慢病毒载体介导转基因小鼠制备技术研究
摘要
一、研究目的及背景
转基因动物模型是联系分子、细胞水平研究和整体动物研究的桥梁。自1980年Gordon[1]首次采用原核显微注射开展动物转基因研究以来,已有十多种主要的转基因动物制作方法,如原核显微注射法、逆转录病毒侵染法、精子介导法、胚胎干细胞法,体细胞核移植法等。这些转基因动物制备方法各有优缺点:原核显微注射法制作转基因动物DNA整合效率低,整合位点不定,技术难度大,应用于高等哺乳动物时的效率很低;精子介导基因转移技术技术存在随机性和不确定性;胚胎干细胞法制备的转基因首建动物一般是嵌合体,不易实现外源基因的自然繁殖传代,且建立干细胞系的难度较大,应用受到限制;体细胞核移植法操作难度大,且外源基因的表达与否受整合位点附近基因的影响。
相对而言,慢病毒侵染法在可操作性、胚胎发育率、移植妊娠率、目的基因整合效率、目的基因表达率、阳性个体的选育等方面比上述几种技术均有显著的优势。1976年,Jaenish[2]首次用逆转录病毒(Retroviruses)感染胚胎制作转基因动物获得成功。逆转录病毒的RNA进入细胞后,逆转录成DNA前病毒,利用逆转录病毒的整合酶及其特异的末端核苷酸序列,将DNA前病毒整合到染色体上,从而将其携带的外源基因也插入到染色体上。慢病毒(lentivirus)属逆转录病毒的亚科,具有逆转录病毒的基本结构gag、pol和env,不同之处在于慢病毒包含4个辅助基因,vif、vpr、nef、vpu和2个调节基因tat和rev。其中调节基因tat和rev分别编码两个反式激活因子Tat蛋白和Rev蛋白, Tat蛋白在慢病毒基因组复制和转录延伸过程中发挥重要作用,Rev蛋白则可促使其基因的表达由早期向晚期转化。HIV-1 DNA前病毒的主要结构基因及其排列形式与其它逆转录病毒相同,为5’LTR-gag-pro-pol-env-3’LTR,其中gag基因编码病毒的核心蛋白,pol基因编码病毒复制所需的酶类,env基因编码病毒的包膜糖蛋白,pro基因则编码切割蛋白前体所需的蛋白酶。慢病毒载体(Lentiviral vector,LV)广泛用于体外细胞的转染和基因治疗的研究。目前用LV成功转染的细胞有神经细胞、视网膜感光细胞、肌细胞,肝细胞、早期胚胎细胞等,用LV制作的转基因动物有小鼠、大鼠、猪,牛等。LV的优势在于:1)转染效率高,能够感染分裂期和静息期细胞,能转染多种组织;2)整合到宿主基因组中的目的基因能持久稳定的表达,不易形成嵌合体,一般能自然繁殖传代;3)LV经改构后,不在宿主细胞内增殖,也不会导致寄主细胞的死亡,免疫源性极小,生物安全性高。LV用于转基因动物模型制备也有一定的局限性:1)一是制备难度较大,不易获得高滴度、高纯度的病毒;2) LV在宿主基因组上的整合一般是随机的,可能干扰插入位点及其附近基因表达;3)虽可通过控制病毒的滴度来调节转入基因的拷贝数,但是不容易实现整合拷贝数的精确控制;4)携带目的基因的容量较小(<10kb),当需要插入大片段DNA时,会限制制备LV的滴度;5) LV前病毒DNA在宿主基因上是多位点随机整合,当转基因启动子的表达需要多拷贝时应选择原核显微注射法。
LV技术与siRNA结合使用,可以相对容易地在转基因动物细胞中对特定基因实现RNAi效应。RNAi效应的核心是一段20-25nt的与靶基因mRNA互补或不完全互补的短RNA,通过它和靶基因mRNA碱基配对引导沉默复合体(RISC)降解mRNA或阻碍其翻译。要实现表达如此短的一段siRNA片段,转基因载体的长度可以控制在很合适的范围内(4~5kb),对插入目的基因容量有限(<10kb)的LV来说十分理想,因而LV在制备RNA干扰转基因动物模型上的优势更加明显。
本实验通过慢病毒介导高效地研制了eGFP转基因小鼠,建立了慢病毒介导的转基因小鼠制备技术体系,并利用该技术制备了树突状细胞(Dentrentic Cells,DCs)特异性干扰Rab32基因表达的RNA干扰小鼠模型,进一步验证了利用该技术体系在小鼠树突状细胞实现对特定基因进行抑制的可行性与有效性。
二、研究内容及结果
在第一部分,本课题开展了通过慢病毒载体介导、结合卵周隙显微注射、以eGFP为报告基因的转基因小鼠制备方法学研究,探讨了通过卵周隙显微注射重组慢病毒对胚胎发育的影响,以及通过此法研制转基因小鼠的效率。结果:1)建立并优化了三质粒慢病毒包装系统,获得了滴度达到1×10~9TU/ml以上的注射用重组慢病毒;通过卵周隙显微注射,胚胎的2-细胞卵裂率为81.8%(1189/1453),与无处理组无显著差异;在2-细胞期,每一视野下胚胎阳性率>90%(1178/1189),说明包装的病毒成功并高效地转染小鼠胚胎;2)将注射后发育至2-细胞的胚胎通过输卵管壶腹部穿刺移植法移植后,假孕母鼠妊娠率为42.8%(12/28) ,很大程度上避免了伞部移植法出血导致的胚胎存活率下降,提高了移植成功率;3)eGFP首建鼠阳性率为60.8%(73/120)、转基因小鼠的总体研制效率(转基因小鼠数/注射胚胎数)为5.0%(73/1453),说明卵周隙显微注射对胚胎2-细胞卵裂率几乎无影响,表明慢病毒载体辅以卵周隙显微注射法制作转基因动物高效可行;4)将eGFP阳性首建小鼠采用近交方法建系,至第6个世代,荧光观察发现超排获得的胚胎全部呈eGFP阳性,且新生鼠阳性率为100%,说明eGFP基因可以通过种系传代。
在第二部分,采用了基于siRNA(Small interfering RNA)的RNA干扰策略、利用第一部分建立的转基因平台制作转基因小鼠模型。转基因RNA干扰载体FcdGW-Rab32的表达产物siRNA-Rab32可在小鼠树突状细胞(dendritic cells,DCs)中特异性干扰Rab32基因,报告基因为eGFP。由于DCs中并没有荧光素酶基因,构建了在DCs中特异性干扰荧光素酶基因的载体质粒FcdGW-FF3作为对照。结果:1) FcdGW-Rab32和FcdGW-FF3组的2-细胞卵裂率分别为79.8%(408/511)、75.8%(317/418),受体妊娠率分别为35.7%(5/14)、33.3%(4/12),移植胚胎体内存活率分别为2.7%(11/408)、2.8%(9/317),转基因首建鼠阳性率分别为54.5%(6/11)、66.7%(6/9),转基因小鼠总体研制效率分别为1.2%(6/511)、1.4%(6/418),除胚胎体内存活率和总体研制效率外,其他数据与第一部分的结果相当;2)经PCR检测和耳廓皮肤真皮层DCs荧光观察结果表明,FcdGW-Rab32和FcdGW-FF3转基因小鼠已被建立,目的基因已经正确整合至转基因小鼠基因组中,且能通过种系传代;3)流式细胞仪检测BMDC表明,eGFP只在小鼠的DCs内特异性表达,说明我们所采用的CD11c启动子具备较好的定向表达能力;4)通过相对荧光定量PCR(Fluorenscent Quantitative PCR,FQ-PCR)对DCs的Rab32基因表达情况进行检测,结果显示Rab32基因的表达下调78.3%,特异性地实现了对DCs的Rab32基因产生Gene knock-down的干扰效应。
三、研究结论
1.本课题成功建立了以慢病毒介导、结合卵周隙显微注射、以eGFP为报告基因的转基因小鼠制备技术体系,获得了可稳定传代的eGFP转基因小鼠,证实该技术体系有较高的转基因效率。
2.将慢病毒介导的转基因动物技术与RNAi技术相结合,首次建立了在小鼠DCs特异性抑制Rab32基因的RNA干扰小鼠模型,验证了该转基因技术体系的在制备转基因小鼠模型的有效性,提示此技术路线在研究树突状细胞抗原交叉递呈的调控方面有一定的应用价值。
1. Background
Transgenic technology is an important technique plateforme for in vivo investigation of gene functions. Currently, the applicable transgene techniques include micro-injection, ES cell-mediated transgenesis, sperm-mediated gene transfer and nuclear transfer. In all the techniques mentioned above, micro-injiection is most widely used and applicable to most animal species. However, this technique is inefficient, costly and requires sophisticated equipments and skills, especially when it comes to large animal species. Lentiviral vector provides an alternative method to produce transgenic animals. Lentiviral vector is a retroviral vecor. After infection of cells, the RNA genome of the viral vector can be reverse transcribed into DNA, and the DNA can be efficiently integrated into the genome of host cells. Besides, in contrast to the congene retroviral vectors, lentivral vector is capable of infecting both deviding and quiescient cells. On the basis of this point, Lois(2002) et al successfully produced transgenic mice with lentiviral vector. In this work, the transgene delivered by lentiviral vector expressed efficiently and was not subject to silence during germline transmission.
Detrentic cell (DC) is the main professional antigen-presentation cells (APC) in human and mammal bodies. In addition to stimulating immune response, DC is also capable of inducing peripheral immune tolerance. Therefore, to understand the precise mechanism of antigene presentation, DC is the suitable target cells. The process of antigen presentation in DC is mediated by vestricle translocation, which is a highly organized process regulated by Rab gene farmily. On the basis of this point, to understand the detailed mechanism of antigen presentation, it is necessary to investigate the roles of Rab genes in the process of antigene presentation. In this work, we established a DC-specific Rab32 knock-down mouse model by lentiviral transgenesis which had significantly reduced Rab32 expression specificanlly in DCs, and laid down the foundation to further investigate the roles of Rab32 protein in antigen presentation.
2. Results
In the first part, methological researches on lentiviral transgenesis was performed. The mouse eggs was collected and subjected to perivitelline space injection (PSI) with concentrated lentiviral vectors. The effects of PSI on egg development were investigated. The results were the following:1) Through the three-plasmd packgeing system, concentrated lentivial suspension of which the TU was >1×109 was prepared; after PSI of the vector, 2-cell cleavage rate of the eggs injected with lentivral vector was 81.8%(1189/1453), with no significant difference from the untreated eggs, indicating that PSI with lentiviral vector had no impactt on the development of the injected eggs; the GFP positive rate (based on counting under microscope) was more than 90%(1178/1189), indicating that the lentiviral vector was properly packaged and capable of transducing mouse eggs efficiently; 2) the 2-cell eggs which had been subjected to PSI were transferred into pseudo-pregnant female mice, and the pregnance rate of the recipients was 42.8%(12/28), which was also comparable to the untreated egg group, suggesting that in vivo development capacity of the injected eggs was not impacted by PSI process either; 3) the transgenic rate of the founder mice was 60.8%(73/120), and the overall transgenic mouse production efficiency (transgenic mice/injected eggs) was 5.0%(73/1453), further indicating that lentiviral vector-mediated transgenesis is an efficient and practicable method for transgenic mouse production; 4) based on the eGFP transgenic founder mice, a transgenic line was established by mating the transgenic founder mice with wild-type mice of the same strain. Results indicated that transgene expression remained stable at least over six generations, suggesting that the expression of transgenes delivered by lentiviral vector can be passed on through germline transmission.
In the second part, based on RNA interference (RNAi) strategy, we produced transgenic knock-down mice targeting Rab32 gene in DCs by lentiviral transgenesis. The vector for expressing siRNA molecules was constructed based on the FUGW vector which was used in the first part. To express the siRNA molecules specifically in DCs, the siRNA coding sequence was cloned downstream the CD11c promoter. To trace the siRNA expression visibly, the eGFP coding sequence was placed downstream the same promoter in the same orientation. The results are the following: 1) the siRNA expression cassette was detected in the genomic DNAs of both the founder mice and their F1 offspring by PCR. Besides, eGFP expression was observed in the DCs located in the skin of ear concha under fluorescent microscope. These results indicated that transgenic mice haboring the siRNA expression cassette was produced. 2) By flow cytometry analysis of Bone Marrow Detrentic Cells (BMDC), it was shown that eGFP was specifically expressed in DCs, indicating that the used CD11c promoter drive siRNA expression specifically in DCs. 3) By Fluorenscent Quantitative PCR ( FQ-PCR) analysis, the Rab32 expression in DCs was shown to be reduced by 78.3%, indicating that the siRNA molecule delivered by lentiviral vector was expressed and knocked down the target gene expression significantly.
Conclusions
1. An efficient and praticable lentiviral transgenesis system, which consists of lentiviral vector package, perivitllent space injection (PSI) and mouse egg manipulation, was successfully established.
2. Using the established lentiviral transgenesis system, transgenic knock-down mice targeting Rab32 specifically in DCs was produced, further indicating that the established lentiviral transgenesis can be applied to expressing both structural genes and siRNA molecules.
转基因动物模型是联系分子、细胞水平研究和整体动物研究的桥梁。自1980年Gordon[1]首次采用原核显微注射开展动物转基因研究以来,已有十多种主要的转基因动物制作方法,如原核显微注射法、逆转录病毒侵染法、精子介导法、胚胎干细胞法,体细胞核移植法等。这些转基因动物制备方法各有优缺点:原核显微注射法制作转基因动物DNA整合效率低,整合位点不定,技术难度大,应用于高等哺乳动物时的效率很低;精子介导基因转移技术技术存在随机性和不确定性;胚胎干细胞法制备的转基因首建动物一般是嵌合体,不易实现外源基因的自然繁殖传代,且建立干细胞系的难度较大,应用受到限制;体细胞核移植法操作难度大,且外源基因的表达与否受整合位点附近基因的影响。
相对而言,慢病毒侵染法在可操作性、胚胎发育率、移植妊娠率、目的基因整合效率、目的基因表达率、阳性个体的选育等方面比上述几种技术均有显著的优势。1976年,Jaenish[2]首次用逆转录病毒(Retroviruses)感染胚胎制作转基因动物获得成功。逆转录病毒的RNA进入细胞后,逆转录成DNA前病毒,利用逆转录病毒的整合酶及其特异的末端核苷酸序列,将DNA前病毒整合到染色体上,从而将其携带的外源基因也插入到染色体上。慢病毒(lentivirus)属逆转录病毒的亚科,具有逆转录病毒的基本结构gag、pol和env,不同之处在于慢病毒包含4个辅助基因,vif、vpr、nef、vpu和2个调节基因tat和rev。其中调节基因tat和rev分别编码两个反式激活因子Tat蛋白和Rev蛋白, Tat蛋白在慢病毒基因组复制和转录延伸过程中发挥重要作用,Rev蛋白则可促使其基因的表达由早期向晚期转化。HIV-1 DNA前病毒的主要结构基因及其排列形式与其它逆转录病毒相同,为5’LTR-gag-pro-pol-env-3’LTR,其中gag基因编码病毒的核心蛋白,pol基因编码病毒复制所需的酶类,env基因编码病毒的包膜糖蛋白,pro基因则编码切割蛋白前体所需的蛋白酶。慢病毒载体(Lentiviral vector,LV)广泛用于体外细胞的转染和基因治疗的研究。目前用LV成功转染的细胞有神经细胞、视网膜感光细胞、肌细胞,肝细胞、早期胚胎细胞等,用LV制作的转基因动物有小鼠、大鼠、猪,牛等。LV的优势在于:1)转染效率高,能够感染分裂期和静息期细胞,能转染多种组织;2)整合到宿主基因组中的目的基因能持久稳定的表达,不易形成嵌合体,一般能自然繁殖传代;3)LV经改构后,不在宿主细胞内增殖,也不会导致寄主细胞的死亡,免疫源性极小,生物安全性高。LV用于转基因动物模型制备也有一定的局限性:1)一是制备难度较大,不易获得高滴度、高纯度的病毒;2) LV在宿主基因组上的整合一般是随机的,可能干扰插入位点及其附近基因表达;3)虽可通过控制病毒的滴度来调节转入基因的拷贝数,但是不容易实现整合拷贝数的精确控制;4)携带目的基因的容量较小(<10kb),当需要插入大片段DNA时,会限制制备LV的滴度;5) LV前病毒DNA在宿主基因上是多位点随机整合,当转基因启动子的表达需要多拷贝时应选择原核显微注射法。
LV技术与siRNA结合使用,可以相对容易地在转基因动物细胞中对特定基因实现RNAi效应。RNAi效应的核心是一段20-25nt的与靶基因mRNA互补或不完全互补的短RNA,通过它和靶基因mRNA碱基配对引导沉默复合体(RISC)降解mRNA或阻碍其翻译。要实现表达如此短的一段siRNA片段,转基因载体的长度可以控制在很合适的范围内(4~5kb),对插入目的基因容量有限(<10kb)的LV来说十分理想,因而LV在制备RNA干扰转基因动物模型上的优势更加明显。
本实验通过慢病毒介导高效地研制了eGFP转基因小鼠,建立了慢病毒介导的转基因小鼠制备技术体系,并利用该技术制备了树突状细胞(Dentrentic Cells,DCs)特异性干扰Rab32基因表达的RNA干扰小鼠模型,进一步验证了利用该技术体系在小鼠树突状细胞实现对特定基因进行抑制的可行性与有效性。
二、研究内容及结果
在第一部分,本课题开展了通过慢病毒载体介导、结合卵周隙显微注射、以eGFP为报告基因的转基因小鼠制备方法学研究,探讨了通过卵周隙显微注射重组慢病毒对胚胎发育的影响,以及通过此法研制转基因小鼠的效率。结果:1)建立并优化了三质粒慢病毒包装系统,获得了滴度达到1×10~9TU/ml以上的注射用重组慢病毒;通过卵周隙显微注射,胚胎的2-细胞卵裂率为81.8%(1189/1453),与无处理组无显著差异;在2-细胞期,每一视野下胚胎阳性率>90%(1178/1189),说明包装的病毒成功并高效地转染小鼠胚胎;2)将注射后发育至2-细胞的胚胎通过输卵管壶腹部穿刺移植法移植后,假孕母鼠妊娠率为42.8%(12/28) ,很大程度上避免了伞部移植法出血导致的胚胎存活率下降,提高了移植成功率;3)eGFP首建鼠阳性率为60.8%(73/120)、转基因小鼠的总体研制效率(转基因小鼠数/注射胚胎数)为5.0%(73/1453),说明卵周隙显微注射对胚胎2-细胞卵裂率几乎无影响,表明慢病毒载体辅以卵周隙显微注射法制作转基因动物高效可行;4)将eGFP阳性首建小鼠采用近交方法建系,至第6个世代,荧光观察发现超排获得的胚胎全部呈eGFP阳性,且新生鼠阳性率为100%,说明eGFP基因可以通过种系传代。
在第二部分,采用了基于siRNA(Small interfering RNA)的RNA干扰策略、利用第一部分建立的转基因平台制作转基因小鼠模型。转基因RNA干扰载体FcdGW-Rab32的表达产物siRNA-Rab32可在小鼠树突状细胞(dendritic cells,DCs)中特异性干扰Rab32基因,报告基因为eGFP。由于DCs中并没有荧光素酶基因,构建了在DCs中特异性干扰荧光素酶基因的载体质粒FcdGW-FF3作为对照。结果:1) FcdGW-Rab32和FcdGW-FF3组的2-细胞卵裂率分别为79.8%(408/511)、75.8%(317/418),受体妊娠率分别为35.7%(5/14)、33.3%(4/12),移植胚胎体内存活率分别为2.7%(11/408)、2.8%(9/317),转基因首建鼠阳性率分别为54.5%(6/11)、66.7%(6/9),转基因小鼠总体研制效率分别为1.2%(6/511)、1.4%(6/418),除胚胎体内存活率和总体研制效率外,其他数据与第一部分的结果相当;2)经PCR检测和耳廓皮肤真皮层DCs荧光观察结果表明,FcdGW-Rab32和FcdGW-FF3转基因小鼠已被建立,目的基因已经正确整合至转基因小鼠基因组中,且能通过种系传代;3)流式细胞仪检测BMDC表明,eGFP只在小鼠的DCs内特异性表达,说明我们所采用的CD11c启动子具备较好的定向表达能力;4)通过相对荧光定量PCR(Fluorenscent Quantitative PCR,FQ-PCR)对DCs的Rab32基因表达情况进行检测,结果显示Rab32基因的表达下调78.3%,特异性地实现了对DCs的Rab32基因产生Gene knock-down的干扰效应。
三、研究结论
1.本课题成功建立了以慢病毒介导、结合卵周隙显微注射、以eGFP为报告基因的转基因小鼠制备技术体系,获得了可稳定传代的eGFP转基因小鼠,证实该技术体系有较高的转基因效率。
2.将慢病毒介导的转基因动物技术与RNAi技术相结合,首次建立了在小鼠DCs特异性抑制Rab32基因的RNA干扰小鼠模型,验证了该转基因技术体系的在制备转基因小鼠模型的有效性,提示此技术路线在研究树突状细胞抗原交叉递呈的调控方面有一定的应用价值。
1. Background
Transgenic technology is an important technique plateforme for in vivo investigation of gene functions. Currently, the applicable transgene techniques include micro-injection, ES cell-mediated transgenesis, sperm-mediated gene transfer and nuclear transfer. In all the techniques mentioned above, micro-injiection is most widely used and applicable to most animal species. However, this technique is inefficient, costly and requires sophisticated equipments and skills, especially when it comes to large animal species. Lentiviral vector provides an alternative method to produce transgenic animals. Lentiviral vector is a retroviral vecor. After infection of cells, the RNA genome of the viral vector can be reverse transcribed into DNA, and the DNA can be efficiently integrated into the genome of host cells. Besides, in contrast to the congene retroviral vectors, lentivral vector is capable of infecting both deviding and quiescient cells. On the basis of this point, Lois(2002) et al successfully produced transgenic mice with lentiviral vector. In this work, the transgene delivered by lentiviral vector expressed efficiently and was not subject to silence during germline transmission.
Detrentic cell (DC) is the main professional antigen-presentation cells (APC) in human and mammal bodies. In addition to stimulating immune response, DC is also capable of inducing peripheral immune tolerance. Therefore, to understand the precise mechanism of antigene presentation, DC is the suitable target cells. The process of antigen presentation in DC is mediated by vestricle translocation, which is a highly organized process regulated by Rab gene farmily. On the basis of this point, to understand the detailed mechanism of antigen presentation, it is necessary to investigate the roles of Rab genes in the process of antigene presentation. In this work, we established a DC-specific Rab32 knock-down mouse model by lentiviral transgenesis which had significantly reduced Rab32 expression specificanlly in DCs, and laid down the foundation to further investigate the roles of Rab32 protein in antigen presentation.
2. Results
In the first part, methological researches on lentiviral transgenesis was performed. The mouse eggs was collected and subjected to perivitelline space injection (PSI) with concentrated lentiviral vectors. The effects of PSI on egg development were investigated. The results were the following:1) Through the three-plasmd packgeing system, concentrated lentivial suspension of which the TU was >1×109 was prepared; after PSI of the vector, 2-cell cleavage rate of the eggs injected with lentivral vector was 81.8%(1189/1453), with no significant difference from the untreated eggs, indicating that PSI with lentiviral vector had no impactt on the development of the injected eggs; the GFP positive rate (based on counting under microscope) was more than 90%(1178/1189), indicating that the lentiviral vector was properly packaged and capable of transducing mouse eggs efficiently; 2) the 2-cell eggs which had been subjected to PSI were transferred into pseudo-pregnant female mice, and the pregnance rate of the recipients was 42.8%(12/28), which was also comparable to the untreated egg group, suggesting that in vivo development capacity of the injected eggs was not impacted by PSI process either; 3) the transgenic rate of the founder mice was 60.8%(73/120), and the overall transgenic mouse production efficiency (transgenic mice/injected eggs) was 5.0%(73/1453), further indicating that lentiviral vector-mediated transgenesis is an efficient and practicable method for transgenic mouse production; 4) based on the eGFP transgenic founder mice, a transgenic line was established by mating the transgenic founder mice with wild-type mice of the same strain. Results indicated that transgene expression remained stable at least over six generations, suggesting that the expression of transgenes delivered by lentiviral vector can be passed on through germline transmission.
In the second part, based on RNA interference (RNAi) strategy, we produced transgenic knock-down mice targeting Rab32 gene in DCs by lentiviral transgenesis. The vector for expressing siRNA molecules was constructed based on the FUGW vector which was used in the first part. To express the siRNA molecules specifically in DCs, the siRNA coding sequence was cloned downstream the CD11c promoter. To trace the siRNA expression visibly, the eGFP coding sequence was placed downstream the same promoter in the same orientation. The results are the following: 1) the siRNA expression cassette was detected in the genomic DNAs of both the founder mice and their F1 offspring by PCR. Besides, eGFP expression was observed in the DCs located in the skin of ear concha under fluorescent microscope. These results indicated that transgenic mice haboring the siRNA expression cassette was produced. 2) By flow cytometry analysis of Bone Marrow Detrentic Cells (BMDC), it was shown that eGFP was specifically expressed in DCs, indicating that the used CD11c promoter drive siRNA expression specifically in DCs. 3) By Fluorenscent Quantitative PCR ( FQ-PCR) analysis, the Rab32 expression in DCs was shown to be reduced by 78.3%, indicating that the siRNA molecule delivered by lentiviral vector was expressed and knocked down the target gene expression significantly.
Conclusions
1. An efficient and praticable lentiviral transgenesis system, which consists of lentiviral vector package, perivitllent space injection (PSI) and mouse egg manipulation, was successfully established.
2. Using the established lentiviral transgenesis system, transgenic knock-down mice targeting Rab32 specifically in DCs was produced, further indicating that the established lentiviral transgenesis can be applied to expressing both structural genes and siRNA molecules.
引文
1. Gordon JW,Scangos AS,Plokin DJ,et al.Genetic transformation o fmouse embryos by microinjection of purified DNA.Proc Natl Acad Sci.USA,1980,77(12):7380-7384.
2. Jaenisch R,Dausman J,Cox V,et al. Infection of developing mouse embryos with murine leukemia virus:tissue specificity and genetic transmission of the virus[J]. Hamatol Bluttransfus,1976,(19):341-356.
3. Lois C, Hong E J, Pease S, et al. Germline and transmission and tissue-specific expression of transgenes delivered by lentiviral vectors[J]. Science, 2002, 295: 868-872.
4. Ikawa M,Tanaka N,Kao WWY,et al.Generation of transgenic Mice using lentiviral vectors:a novel preclinical assessment of Lentiviral vectors for gene therapy.Mol Ther,2003,8(4):666~ 673.
5. Andreas Hofmann,Barbara Kessler,Sonja Ewerling,et al.Efficient transgenesis in farm animals by lentiviral vectors. EMBO reports,2003,4(11):1054-1060.
6. Prasher DC , Eckenrode VK, Ward WW, et al . Primary structure of the Aequorea victoria green fluorescent protein [ J ] . Gene ,1992 ,111 (2) :229 - 233.
7. Inouye S , Tsuji FI . Evidence for redox forms of t he Aequorea green fluorescent protein [ J ] . FEBS Lett , 1994 , 351 ( 2) : 211 -214.
8. Chalfie M , Tu Y, Euskirchen G, et al . Green fluorescent protein as a marker for gene expression [ J ] . Science ,1994 ,263 ( 5148) :802 - 805.
9. Cheng L , Fu J , Tsukamoto A , et al . Use of green flurescent protein variant s to monitor gene t ransfer and expression in mammaliances[J ] . Nat Biotechnol ,1996 ,14 (5) : 606 - 609.
10. Nicole Maria Seibel, Jihane Eljouni, Marcus Michael Nalaskowski ,et al.Nuclear localization of enhanced green fluorescent protein homomultimers[J].Analytical Biochemistry, 2007(368):95-99.
11. Tomomi Sakai,Hidehito Tochio,Kosuke Inomata,et al.Fluoroscopic assessment of protein leakage during Xenopus oocytes in-cell NMR experiment by co-injected eGFP[J]. Analytical Biochemistry,2007(371):247-249.
12. Yoshihiro Hayashi, Katsumi Toda,Toshiji Saibara,et al. Assessment of anti-estrogenic activity of tamoxifen in transgenic mice expressing an enhanced green fluorescent protein gene regulated by estrogen response element[J].Biochimica et Biophysica Acta,2006(1760):164-171.
13. Fiona Rae,Kyra Woods,Tedjo Sasmono,et al.Characterisation and trophic functions of murineembryonic macrophages based upon the use of a Csf1r-eGFP transgene reporter[J].Developmental Biology,2007(308):232-246.
14.李小月,何金生,宋蔚等.增强型绿色荧光蛋白真核表达载体的构建和表达.中国寄生虫病防治杂志.2005(18)1:12-15.
15. Andras Nagy,et al. Manipulating the Mouse Embryo:a Laboratory Manual,third Edition.
16.魏泓,主编.医学实验动物学.四川科学技术出版社,1999:451-456.
17.王勇,刘勤,倪勇等.通过双原核显微注射提高转基因小鼠研制效率的实验研究.中国实验动物学报,2005,13(3):159-162.
18. Brigid Hogan, Rosa Beddington, Frank Costantini et al. Mouse embryo manipulation, second edition. Cold Spring Laboratory Press, 1994: 390-397.
19.陈占昆,吕厚山,蒋东芳等。携带HLA-B2704基因转基因小鼠技术的建立。中国生物化学与分子生物学报,2002,18(4):245-249.
20.张德福,陈茵,王英等。影响小鼠转基因原核胚胎体外发育的若干因素。中国兽医学报,2001,21(3):255-257.
21.王龙,杨桦,徐国江等.显微注射法制备遗传工程小鼠模型的研究.中国生物工程杂志.2003.11(23):63-67.
22.易东主编,军事医学统计学(第一版),军事医学科学出版社,2000.8:84-88.
23. Ralph L Brinster,Howard Y Chen,Myrna E Trumbauer,et al .Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci USA,1989,82 :4438～4442.
24. Akiko Hasegawa Ph.D., Nahoko Mochida M.Sc., Toshitada Ogasawara M.D.,et al. Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization[J]. Fertility and Sterility, 2006,86(Supplement 1):1182-1192.
25. Nagashima H, Fujimura T, Takahagi Y, et al. Development of efficient strategies for the production of genetically modified pigs. Theriogenology, 2003; 59(1): 95-106.
26. Wolf E,Schemthaner W,Zakhartchenko V,et al.Transgenic technology in farm animals-progress and perspectives[J].Exp Physiol,2000,85(6):615-625.
27. Plasterk.R.H.A et al. RNA silencing: the genome immune system. Science, 2002, 296:.1263-1265.
28. Gregory. J.Hannon. RNAi interference. Nature, 2002, 418:244-251.
29. Zamore. P.D et al. Ancient pathways in programmed by small RNAs. Science, 2002, 296:1265-1269.
30. Fire.A et al. RNA-triggered gene silencing. Trends in Genetics, 1999, 15:358-363.
31. Sui.G.C et al. A DNA-based RNAi technology to suppress gene expression in mammalian cells. Pro.Natl.Acad.Sci.USA, 2002, 99:5515-5520.
32. Brumelkamp.T.R et al. A system for stable expression of short-interfering RNAs in mammalian cells. Science, 2002, 96:550-553.
33. Lee.N.S et al. Expression of small interfering RNAs targeted against HIV rev transcripts in human cells. Nature Biotechnology, 2002,20:500-504.
34. Miyagishi.M and Taira. K. U6 promoter-driven siRNAs with four uridine 3’overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnology, 2002,20:497-500.
35. Elbashir.S.M et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411:494-498.
36. Calpen. N.J et al. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA, 2001, 98:9742-9749.
37. Elbashir.S.M et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosphila Melanogaster embryo lysate. EMBO. J, 2001, 20:6877-6888.
38. Zeng.Y et al. Both natural and designed mico-RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol.Cell,2 002, 9:1327-1333
39. Mcmanus. M.T et al. Gene silencing using micro-RNA designed hairpins. RNA, 2002, 8:842-850.
40. Thu-Thao.T.Pham, Douglas.S.Conklin, Gregory.J.Hanno et al. RNA interference in adult mice. Nature, 2002, 418:39-40.
41. Hidetoshi Hasuwaa, Kazuhiro Kasedab, Thorbjorg Einarsdottira, Masaru Okabea. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Letters, 2002, 532: 227-230.
42. Toshie Shinagawa and Shunsuke Ishii. Generation of Ski-knowdown mice by expression a long doub-stranded RNA from an RNA polymerase II promoter. Gene and Development, 2003, 17:1340-1345.
43. Pereira-Leal JB,Seabra MC.The mammalian Rab family of small GT-Pases:definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily .J Mol Biol,2000,301(4):1077-1087.
44. Stenmark H, Olkkonen VM. The Rab GTPase family. Genome Biol, 2001,2(5):3007.
45. Cohen-Solal, K.A., Sood, R., Marin, Y., Crespo-Carbone, S.M.,Sinsimer, D., Martino, J.J., et al., 2003. Identification and characterization of mouse Rab32 by mRNA and protein expression analysis.Biochimica et Biophysica Acta 1651(2003): 68–75.
46. 551. Jana Voigt, Jun-An Chen, Mike Gilchrist, Enrique Amaya, Nancy Papalopulu. Expression cloning screening of a unique and full-length set of cDNA clones is an efficient method for identifying genes involved in Xenopus neurogenesis. Mechanisms of Development,122 (2005):289–306.
47. Wasmeier C,Romao M,Plowright L,Bennett DC,Raposo G,Seabra MC .Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes.J Cell Biol. 2006 Oct 23;175(2):271-281. Epub 2006 Oct 16.
48. Stegmeier F, Hu G, Rickles RJ, et al. A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA, 2005, 102: 13212-13217.
49. Shin KJ, Wall EA, Zavzavadjian JR, et al. A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci USA, 2006, 103: 13759-13764.
1.王建辰,马保华.转基因技术在畜牧兽医中的发展前景.动物医学进展,2000,21(1):1-8.
2. Wall R J. New gene transfer methods[J]. Theriogenology, 2002, 57(1): 189-201.
3. Gordon JW,Scangos AS,Plokin DJ,et al.Genetic transformation o fmouse embryos by microinjection of purified DNA.Proc Natl Acad Sci.USA,1980,77(12):7380-7384.
4. Gordon JW,Ruddle FH.Integration and stable germ line trans-mission of gene injected into mouse pronuclei.Science,1981,214:1244-1246.
5. Brinster RL,Chen HY, Trumbauer ME,etal. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell,1981,27:223-231.
6. Costantini F,Lacy E. Introduction of a rabbit 5-globin gene into the mouse germ line.Nature,1981,294:92-94.
7. Palmiter RD,Brinster RL,Hammer RE,et al.Dramatic grouth of mice that develop from eggs microinjected with metallothioneingrouth hormone fusion genes[J].Biotechnology, 1992,24429-33.
8. Love J,Gribbin C,Mather C,et al.Transgenic birds by DNA microinjection[J]. Biotechnology, 1994,12(1):60-3.
9. Wolf E,Schemthaner W,Zakhartchenko V,et al.Transgenic technology in farm animals-progress and perspectives[J].Exp Physiol,2000,85(6):615-625.
10. Nagashima H, Fujimura T, Takahagi Y, et al. Development of efficient strategies for the production of genetically modified pigs. Theriogenology, 2003; 59(1): 95-106.
11. Jaenisch R,Mintz B.Simian virus 40 DNA sequence in DNAofhealthy adult mice derived from preimplantation blastocytes injectied with viral DNA[J].Proc Natl Acad Sci USA,1974,1250-1254.
12. Jaenisch R, Dausman J, Cox V, et al. Infection of developing mouse embryos with murine leukemia virus: tissue specificity and genetic transmission of the virus[J]. Hamatol Bluttransfus,1976, 19:341-356.
13. Robertson E,Bradley A,Kuehn M,et al.Germline transmission of gene introduced into cultured pluripotential cell by retroviral vectior[J].nature,1986,323:445-448.
14. Crittenden LB,Salter DW.Expression of retroviral genes in transgenic chickens[J].J Reprod Ferti(suppl),1990,41,163-167.
15. Nagano M,Shinohara T,Mary R.Retrovirus-mediated gene delivery into male germ line stemcells.FEBSLetters,2000,475:7~10.
16. Chan A W,Homan E J,Ballou L U,et al.Transgenic cattle produced by reverse-transcribed gene transfer in oocytes.Proc Natl Acad Sci USA,1998, 95(24):14028~14033.
17. Cabot R A,Kuhholzer B,ChanA W,et al,Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector.Anim Biotechnol,2001,12(2):205~214.
18. Kyle E Orwig,Mary R Avarbock, Ralph L Brinster.Retrovirus-mediated modification of male germline stem cells in rats.Biology of Reproduction,2002,67:874~879.
19. De Miguel M P,Donovan P J. Determinants of retroviral-mediated gene delivery to mouse spermatogonia.Biol Reprod,2003,68(3):860~866.
20. Trarbach T, Greifenberg S, Bardenheuer W, et al. Optimized retroviral transduction protocol for human progenitor cells utilizing fibronectin fragments. Cytotherapy, 2000, 2(6):429-38.
21. Herraiz M, Beraza N, Solano A, et al. Liver failure caused by herpes simplex virus thymidine kinase plus ganciclovir therapy is associated with mitochondrial dysfunction and mitochondrial DNA depletion. Hum Gene Ther, 2003 , 14(5):463-72.
22. Solaiman F,Zink M A,Xu G,et al.Modular retro-vector for transgenic and the rapeutic use.Molecular Reprod Develop,2000,56:309~315.
23. Fuller M, Anson DS. Helper plasmids for production of HIV-1-derived vectors.Hum Gene Ther 2001: 12(17):2081-2093.
24. Bartosch B, Cosset FL. Strategies for retargeted gene delivery using vectors derived from lentiviruses. Curr Gene Ther 2004: 4(4):427-443.
25. Deglon N, Tseng JL, Bensadoun JC, Zurn AD, Arsenijevic Y, Pereira de AL et al.Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson's disease. Hum Gene Ther 2000: 11(1):179-190.
26. Humeau LM, Binder GK, Lu X, Slepushkin V, Merling R, Echeagaray P et al.Efficient lentiviral vector-mediated control of HIV-1 replication in CD4 lymphocytes from diverse HIV+ infected patients grouped according to CD4 count and viral load. Mol Ther 2004: 9(6):902-913.
27. Pandya S, Klimatcheva E, Planelles V. Lentivirus and foamy virus vectors: novel gene therapy tools. Expert Opin Biol Ther 2001: 1(1):17-40.
28. Vigna E, Naldini L. Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy. J Gene Med 2000: 2(5):308-316.
29. Naldini L,Blomer U,Gallay P,et al.In vivo gene delivery and stable transduction of nondividing cellsby alentiviral vector.Science,1996,272:263~267.
30. Dull T,Zuffery R,Kelly M,et al,Athord-generation lentivirus Vector with a conditional packaging system.JournalofVirology,1998,72(11):8463~8471.
31. Lu X, Humeau L, Slepushkin V, Binder G, Yu Q, Slepushkina T et al. Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol. J Gene Med 2004: 6(9):963-973.
32. Ramezani A, Hawley TS, Hawley RG. Performance- and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold attachment region and the chicken beta-globin insulator. Blood 2003: 101(12):4717-4724.
33. Kappes JC, Wu X. Safety considerations in vector development. Somat Cell Mol Genet 2001: 26(1-6):147-158.
34. Kappes JC, Wu X, Wakefield JK. Production of trans-lentiviral vector with predictable safety. Methods Mol Med 2003: 76:449-465.
35. Wu X, Wakefield JK, Liu H, Xiao H, Kralovics R, Prchal JT et al. Development of a novel trans-lentiviral vector that affords predictable safety. Mol Ther 2000: 2(1):47-55.
36. Brun S, Faucon-Biguet N, Mallet J. Optimization of transgene expression at the posttranscriptional level in neural cells: implications for gene therapy. Mol Ther 2003:7(6):782-789.
37. Hawley RG. Progress toward vector design for hematopoietic stem cell gene therapy. Curr Gene Ther 2001: 1(1):1-17.
38. Leurs C, Jansen M, Pollok KE, Heinkelein M, Schmidt M, Wissler M et al. Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells. Hum Gene Ther 2003: 14(6):509-519.
39. Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A 1996: 93(21):11382-11388.
40. Nakajima T, Nakamaru K, Ido E, Terao K, Hayami M, Hasegawa M. Development of novel simian immunodeficiency virus vectors carrying a dual gene expression system. Hum Gene Ther 2000: 11(13):1863-1874.
41. Pandya S, Boris-Lawrie K, Leung NJ, Akkina R, Planelles V. Development of an Rev-independent, minimal simian immunodeficiency virus-derived vector system. Hum Gene Ther 2001: 12(7):847-857.
42. Poeschla EM, Wong-Staal F, Looney DJ. Efficient transduction of nondividing human cells by felineimmunodeficiency virus lentiviral vectors. Nat Med 1998:4(3):354-357.
43. Medina MF, Kobinger GP, Rux J, Gasmi M, Looney DJ, Bates P et al. Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung. Mol Ther 2003: 8(5):777-789.
44. Metharom P, Takyar S, Xia HH, Ellem KA, Macmillan J, Shepherd RW et al. Novel bovine lentiviral vectors based on Jembrana disease virus. J Gene Med 2000: 2(3):176-185.
45. Chang LJ, Urlacher V, Iwakuma T, Cui Y, Zucali J. Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system. Gene Ther 1999: 6(5):715-728.
46. Connolly JB. Lentiviruses in gene therapy clinical research. Gene Ther 2002: 9(24):1730-1734.
47. Kankkonen HM, Vahakangas E, Marr RA, Pakkanen T, Laurema A, Leppanen P et al. Long-term lowering of plasma cholesterol levels in LDL-receptor-deficient WHHL rabbits by gene therapy. Mol Ther 2004: 9(4):548-556.
48. Wu X, Wakefield JK, Liu H, Kappes JC. Analysis of lenti- and trans-lentiviral vector genetic recombination. Dev Biol (Basel) 2001: 106:237-248.
49. Lois C, Hong E J, Pease S, et al. Germline and transmission and tissue-specific expression of transgenes delivered by lentiviral vectors[J]. Science, 2002, 295: 868-872.
50. Pfeifer P,Ikawa M,Dayn Y,e tal.Transgenesis by lentiviral vectors:lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos.Proc Natl Acad Sci USA,2002,99:2140~2145.
51. Hamra F K.Production of transgenic rats by lentiviral transduction of male germ-line stem cells.Proc Natl Acad Sci USA,2002,99(23):14931~14936.
52. Ikawa M,Tanaka N,Kao WWY,et al.Generation of transgenic Mice using lentiviral vectors:a novel preclinical assessment of Lentiviral vectors for gene therapy.MolTher,2003,8(4):666~673.
53. Hofmann A, Kessler B, Ewerling S, et al. Efficient transgenesis in farm animals by lentiviral vectors[J]. EMBO Rep, 2003, 4(11):1054-1060.
54. Brackett BG, Boranska W, Sawicki W, et al. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc Nat Acad Sci USA, 1971, 68(1):353-357.
55. Lavitrano M, Camaioni A, Fazio V M, et al. Sperm cells as vector for introduction foreign DNA into eggs: genetic transformation of mice. Cell, 1989, 57: 717-723.
56. Brinster R N, Sandgren E P, Behringer R R, et al. No simple solution for making transgenic mice. Cell, 1989, 59: 239-241.
57. Sperandio S, Lulli V, Bacci M L, et al. Sperm– mediated DNA transfer in bovine and swine species. Anim Biotechnol, 1996, 7: 59-77.
58.武坚,刘明军,李文蓉,等.精子载体介导法生产转基因绵羊的研究.草食家畜,2001,增刊,186-202.
59. Rottmann O, Antes R, Hofer P, et al. Liposome– mediated gene transfer via sperm cells—high transfer efficiency and persistence of transgenes by use of liposomes and sperm cells and a murine amplification element. J Anim Breed Genet, 1996, 113: 401-411.
60. Kuznetsov A V, Kuznetsov I V, Schit I Y. DNA intereaction with rabbit sperm cells and its transfer into ova in vitro and in vivo. Mol Reprod Dev, 2000; 56(s):292-297.
61. Maione B, Lavitrano M, Spadafora C, et al. Sperm-mediated gene transfer in mice. Mol Reprod Dev, 1998 , 50(4):406-409.
62. Nakanishi A, Iritani A. Gene transfer in the chicken by sperm mediated methods. Mol Reprod Dev, 1993,36(2): 258-261.
63. Milne C P, Elschen F A, Collis J E, et al. Preliminary evidence for honeybee sperm mediated DNA transfer. International Symposium on Molecular Insect Science, 1989, 120-125.
64. Shamila Y, Mathavan S. Sperm– mediated gene transfer in the silkworm bombyx mori. Arch Insect Biochem Physiol, 1998, 37: 168-177.
65. Perry AC, Wakayama T, Kishikawa H, et al. Mammalian transgenesis by. intracytoplasmic sperm injection Science, 1999 , 284: 1180-1183.
66. Huang Z Y, Tamura M, Sakurai T, et al. In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially loclizaed jellyfish fluorescent protein gene. FEBS Letters, 2000, 487: 248-251.
67. Lavitrano M, Bacci ML, Forni M,et al. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation. Proc Natl Acad Sci U S A. 2002; 99(22):14230-14235.
68.黄伟民,赖良学,乔桂林,等.小鼠输精管内转染发建立人组织纤溶酶原激活剂(tPA)乳腺定位表达生物反应器的研究.实验生物学报,1999,32:227-231.
69.黄伟民,赖良学,乔桂林,等.脂质体介导外源基因体外转染精子的研究.第一军医大学学报,2000,20:216-218.
70. Wang HJ, Lin AX, Zhang ZC, et al. Expression of porcine growth hormone gene in transgenic rabbits as reported by green fluorescent protein. Anim Biotechnol, 2001 , 12(2):101-110.
71. Lavitrano. M, Bacci. M. L, Forni. M et al. Sperm mediated gene transfer in pig: Selection of donor boars and optimization of DNA uptake. Mol. Reprod. Dev, 2003, 64(3):284-291.
72. Lavitrano. M, Maione. B, Forte E et al. The Interaction of Sperm Cells with Exogenous DNA: A Role of CD4 and Major Histocompatibility Complex Class II Molecules. Exp. Cell.Res, 1997, 233(1): 56-62.
73. Celebi C, Auvray P, Benvegnu T, et al. Transient transmission of a transgene in mouse offspring following in vivo transfection of male germ cells. Mol Reprod Dev, 2002 , 62(4):477-482.
74. Jesuthasan S, Subburaju S. Gene transfer into zebrafish by sperm nuclear transplantation. Dev Biol, 2002, 242(2):88-95.
75. Chan PJ. Sperm-mediated DNA transfer to cells of the uterus and embryo. Mol Reprod Dev. 2000 Jun;56(2 Suppl):316-318.
76. Giuili G, Tomljenovic A, Labrecque N, et al. Murine spermatogonial stem cells: targeted transgene expression and purification in an active state. EMBO Rep, 2002; 3(8):753-759.
77. Evans MJ,Kaufman MH.Establishment in culture of pluripotential cells from mouse embryos.Nature,1981,292:154-156.
78. Martin GR.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.Proc Natl Acad Sci,1981,78:7634-7638.
79. Gerald D ,Fischbach and Ruth L , et al . Stem cells :science ,policy ,and ethics[J ] . J Clin Invest ,2004 ,114(10) :136421370.
80. Bradlry A,Evans M,Kaufman MH,et al.Formatin of germ-line chimaeras from embryo-derived teratocarcinoma cell lines[J].Nature,1984,309:255-256.
81. Gossler A,Doetachman T,Korn R,et al.Transgesis by means of blastocyst-derived embryonic stem cell lines[J].Proc Natal Acad USA,1986,83:9065-9069.
82. Thompson S,Clarke AR,Pow AM,et al.Germline transmission and expression of a corrected HPRT gene produced genetargetion in embryonic stem cell[J].Cell, 1989,56:313-321.
83.周江,杨晓.Smad2条件打靶种系嵌合体小鼠的获得[J].中国科学(C辑),2001,31(4):335.
84. Nakano T. Establishment, maintenance and differentiation induction of embryonic stem cells] Nippon Rinsho, 2003, 61(3):385-9.
85. Kaufman DS. A scientific rationale for human embryonic stem cell research. Yale J Health Policy Law Ethics, 2001, 2(1):109, 177-87.
86. Wilmut I, Schnieke AE, McWhir J, et al. Viable offspring derives fromfetal and admammliancell[J].Nature, 1997,385:810-813.
87. McCcreath KJ, Howcraft J, Campbell KH, et al .Production ofgene-targeted sheep by nucleartransfer from cultured somatic cells [J].Nature,2000,405:810-813.
88. Alexander B, Esmai LB, David TM,et al. Production of goat by somatic cell nuclear transfer.Nature Biotechnology,1999,17(5): 456-461.
89. Wakayama T, Pery AC, Zuccotti M,et al. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.Nature,1998,394(6691):369-374.
90. Kato Y, Tani T, Sotomaru Y,et al. Eight calves cloned from somatic cells of a single adult.Science, 1998,282(5396):2095-2098.
91. Cibelli JB, Stice SL, Golueke PJ, et al. Cloned transgenic calves produced from nonquiescent fetal fibroblasts[J].Science,1998,200:2130-2133.
92. Zakhartchenko V, Alberio R, Stojkovic M,et al. Adult cloning in cattle:potential of nuclei from a permanent cell line and from primary cultures.Mol Reprod Dev,1999,54(3):264-272.
93. Shiga K, Fujita T, Hirose K,et al.Production of calves by transfer of nuclear from cultured somatic cells obtained from Japanese black bull.Theriogenology, 1999,54(3):527-535.
94. Polejaeva IA,Chen SH,Vaught TD,et al.Cloned pigs produced by nuclear transfer from adult somatic cells.Nature,2000,407(6800):86-90.
95. Bondioli K, Ramsoondar J, William B, et al. Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar[J].Mol Reprod Dev,2001,60(2):189-195.
96.张德福,王建荣.生物工程进展[J].转基因克隆动物技术及应用,2000,20(5):25-27.
97. Anderson GB, Seidel GE. Cloning for profit.Science, 1998,280: 1400-1401.
98.李汶,卢光绣.人类植入前胚胎单细胞卵裂丘=球性别鉴定的双色荧光原位杂交技术.中华医学遗传学杂志,1999,16:255-258.
99.李建勇,过宇,薛永权,等.双色荧光原位杂交技术在异性间造血干细胞移植中的应用.中华血液学杂志,2000,18:465-468.
100. Hyttinen JM, Peura T, Tolvanen M, et al. Detection of microinjected genes in bovine preimplantation embryos with combined DNA digestion and polymerase chain reaction. Mol Reprod Dev, 1996, 43(2):150-7.
101. Rieth A, Pothier F, Gagne M, et al. Use of bovine satellite sequences to increase transgene integration by homologous recombination in bovine embryos. Mol Reprod Dev, 1999 , 53(1):1-7.
102. Ju B, Chong SW, He J, et al. Recapitulation of fast skeletal muscle development in zebrafish bytransgenic expression of GFP under the mylz2 promoter. Dev Dyn, 2003 , 227(1):14-26.
103. Tian L, Levee V, Mentag R, et al. Green fluorescent protein as a tool for monitoring transgene expression in forest tree species. Tree Physiol, 1999 , 19(8):541-546.
104. Tran ND, Liu X, Yan Z, et al. Efficiency of chimeraplast gene targeting by direct nuclear injection using a GFP recovery assay. Mol Ther, 2003, 7(2):248-53.
105. Nicole Maria Seibel, Jihane Eljouni, Marcus Michael Nalaskowski ,et al.Nuclear localization of enhanced green fluorescent protein homomultimers[J].Analytical Biochemistry, 2007(368):95-99.
106. Tomomi Sakai,Hidehito Tochio,Kosuke Inomata,et al.Fluoroscopic assessment of protein leakage during Xenopus oocytes in-cell NMR experiment by co-injected eGFP[J]. Analytical Biochemistry,2007(371):247-249.
2. Jaenisch R,Dausman J,Cox V,et al. Infection of developing mouse embryos with murine leukemia virus:tissue specificity and genetic transmission of the virus[J]. Hamatol Bluttransfus,1976,(19):341-356.
3. Lois C, Hong E J, Pease S, et al. Germline and transmission and tissue-specific expression of transgenes delivered by lentiviral vectors[J]. Science, 2002, 295: 868-872.
4. Ikawa M,Tanaka N,Kao WWY,et al.Generation of transgenic Mice using lentiviral vectors:a novel preclinical assessment of Lentiviral vectors for gene therapy.Mol Ther,2003,8(4):666~ 673.
5. Andreas Hofmann,Barbara Kessler,Sonja Ewerling,et al.Efficient transgenesis in farm animals by lentiviral vectors. EMBO reports,2003,4(11):1054-1060.
6. Prasher DC , Eckenrode VK, Ward WW, et al . Primary structure of the Aequorea victoria green fluorescent protein [ J ] . Gene ,1992 ,111 (2) :229 - 233.
7. Inouye S , Tsuji FI . Evidence for redox forms of t he Aequorea green fluorescent protein [ J ] . FEBS Lett , 1994 , 351 ( 2) : 211 -214.
8. Chalfie M , Tu Y, Euskirchen G, et al . Green fluorescent protein as a marker for gene expression [ J ] . Science ,1994 ,263 ( 5148) :802 - 805.
9. Cheng L , Fu J , Tsukamoto A , et al . Use of green flurescent protein variant s to monitor gene t ransfer and expression in mammaliances[J ] . Nat Biotechnol ,1996 ,14 (5) : 606 - 609.
10. Nicole Maria Seibel, Jihane Eljouni, Marcus Michael Nalaskowski ,et al.Nuclear localization of enhanced green fluorescent protein homomultimers[J].Analytical Biochemistry, 2007(368):95-99.
11. Tomomi Sakai,Hidehito Tochio,Kosuke Inomata,et al.Fluoroscopic assessment of protein leakage during Xenopus oocytes in-cell NMR experiment by co-injected eGFP[J]. Analytical Biochemistry,2007(371):247-249.
12. Yoshihiro Hayashi, Katsumi Toda,Toshiji Saibara,et al. Assessment of anti-estrogenic activity of tamoxifen in transgenic mice expressing an enhanced green fluorescent protein gene regulated by estrogen response element[J].Biochimica et Biophysica Acta,2006(1760):164-171.
13. Fiona Rae,Kyra Woods,Tedjo Sasmono,et al.Characterisation and trophic functions of murineembryonic macrophages based upon the use of a Csf1r-eGFP transgene reporter[J].Developmental Biology,2007(308):232-246.
14.李小月,何金生,宋蔚等.增强型绿色荧光蛋白真核表达载体的构建和表达.中国寄生虫病防治杂志.2005(18)1:12-15.
15. Andras Nagy,et al. Manipulating the Mouse Embryo:a Laboratory Manual,third Edition.
16.魏泓,主编.医学实验动物学.四川科学技术出版社,1999:451-456.
17.王勇,刘勤,倪勇等.通过双原核显微注射提高转基因小鼠研制效率的实验研究.中国实验动物学报,2005,13(3):159-162.
18. Brigid Hogan, Rosa Beddington, Frank Costantini et al. Mouse embryo manipulation, second edition. Cold Spring Laboratory Press, 1994: 390-397.
19.陈占昆,吕厚山,蒋东芳等。携带HLA-B2704基因转基因小鼠技术的建立。中国生物化学与分子生物学报,2002,18(4):245-249.
20.张德福,陈茵,王英等。影响小鼠转基因原核胚胎体外发育的若干因素。中国兽医学报,2001,21(3):255-257.
21.王龙,杨桦,徐国江等.显微注射法制备遗传工程小鼠模型的研究.中国生物工程杂志.2003.11(23):63-67.
22.易东主编,军事医学统计学(第一版),军事医学科学出版社,2000.8:84-88.
23. Ralph L Brinster,Howard Y Chen,Myrna E Trumbauer,et al .Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci USA,1989,82 :4438～4442.
24. Akiko Hasegawa Ph.D., Nahoko Mochida M.Sc., Toshitada Ogasawara M.D.,et al. Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization[J]. Fertility and Sterility, 2006,86(Supplement 1):1182-1192.
25. Nagashima H, Fujimura T, Takahagi Y, et al. Development of efficient strategies for the production of genetically modified pigs. Theriogenology, 2003; 59(1): 95-106.
26. Wolf E,Schemthaner W,Zakhartchenko V,et al.Transgenic technology in farm animals-progress and perspectives[J].Exp Physiol,2000,85(6):615-625.
27. Plasterk.R.H.A et al. RNA silencing: the genome immune system. Science, 2002, 296:.1263-1265.
28. Gregory. J.Hannon. RNAi interference. Nature, 2002, 418:244-251.
29. Zamore. P.D et al. Ancient pathways in programmed by small RNAs. Science, 2002, 296:1265-1269.
30. Fire.A et al. RNA-triggered gene silencing. Trends in Genetics, 1999, 15:358-363.
31. Sui.G.C et al. A DNA-based RNAi technology to suppress gene expression in mammalian cells. Pro.Natl.Acad.Sci.USA, 2002, 99:5515-5520.
32. Brumelkamp.T.R et al. A system for stable expression of short-interfering RNAs in mammalian cells. Science, 2002, 96:550-553.
33. Lee.N.S et al. Expression of small interfering RNAs targeted against HIV rev transcripts in human cells. Nature Biotechnology, 2002,20:500-504.
34. Miyagishi.M and Taira. K. U6 promoter-driven siRNAs with four uridine 3’overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnology, 2002,20:497-500.
35. Elbashir.S.M et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411:494-498.
36. Calpen. N.J et al. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA, 2001, 98:9742-9749.
37. Elbashir.S.M et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosphila Melanogaster embryo lysate. EMBO. J, 2001, 20:6877-6888.
38. Zeng.Y et al. Both natural and designed mico-RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol.Cell,2 002, 9:1327-1333
39. Mcmanus. M.T et al. Gene silencing using micro-RNA designed hairpins. RNA, 2002, 8:842-850.
40. Thu-Thao.T.Pham, Douglas.S.Conklin, Gregory.J.Hanno et al. RNA interference in adult mice. Nature, 2002, 418:39-40.
41. Hidetoshi Hasuwaa, Kazuhiro Kasedab, Thorbjorg Einarsdottira, Masaru Okabea. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Letters, 2002, 532: 227-230.
42. Toshie Shinagawa and Shunsuke Ishii. Generation of Ski-knowdown mice by expression a long doub-stranded RNA from an RNA polymerase II promoter. Gene and Development, 2003, 17:1340-1345.
43. Pereira-Leal JB,Seabra MC.The mammalian Rab family of small GT-Pases:definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily .J Mol Biol,2000,301(4):1077-1087.
44. Stenmark H, Olkkonen VM. The Rab GTPase family. Genome Biol, 2001,2(5):3007.
45. Cohen-Solal, K.A., Sood, R., Marin, Y., Crespo-Carbone, S.M.,Sinsimer, D., Martino, J.J., et al., 2003. Identification and characterization of mouse Rab32 by mRNA and protein expression analysis.Biochimica et Biophysica Acta 1651(2003): 68–75.
46. 551. Jana Voigt, Jun-An Chen, Mike Gilchrist, Enrique Amaya, Nancy Papalopulu. Expression cloning screening of a unique and full-length set of cDNA clones is an efficient method for identifying genes involved in Xenopus neurogenesis. Mechanisms of Development,122 (2005):289–306.
47. Wasmeier C,Romao M,Plowright L,Bennett DC,Raposo G,Seabra MC .Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes.J Cell Biol. 2006 Oct 23;175(2):271-281. Epub 2006 Oct 16.
48. Stegmeier F, Hu G, Rickles RJ, et al. A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA, 2005, 102: 13212-13217.
49. Shin KJ, Wall EA, Zavzavadjian JR, et al. A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci USA, 2006, 103: 13759-13764.
1.王建辰,马保华.转基因技术在畜牧兽医中的发展前景.动物医学进展,2000,21(1):1-8.
2. Wall R J. New gene transfer methods[J]. Theriogenology, 2002, 57(1): 189-201.
3. Gordon JW,Scangos AS,Plokin DJ,et al.Genetic transformation o fmouse embryos by microinjection of purified DNA.Proc Natl Acad Sci.USA,1980,77(12):7380-7384.
4. Gordon JW,Ruddle FH.Integration and stable germ line trans-mission of gene injected into mouse pronuclei.Science,1981,214:1244-1246.
5. Brinster RL,Chen HY, Trumbauer ME,etal. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell,1981,27:223-231.
6. Costantini F,Lacy E. Introduction of a rabbit 5-globin gene into the mouse germ line.Nature,1981,294:92-94.
7. Palmiter RD,Brinster RL,Hammer RE,et al.Dramatic grouth of mice that develop from eggs microinjected with metallothioneingrouth hormone fusion genes[J].Biotechnology, 1992,24429-33.
8. Love J,Gribbin C,Mather C,et al.Transgenic birds by DNA microinjection[J]. Biotechnology, 1994,12(1):60-3.
9. Wolf E,Schemthaner W,Zakhartchenko V,et al.Transgenic technology in farm animals-progress and perspectives[J].Exp Physiol,2000,85(6):615-625.
10. Nagashima H, Fujimura T, Takahagi Y, et al. Development of efficient strategies for the production of genetically modified pigs. Theriogenology, 2003; 59(1): 95-106.
11. Jaenisch R,Mintz B.Simian virus 40 DNA sequence in DNAofhealthy adult mice derived from preimplantation blastocytes injectied with viral DNA[J].Proc Natl Acad Sci USA,1974,1250-1254.
12. Jaenisch R, Dausman J, Cox V, et al. Infection of developing mouse embryos with murine leukemia virus: tissue specificity and genetic transmission of the virus[J]. Hamatol Bluttransfus,1976, 19:341-356.
13. Robertson E,Bradley A,Kuehn M,et al.Germline transmission of gene introduced into cultured pluripotential cell by retroviral vectior[J].nature,1986,323:445-448.
14. Crittenden LB,Salter DW.Expression of retroviral genes in transgenic chickens[J].J Reprod Ferti(suppl),1990,41,163-167.
15. Nagano M,Shinohara T,Mary R.Retrovirus-mediated gene delivery into male germ line stemcells.FEBSLetters,2000,475:7~10.
16. Chan A W,Homan E J,Ballou L U,et al.Transgenic cattle produced by reverse-transcribed gene transfer in oocytes.Proc Natl Acad Sci USA,1998, 95(24):14028~14033.
17. Cabot R A,Kuhholzer B,ChanA W,et al,Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector.Anim Biotechnol,2001,12(2):205~214.
18. Kyle E Orwig,Mary R Avarbock, Ralph L Brinster.Retrovirus-mediated modification of male germline stem cells in rats.Biology of Reproduction,2002,67:874~879.
19. De Miguel M P,Donovan P J. Determinants of retroviral-mediated gene delivery to mouse spermatogonia.Biol Reprod,2003,68(3):860~866.
20. Trarbach T, Greifenberg S, Bardenheuer W, et al. Optimized retroviral transduction protocol for human progenitor cells utilizing fibronectin fragments. Cytotherapy, 2000, 2(6):429-38.
21. Herraiz M, Beraza N, Solano A, et al. Liver failure caused by herpes simplex virus thymidine kinase plus ganciclovir therapy is associated with mitochondrial dysfunction and mitochondrial DNA depletion. Hum Gene Ther, 2003 , 14(5):463-72.
22. Solaiman F,Zink M A,Xu G,et al.Modular retro-vector for transgenic and the rapeutic use.Molecular Reprod Develop,2000,56:309~315.
23. Fuller M, Anson DS. Helper plasmids for production of HIV-1-derived vectors.Hum Gene Ther 2001: 12(17):2081-2093.
24. Bartosch B, Cosset FL. Strategies for retargeted gene delivery using vectors derived from lentiviruses. Curr Gene Ther 2004: 4(4):427-443.
25. Deglon N, Tseng JL, Bensadoun JC, Zurn AD, Arsenijevic Y, Pereira de AL et al.Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson's disease. Hum Gene Ther 2000: 11(1):179-190.
26. Humeau LM, Binder GK, Lu X, Slepushkin V, Merling R, Echeagaray P et al.Efficient lentiviral vector-mediated control of HIV-1 replication in CD4 lymphocytes from diverse HIV+ infected patients grouped according to CD4 count and viral load. Mol Ther 2004: 9(6):902-913.
27. Pandya S, Klimatcheva E, Planelles V. Lentivirus and foamy virus vectors: novel gene therapy tools. Expert Opin Biol Ther 2001: 1(1):17-40.
28. Vigna E, Naldini L. Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy. J Gene Med 2000: 2(5):308-316.
29. Naldini L,Blomer U,Gallay P,et al.In vivo gene delivery and stable transduction of nondividing cellsby alentiviral vector.Science,1996,272:263~267.
30. Dull T,Zuffery R,Kelly M,et al,Athord-generation lentivirus Vector with a conditional packaging system.JournalofVirology,1998,72(11):8463~8471.
31. Lu X, Humeau L, Slepushkin V, Binder G, Yu Q, Slepushkina T et al. Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol. J Gene Med 2004: 6(9):963-973.
32. Ramezani A, Hawley TS, Hawley RG. Performance- and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold attachment region and the chicken beta-globin insulator. Blood 2003: 101(12):4717-4724.
33. Kappes JC, Wu X. Safety considerations in vector development. Somat Cell Mol Genet 2001: 26(1-6):147-158.
34. Kappes JC, Wu X, Wakefield JK. Production of trans-lentiviral vector with predictable safety. Methods Mol Med 2003: 76:449-465.
35. Wu X, Wakefield JK, Liu H, Xiao H, Kralovics R, Prchal JT et al. Development of a novel trans-lentiviral vector that affords predictable safety. Mol Ther 2000: 2(1):47-55.
36. Brun S, Faucon-Biguet N, Mallet J. Optimization of transgene expression at the posttranscriptional level in neural cells: implications for gene therapy. Mol Ther 2003:7(6):782-789.
37. Hawley RG. Progress toward vector design for hematopoietic stem cell gene therapy. Curr Gene Ther 2001: 1(1):1-17.
38. Leurs C, Jansen M, Pollok KE, Heinkelein M, Schmidt M, Wissler M et al. Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells. Hum Gene Ther 2003: 14(6):509-519.
39. Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A 1996: 93(21):11382-11388.
40. Nakajima T, Nakamaru K, Ido E, Terao K, Hayami M, Hasegawa M. Development of novel simian immunodeficiency virus vectors carrying a dual gene expression system. Hum Gene Ther 2000: 11(13):1863-1874.
41. Pandya S, Boris-Lawrie K, Leung NJ, Akkina R, Planelles V. Development of an Rev-independent, minimal simian immunodeficiency virus-derived vector system. Hum Gene Ther 2001: 12(7):847-857.
42. Poeschla EM, Wong-Staal F, Looney DJ. Efficient transduction of nondividing human cells by felineimmunodeficiency virus lentiviral vectors. Nat Med 1998:4(3):354-357.
43. Medina MF, Kobinger GP, Rux J, Gasmi M, Looney DJ, Bates P et al. Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung. Mol Ther 2003: 8(5):777-789.
44. Metharom P, Takyar S, Xia HH, Ellem KA, Macmillan J, Shepherd RW et al. Novel bovine lentiviral vectors based on Jembrana disease virus. J Gene Med 2000: 2(3):176-185.
45. Chang LJ, Urlacher V, Iwakuma T, Cui Y, Zucali J. Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system. Gene Ther 1999: 6(5):715-728.
46. Connolly JB. Lentiviruses in gene therapy clinical research. Gene Ther 2002: 9(24):1730-1734.
47. Kankkonen HM, Vahakangas E, Marr RA, Pakkanen T, Laurema A, Leppanen P et al. Long-term lowering of plasma cholesterol levels in LDL-receptor-deficient WHHL rabbits by gene therapy. Mol Ther 2004: 9(4):548-556.
48. Wu X, Wakefield JK, Liu H, Kappes JC. Analysis of lenti- and trans-lentiviral vector genetic recombination. Dev Biol (Basel) 2001: 106:237-248.
49. Lois C, Hong E J, Pease S, et al. Germline and transmission and tissue-specific expression of transgenes delivered by lentiviral vectors[J]. Science, 2002, 295: 868-872.
50. Pfeifer P,Ikawa M,Dayn Y,e tal.Transgenesis by lentiviral vectors:lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos.Proc Natl Acad Sci USA,2002,99:2140~2145.
51. Hamra F K.Production of transgenic rats by lentiviral transduction of male germ-line stem cells.Proc Natl Acad Sci USA,2002,99(23):14931~14936.
52. Ikawa M,Tanaka N,Kao WWY,et al.Generation of transgenic Mice using lentiviral vectors:a novel preclinical assessment of Lentiviral vectors for gene therapy.MolTher,2003,8(4):666~673.
53. Hofmann A, Kessler B, Ewerling S, et al. Efficient transgenesis in farm animals by lentiviral vectors[J]. EMBO Rep, 2003, 4(11):1054-1060.
54. Brackett BG, Boranska W, Sawicki W, et al. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc Nat Acad Sci USA, 1971, 68(1):353-357.
55. Lavitrano M, Camaioni A, Fazio V M, et al. Sperm cells as vector for introduction foreign DNA into eggs: genetic transformation of mice. Cell, 1989, 57: 717-723.
56. Brinster R N, Sandgren E P, Behringer R R, et al. No simple solution for making transgenic mice. Cell, 1989, 59: 239-241.
57. Sperandio S, Lulli V, Bacci M L, et al. Sperm– mediated DNA transfer in bovine and swine species. Anim Biotechnol, 1996, 7: 59-77.
58.武坚,刘明军,李文蓉,等.精子载体介导法生产转基因绵羊的研究.草食家畜,2001,增刊,186-202.
59. Rottmann O, Antes R, Hofer P, et al. Liposome– mediated gene transfer via sperm cells—high transfer efficiency and persistence of transgenes by use of liposomes and sperm cells and a murine amplification element. J Anim Breed Genet, 1996, 113: 401-411.
60. Kuznetsov A V, Kuznetsov I V, Schit I Y. DNA intereaction with rabbit sperm cells and its transfer into ova in vitro and in vivo. Mol Reprod Dev, 2000; 56(s):292-297.
61. Maione B, Lavitrano M, Spadafora C, et al. Sperm-mediated gene transfer in mice. Mol Reprod Dev, 1998 , 50(4):406-409.
62. Nakanishi A, Iritani A. Gene transfer in the chicken by sperm mediated methods. Mol Reprod Dev, 1993,36(2): 258-261.
63. Milne C P, Elschen F A, Collis J E, et al. Preliminary evidence for honeybee sperm mediated DNA transfer. International Symposium on Molecular Insect Science, 1989, 120-125.
64. Shamila Y, Mathavan S. Sperm– mediated gene transfer in the silkworm bombyx mori. Arch Insect Biochem Physiol, 1998, 37: 168-177.
65. Perry AC, Wakayama T, Kishikawa H, et al. Mammalian transgenesis by. intracytoplasmic sperm injection Science, 1999 , 284: 1180-1183.
66. Huang Z Y, Tamura M, Sakurai T, et al. In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially loclizaed jellyfish fluorescent protein gene. FEBS Letters, 2000, 487: 248-251.
67. Lavitrano M, Bacci ML, Forni M,et al. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation. Proc Natl Acad Sci U S A. 2002; 99(22):14230-14235.
68.黄伟民,赖良学,乔桂林,等.小鼠输精管内转染发建立人组织纤溶酶原激活剂(tPA)乳腺定位表达生物反应器的研究.实验生物学报,1999,32:227-231.
69.黄伟民,赖良学,乔桂林,等.脂质体介导外源基因体外转染精子的研究.第一军医大学学报,2000,20:216-218.
70. Wang HJ, Lin AX, Zhang ZC, et al. Expression of porcine growth hormone gene in transgenic rabbits as reported by green fluorescent protein. Anim Biotechnol, 2001 , 12(2):101-110.
71. Lavitrano. M, Bacci. M. L, Forni. M et al. Sperm mediated gene transfer in pig: Selection of donor boars and optimization of DNA uptake. Mol. Reprod. Dev, 2003, 64(3):284-291.
72. Lavitrano. M, Maione. B, Forte E et al. The Interaction of Sperm Cells with Exogenous DNA: A Role of CD4 and Major Histocompatibility Complex Class II Molecules. Exp. Cell.Res, 1997, 233(1): 56-62.
73. Celebi C, Auvray P, Benvegnu T, et al. Transient transmission of a transgene in mouse offspring following in vivo transfection of male germ cells. Mol Reprod Dev, 2002 , 62(4):477-482.
74. Jesuthasan S, Subburaju S. Gene transfer into zebrafish by sperm nuclear transplantation. Dev Biol, 2002, 242(2):88-95.
75. Chan PJ. Sperm-mediated DNA transfer to cells of the uterus and embryo. Mol Reprod Dev. 2000 Jun;56(2 Suppl):316-318.
76. Giuili G, Tomljenovic A, Labrecque N, et al. Murine spermatogonial stem cells: targeted transgene expression and purification in an active state. EMBO Rep, 2002; 3(8):753-759.
77. Evans MJ,Kaufman MH.Establishment in culture of pluripotential cells from mouse embryos.Nature,1981,292:154-156.
78. Martin GR.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.Proc Natl Acad Sci,1981,78:7634-7638.
79. Gerald D ,Fischbach and Ruth L , et al . Stem cells :science ,policy ,and ethics[J ] . J Clin Invest ,2004 ,114(10) :136421370.
80. Bradlry A,Evans M,Kaufman MH,et al.Formatin of germ-line chimaeras from embryo-derived teratocarcinoma cell lines[J].Nature,1984,309:255-256.
81. Gossler A,Doetachman T,Korn R,et al.Transgesis by means of blastocyst-derived embryonic stem cell lines[J].Proc Natal Acad USA,1986,83:9065-9069.
82. Thompson S,Clarke AR,Pow AM,et al.Germline transmission and expression of a corrected HPRT gene produced genetargetion in embryonic stem cell[J].Cell, 1989,56:313-321.
83.周江,杨晓.Smad2条件打靶种系嵌合体小鼠的获得[J].中国科学(C辑),2001,31(4):335.
84. Nakano T. Establishment, maintenance and differentiation induction of embryonic stem cells] Nippon Rinsho, 2003, 61(3):385-9.
85. Kaufman DS. A scientific rationale for human embryonic stem cell research. Yale J Health Policy Law Ethics, 2001, 2(1):109, 177-87.
86. Wilmut I, Schnieke AE, McWhir J, et al. Viable offspring derives fromfetal and admammliancell[J].Nature, 1997,385:810-813.
87. McCcreath KJ, Howcraft J, Campbell KH, et al .Production ofgene-targeted sheep by nucleartransfer from cultured somatic cells [J].Nature,2000,405:810-813.
88. Alexander B, Esmai LB, David TM,et al. Production of goat by somatic cell nuclear transfer.Nature Biotechnology,1999,17(5): 456-461.
89. Wakayama T, Pery AC, Zuccotti M,et al. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.Nature,1998,394(6691):369-374.
90. Kato Y, Tani T, Sotomaru Y,et al. Eight calves cloned from somatic cells of a single adult.Science, 1998,282(5396):2095-2098.
91. Cibelli JB, Stice SL, Golueke PJ, et al. Cloned transgenic calves produced from nonquiescent fetal fibroblasts[J].Science,1998,200:2130-2133.
92. Zakhartchenko V, Alberio R, Stojkovic M,et al. Adult cloning in cattle:potential of nuclei from a permanent cell line and from primary cultures.Mol Reprod Dev,1999,54(3):264-272.
93. Shiga K, Fujita T, Hirose K,et al.Production of calves by transfer of nuclear from cultured somatic cells obtained from Japanese black bull.Theriogenology, 1999,54(3):527-535.
94. Polejaeva IA,Chen SH,Vaught TD,et al.Cloned pigs produced by nuclear transfer from adult somatic cells.Nature,2000,407(6800):86-90.
95. Bondioli K, Ramsoondar J, William B, et al. Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar[J].Mol Reprod Dev,2001,60(2):189-195.
96.张德福,王建荣.生物工程进展[J].转基因克隆动物技术及应用,2000,20(5):25-27.
97. Anderson GB, Seidel GE. Cloning for profit.Science, 1998,280: 1400-1401.
98.李汶,卢光绣.人类植入前胚胎单细胞卵裂丘=球性别鉴定的双色荧光原位杂交技术.中华医学遗传学杂志,1999,16:255-258.
99.李建勇,过宇,薛永权,等.双色荧光原位杂交技术在异性间造血干细胞移植中的应用.中华血液学杂志,2000,18:465-468.
100. Hyttinen JM, Peura T, Tolvanen M, et al. Detection of microinjected genes in bovine preimplantation embryos with combined DNA digestion and polymerase chain reaction. Mol Reprod Dev, 1996, 43(2):150-7.
101. Rieth A, Pothier F, Gagne M, et al. Use of bovine satellite sequences to increase transgene integration by homologous recombination in bovine embryos. Mol Reprod Dev, 1999 , 53(1):1-7.
102. Ju B, Chong SW, He J, et al. Recapitulation of fast skeletal muscle development in zebrafish bytransgenic expression of GFP under the mylz2 promoter. Dev Dyn, 2003 , 227(1):14-26.
103. Tian L, Levee V, Mentag R, et al. Green fluorescent protein as a tool for monitoring transgene expression in forest tree species. Tree Physiol, 1999 , 19(8):541-546.
104. Tran ND, Liu X, Yan Z, et al. Efficiency of chimeraplast gene targeting by direct nuclear injection using a GFP recovery assay. Mol Ther, 2003, 7(2):248-53.
105. Nicole Maria Seibel, Jihane Eljouni, Marcus Michael Nalaskowski ,et al.Nuclear localization of enhanced green fluorescent protein homomultimers[J].Analytical Biochemistry, 2007(368):95-99.
106. Tomomi Sakai,Hidehito Tochio,Kosuke Inomata,et al.Fluoroscopic assessment of protein leakage during Xenopus oocytes in-cell NMR experiment by co-injected eGFP[J]. Analytical Biochemistry,2007(371):247-249.