J].BMC Genetics,2000,1:1-7
[12]Cha-Molstad H,Young DP,Kushner I,et al.The interaction of C-Rel with C/EBPbeta enhances C/EBPbeta binding to the C-reactive protein gene promoter[J].Molecular Immunology,2007,44:2933-2942
[13]Sun YL,Lin CS,Chou YC,Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows[J].Cell Biology International,2005,29:576-582
[14]Barash I,Faerman A,Richenstein M,etal,In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with β-lactoglobulin and whey acidic protein promoters.Mol Repro.Dev.1999,52:241-252
[15]徐肖江,王连水,丁达夫,从酵母表达时间序列估计基因调控网络[J].生物化学和生物物理学报,2003,35:707-716