猪PNAS-4基因的结构与功能研究及α1，3-GT基因敲除载体的构建
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摘要
贯穿于机体整个生命周期的细胞凋亡对机体的发育、存活及维持正常生理功能都有着重要意义，尤其是在个体发育、细胞分化、组织器官形成过程中都发挥着重要作用。而骨骼肌在不同的生长发育过程中涉及大批基因的差异表达及网络调控，而这些差异表达的基因很可能在生长发育过程中调控着某些细胞的增殖与凋亡。
根据该推理假设，本研究从构建的猪胎儿骨胳肌cDNA文库中挑选出一个在组织及不同发育时期都有差异表达的未知基因作为研究对象，并通过分子生物学、细胞生物学以及生物信息学等方法从核酸及蛋白质两个水平上进行了结构与功能研究，并取得了如下研究结果：
1．猪的PNAS-4基因cDNA全长序列的获得
根据已获得的该基因部分信息，采用电脑克隆策略结合RACE技术，获得了猪的PNAS-4基因的5’非翻译区序列，然后通过PCR方法填补了序列中一个82bp的Gap，从而得到该基因的cDNA全长4133bp，并提交GenBank数据库，GenBank收录号DQ435075；
2．cDNA序列上的功能元件分析
对猪的PNAS-4 cDNA序列进行分析，发现该基因的在5’UTR区有一内源性核糖体进入位点(IRES)，而在3’UTR区除了有两个多(聚)腺苷酸加尾信号(AATAAA)外，还存在着两个功能域框Brd-Box和GY-Box，其中Brd-Box通过影响转录的稳定性和翻译的效率来介导转录后负调控；
3．人的同源基因clorf121的启动子区域调控因子的分析
利用人的同源基因clorf121的启动子区域进行网络调控分析时发现，有一个CpG岛附近有着较多的转录因子(Sp1、AP-1)的结合点，说明该基因具有较强的转录效率，另外还发现在启动子区域有较多的抑凋亡因子结合位点。根据其CpG岛的GC含量高及无TATA box的特征推测该基因是一个无处不表达的持家基因；
4．推导的该编码的基因蛋白质特点
由该基因CDS序列推导出猪的PNAS-4编码着是一个含有194个氨基酸残基，是一个既无信号肽又无跨膜结构的非分泌性蛋白质；并对其保守的功能域DUF862进行了三维结构的模拟，发现其有三个可能的蛋白质结合域以及三个裸露在外的氨基酸残基；同源比对后，发现该基因在哺乳动物之间同源性极高达96-98％，与家禽及非洲蟾蜍分别有着94％和74％的相似性，甚至拟南芥的该蛋白质与猪也有着45％的相似性；
5．多态检测及连锁不平衡分析
利用比较基因组学通过PCR方法获得猪的该基因第二内含子全长，并提交GenBank数据库，GenBank收录号DQ406743。通过PCR-RFLP及DHPLC技术对第二内含子及3’UTR区共计四个SNPs位点进行多态分析，发现这四个多态位点在不同品种中表现出等位基因频率的差异。这四个位点虽相隔20Kb，但却不同程度地相互处于连锁不平衡状态。通过性状关联分析，发现该基因不同基因型个体在内脂率、板油率、腿臀肉骨率、胴体直长、眼肌面积以及血红蛋白浓度等性状存在显著性差异(P＜0.05)；
6．应用定量PCR进行时空表达谱分析
通过real-time PCR方法研究了猪的PNAS-4基因在成年猪15个组织中的表达情况，发现该基因在所研究的组织中均有表达，但存在表达量的差异，其中，骨胳肌和淋巴组织中表达量最高，肾和大脑中表达量最低。另外还发现该基因在癌化的猪肾上皮细胞中的表达量要比正常肾组织要高。而在胚胎发育时期，该基因在骨胳肌中的表达呈现下调表达模式，但出生后呈现出上调表达模式。将该基因在胚胎时期的表达模式进行比较，发现该基因在中外猪种骨骼肌的发育过程中并不存在差异表达；
7．该基因的原核表达
按照常规的实验方法对该基因进行原核表达，并没有发现特异性条带，推测可能是由于大肠杆菌密码子的偏爱造成了该基因在原核生物中的表达障碍；
8．亚细胞定位
利用绿色荧光表达载体pEGFP-C1与该基因编码框进行融合表达，转染并染色后通过共聚焦显微镜进行观察，发现猪的PNAS-4基因的蛋白质CGI-146明显分布于高尔基体，说明该蛋白质存在于高尔基体内并发挥其生物学功能；
9．通过真核表达载体pcDNA3.1进行超表达
将该基因编码框插入到真核超表达载体peDNA3.1中，进行细胞转染后，利用倒置显微镜在普通视野下观察发现，随着转染后时间的延长，越来越多的细胞出现萎缩，浓缩等凋亡现象；通过细胞凋亡检测试剂盒染色后，在倒置荧光显微镜FITC和若丹明双重滤镜下观察到凋亡早期、中期及晚期细胞，从而初步确定了该基因具有促凋亡的特性。
器官移植作为20世纪医学领域发展起来的新兴技术已经成为治疗器官功能衰竭终末期的首要治疗手段。全世界每年有数万名器官功能衰竭患者通过器官移植而获新生。然而供体器官的严重不足，使得许多病人在等待中死去，而物种间异种移植则成为了解决该医学问题的最好方法。
进行猪-人异种器官移植，首先要解决的问题是克服超急性排斥反应(HAR)。它是由猪的血管内皮细胞表面α-Gal抗原与人体内的天然抗体结合，从而激活补体级联反应而发生的。经研究证实，该抗原是由一组具有Galα(1，3)Gal双糖末端的糖蛋白或糖脂组成的，它的形成依赖于α1，3-半乳糖基转移酶(α1，3-GT)的催化。因此利用生物工程技术敲除该基因将能在很大程度上有效地克服HAR。
基因打靶是80年代发展起来的一项重要的分子生物学技术。它是通过外源DNA与受体细胞基因组中的同源序列之间发生重组，从而定向改变细胞或生物个体遗传信息的实验手段。目前，该技术已先后在小鼠、绵羊、山羊、牛和猪的体细胞中获得成功。
本研究利用现有的α1，3-GT基因序列信息，成功构建了正负双筛选基因打靶载体，期望在后续实验中通过体细胞基因打靶技术获得敲除α1，3-GT基因的高近交五指山猪品系，从而为我国的异种器官移植提供更为理想的实验动物。
利用长距离PCR(LR-PCR)方法，从高近交系五指山猪的基因组中成功得到了5.4Kb、1.6Kb的扩增产物。其中5.4Kb的DNA片段包括了α1，3-GT完整的第8内含子；1.6Kb的DNA片段覆盖了大部分第9外显子。考虑到5.4Kb的DNA片段中存在限制性内切酶SalⅠ位点，于是通过PCR方法选取其中的5.1Kb DNA片段作为打靶载体的5’同源臂，并将1.6Kb的DNA片段作为3’同源臂，以neo为正选择标记基因，以HVS-tk为负选择标记基因，构建敲除猪α1，3-GT基因部分序列打靶载体PLS-pPNT，全长约15.2Kb，并经测序验证，从而为后续实验打下了良好基础。
Part I Analysis of structure and function of porcine PNAS-4 Being involved in the whole lifecycle of organism, apoptosis, which is ubiquitous and also normal of cell death, is known to play an important role during organism development, survival and maintain normal physiological function especially in embryonic development, cell proliferation, differentiation, formation of apparatus. In different developmental stages, the skeletal muscle involves a great number of differential expression genes which are regulating proliferation and apoptosis for certain cells in the developmental process.In our previous work, a novel gene with differential expression in different tissues and different developmental stages was isolated by screening from a full-length cDNA library constructed with the 55-day-old pig fetus skeletal muscle. In order to study the structure and function of this gene from nucleic acid and protein level, several methods including molecular biology, cytobiology as well as bioinformatics was employed in experiment. And the main results are as follows:1. Obtaining the Full-length cDNA of porcine PNAS-4The full-length 4133bp cDNA sequence of porcine PNAS-4 (GenBank accession number: DQ435075) was obtained by PCR and 5'RACE.2. Functional elements located in the cDNAA functional element called Internal Ribosome Entry Site (IRES) was found in the 5' UTR. Two boxes named Brd-Box and GY-Box located in the 3' UTR of the porcine gene containing two consensus polyadenylation signals (AATAAA) located upstream of the poly (A) stretch. And the Brd-Box plays an important role in negative regulation for the post- transcription via its influence on the stability and translation efficiency.3. Regulation factors in the promoter of human homologous geneA lot of binding site of transcription factors such as Sp1 and AP-1 were discovered nearby the CpG island after analysis of network regulation for the promoter of human clorf121 which is homologous to porcine PNAS-4, indicating the transcription efficiency will be higher than others. In addition, there are many binding sites of apoptosis repressor in the human promoter. This promoter is GC rich and lacks a TATA box as is typical for promoters of ubiquitously expressed "housekeeping genes".4. Characterization of predicted protein sequences
The conceptual translation product of the porcine PNAS-4 transcript encodes 194 amino acids residues, which is a non-secretary protein without signal peptide, or transmembrane regions. Three-dimensional simulation structure was carried out according to the Putative conserved domain related to Eukaryotie protein of unknown function (DUF862), which comprises three possible protein binding domains and four amino acids residues exposing outside. Comparison to seven amino acids sequences of different species reported in GenBank, the porcine putative protein has a high level identity (96-98%) in mammals, and furthermore, it shares 74% and 45% similarity with Xenopus laevis and Arabidopsis thaliana, respectively.
5. Detection of Polymorphisms and analysis of linkage disequilibria
The complete second intron comprising of 3.2kb in length (GenBank accession no. DQ406743) was obtained according to the human clorf121 genome structure. After analysis of four mutation site located on the second intron and 3'UTR respectively using PCR-RFLP and DHPLC methods, the allele frequency of the four SNPs displays variable in different pig breeds. And the four mutation sites appear linkage disequilibria in single population though they are approximately 21Kb apart from each other. There are significant associations between the genotype of PNAS-4 with phenotypes of Percentage of Leaf Fat, Internal Fat Rate, Carcass Length, Rib Numbers, Loin-muscle Area, Ratio of M vs S in Ham and Hemoglobin Concentration (P＜0.05).
6. Temporal and spatial express pattern
Real-time Q-PCR was performed to analyze tissue-specific expression of porcine PNAS-4 transcript using the housekeeping geneβ-actin as endogenous control. Relative mRNA expression of PNAS-4 was high in the lymph as well as in skeletal muscle, and with weakest signal observed in kidney and brain. In addition, the higher mRNA expression was found in cancerization cell than the normal kidney tissue. For temporal expression analysis, the porcine PNAS-4 appears down-regulated pattern during embryonic development while as up-regulated pattern after birth. And in the process of skeletal muscle development, there is no differential expression between indigenous and exotic pig breeds for this novel gone.
7. Expression P4-p28c in E. coli
A prokaryotic expression plasmids P4-pET28c was Constructed, and transferred into E. coli BL21 (DE3) expression strain. As a result from codon preference, no special signal peptide was found via conventional methods including SDS-PAGE.
8. The porcine CGI-146 intracellular distribution
The intracellular distribution of porcine CGI-146 (the product of PNAS-4) in PK-15 cells was analyzed by fluorescence and confocal analysis of transiently transfected with P4-pEGFP-C1. After stained with MitoTracker Red and Hoechst33342, CGI-146-pEGFP fusion proteins were predominantly detected in Golgi apparatus, which was identified using the homo-functional plasma P4-pEGFP-N3 as the complementation.
9. Overexpression of the porcine CGI-146
Transiently transfection of PK-15 cells with the overexressed plasmid P4-pcDNA3.1 which express 21.4kDa non-tagged protein, the cell shrinkage and the less well defined cell surface was observed in the normal view with normal optical microscope. After the transfected cells were stained with Annexin V and Propidium Iodide, the apoptosis (annexin V-positive/propidium iodide-negative) were observed by the fluorescence microscope with barely vector alone as controls.
As a new medical technology born in 20th century, clinical transplantation has become one of the important and powerful treatment methods for end-stage organ failure. There are tens of thousands patients coming to a new life by this medical technology every year in all over the world. However many patients die in the course of waiting because the donor organs are serious insufficiency. Therefore the Xenotransplantation has become to the most appropriate method to solve the urgently problem of medicine.
The major obstacle to success is the hyperacute rejection (HAR) in pig-to-human discordant xenotransplantation, which attribute to the presence of the Galactose-α1,3-galactose (αGal) epitopes on the surface of pig cells and tissues. And the Synthesis of theα1,3-Gal epitope is catalyzed by the enzymeα1,3-galactosyltransferase (α1, 3-GT, GGAT1), but humans, apes, and Old World monkeys have lost the corresponding galactosyltransferase activity because only with a non-functional copy of theα1,3-GT gene in the course of evolution and therefore produce a large quantity of natural antibodies to the epitope, which are responsible for hyperacute rejection of porcine organs. Consequently, the genetic knockout of the a1,3-galactosyltransferase locus in pigs would provide permanent and complete protection against the HAR at extant range using gene engineering technology.
Gene targeting is an important genetic manipulation technology, which defined as the introduction of site-specific modifications into the genome by homologous recombination, has revolutionarized the field of mouse genetics and allowed the analysis of diverse aspects of gene function in vivo. In present, the cloning of sheep, goat, cattle, and pigs by somatic cell nuclear transfer provides a feasible means of disrupting or deleting theα1,3-GT in mammals other than mice.
According to the porcineα1,3-GT fragment obtained from the NCBI, a gene targeting vector, which comprise of positive and negative selection, was used to establish the highly inbred Wuzhishan miniature pig (WZSP) line knocked out theα1,3-GT gene by the method of gene targeting in somatic cells. At the same time, to offer the possible experimental animals used for Xenotransplantation in our country.
Using the long-range (LR) PCR method, we have cloned two DNA fragments which are 5.4Kb and 1.6Kb respectively from the highly inbred WZSP genome. The 5.4Kb DNA fragment includes the whole eighth intron of porcineα1,3-GT gene, and the other spans the most part of exon 9, which are cloned to vector for sequencing. And sequencing analysis showed that the two fragment are genomic DNA of the porcineα1,3-GT gene.
Because there is a restriction digestion enzyme SalⅠlocated in the former of 5.4Kb DNA fragment, 5.1Kb was amplified for 5'arm of targeting vector using conventional PCR method while the 1.6Kb DNA fragment as the 3'arm. And then, the construction taregeting vector containing 15.2 Kb used for knock-out porcineα1,3-GT gene was completed and conformed by sequencing, in which the neomycin phosphotransferase (neo') and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selection markers.
根据该推理假设，本研究从构建的猪胎儿骨胳肌cDNA文库中挑选出一个在组织及不同发育时期都有差异表达的未知基因作为研究对象，并通过分子生物学、细胞生物学以及生物信息学等方法从核酸及蛋白质两个水平上进行了结构与功能研究，并取得了如下研究结果：
1．猪的PNAS-4基因cDNA全长序列的获得
根据已获得的该基因部分信息，采用电脑克隆策略结合RACE技术，获得了猪的PNAS-4基因的5’非翻译区序列，然后通过PCR方法填补了序列中一个82bp的Gap，从而得到该基因的cDNA全长4133bp，并提交GenBank数据库，GenBank收录号DQ435075；
2．cDNA序列上的功能元件分析
对猪的PNAS-4 cDNA序列进行分析，发现该基因的在5’UTR区有一内源性核糖体进入位点(IRES)，而在3’UTR区除了有两个多(聚)腺苷酸加尾信号(AATAAA)外，还存在着两个功能域框Brd-Box和GY-Box，其中Brd-Box通过影响转录的稳定性和翻译的效率来介导转录后负调控；
3．人的同源基因clorf121的启动子区域调控因子的分析
利用人的同源基因clorf121的启动子区域进行网络调控分析时发现，有一个CpG岛附近有着较多的转录因子(Sp1、AP-1)的结合点，说明该基因具有较强的转录效率，另外还发现在启动子区域有较多的抑凋亡因子结合位点。根据其CpG岛的GC含量高及无TATA box的特征推测该基因是一个无处不表达的持家基因；
4．推导的该编码的基因蛋白质特点
由该基因CDS序列推导出猪的PNAS-4编码着是一个含有194个氨基酸残基，是一个既无信号肽又无跨膜结构的非分泌性蛋白质；并对其保守的功能域DUF862进行了三维结构的模拟，发现其有三个可能的蛋白质结合域以及三个裸露在外的氨基酸残基；同源比对后，发现该基因在哺乳动物之间同源性极高达96-98％，与家禽及非洲蟾蜍分别有着94％和74％的相似性，甚至拟南芥的该蛋白质与猪也有着45％的相似性；
5．多态检测及连锁不平衡分析
利用比较基因组学通过PCR方法获得猪的该基因第二内含子全长，并提交GenBank数据库，GenBank收录号DQ406743。通过PCR-RFLP及DHPLC技术对第二内含子及3’UTR区共计四个SNPs位点进行多态分析，发现这四个多态位点在不同品种中表现出等位基因频率的差异。这四个位点虽相隔20Kb，但却不同程度地相互处于连锁不平衡状态。通过性状关联分析，发现该基因不同基因型个体在内脂率、板油率、腿臀肉骨率、胴体直长、眼肌面积以及血红蛋白浓度等性状存在显著性差异(P＜0.05)；
6．应用定量PCR进行时空表达谱分析
通过real-time PCR方法研究了猪的PNAS-4基因在成年猪15个组织中的表达情况，发现该基因在所研究的组织中均有表达，但存在表达量的差异，其中，骨胳肌和淋巴组织中表达量最高，肾和大脑中表达量最低。另外还发现该基因在癌化的猪肾上皮细胞中的表达量要比正常肾组织要高。而在胚胎发育时期，该基因在骨胳肌中的表达呈现下调表达模式，但出生后呈现出上调表达模式。将该基因在胚胎时期的表达模式进行比较，发现该基因在中外猪种骨骼肌的发育过程中并不存在差异表达；
7．该基因的原核表达
按照常规的实验方法对该基因进行原核表达，并没有发现特异性条带，推测可能是由于大肠杆菌密码子的偏爱造成了该基因在原核生物中的表达障碍；
8．亚细胞定位
利用绿色荧光表达载体pEGFP-C1与该基因编码框进行融合表达，转染并染色后通过共聚焦显微镜进行观察，发现猪的PNAS-4基因的蛋白质CGI-146明显分布于高尔基体，说明该蛋白质存在于高尔基体内并发挥其生物学功能；
9．通过真核表达载体pcDNA3.1进行超表达
将该基因编码框插入到真核超表达载体peDNA3.1中，进行细胞转染后，利用倒置显微镜在普通视野下观察发现，随着转染后时间的延长，越来越多的细胞出现萎缩，浓缩等凋亡现象；通过细胞凋亡检测试剂盒染色后，在倒置荧光显微镜FITC和若丹明双重滤镜下观察到凋亡早期、中期及晚期细胞，从而初步确定了该基因具有促凋亡的特性。
器官移植作为20世纪医学领域发展起来的新兴技术已经成为治疗器官功能衰竭终末期的首要治疗手段。全世界每年有数万名器官功能衰竭患者通过器官移植而获新生。然而供体器官的严重不足，使得许多病人在等待中死去，而物种间异种移植则成为了解决该医学问题的最好方法。
进行猪-人异种器官移植，首先要解决的问题是克服超急性排斥反应(HAR)。它是由猪的血管内皮细胞表面α-Gal抗原与人体内的天然抗体结合，从而激活补体级联反应而发生的。经研究证实，该抗原是由一组具有Galα(1，3)Gal双糖末端的糖蛋白或糖脂组成的，它的形成依赖于α1，3-半乳糖基转移酶(α1，3-GT)的催化。因此利用生物工程技术敲除该基因将能在很大程度上有效地克服HAR。
基因打靶是80年代发展起来的一项重要的分子生物学技术。它是通过外源DNA与受体细胞基因组中的同源序列之间发生重组，从而定向改变细胞或生物个体遗传信息的实验手段。目前，该技术已先后在小鼠、绵羊、山羊、牛和猪的体细胞中获得成功。
本研究利用现有的α1，3-GT基因序列信息，成功构建了正负双筛选基因打靶载体，期望在后续实验中通过体细胞基因打靶技术获得敲除α1，3-GT基因的高近交五指山猪品系，从而为我国的异种器官移植提供更为理想的实验动物。
利用长距离PCR(LR-PCR)方法，从高近交系五指山猪的基因组中成功得到了5.4Kb、1.6Kb的扩增产物。其中5.4Kb的DNA片段包括了α1，3-GT完整的第8内含子；1.6Kb的DNA片段覆盖了大部分第9外显子。考虑到5.4Kb的DNA片段中存在限制性内切酶SalⅠ位点，于是通过PCR方法选取其中的5.1Kb DNA片段作为打靶载体的5’同源臂，并将1.6Kb的DNA片段作为3’同源臂，以neo为正选择标记基因，以HVS-tk为负选择标记基因，构建敲除猪α1，3-GT基因部分序列打靶载体PLS-pPNT，全长约15.2Kb，并经测序验证，从而为后续实验打下了良好基础。
Part I Analysis of structure and function of porcine PNAS-4 Being involved in the whole lifecycle of organism, apoptosis, which is ubiquitous and also normal of cell death, is known to play an important role during organism development, survival and maintain normal physiological function especially in embryonic development, cell proliferation, differentiation, formation of apparatus. In different developmental stages, the skeletal muscle involves a great number of differential expression genes which are regulating proliferation and apoptosis for certain cells in the developmental process.In our previous work, a novel gene with differential expression in different tissues and different developmental stages was isolated by screening from a full-length cDNA library constructed with the 55-day-old pig fetus skeletal muscle. In order to study the structure and function of this gene from nucleic acid and protein level, several methods including molecular biology, cytobiology as well as bioinformatics was employed in experiment. And the main results are as follows:1. Obtaining the Full-length cDNA of porcine PNAS-4The full-length 4133bp cDNA sequence of porcine PNAS-4 (GenBank accession number: DQ435075) was obtained by PCR and 5'RACE.2. Functional elements located in the cDNAA functional element called Internal Ribosome Entry Site (IRES) was found in the 5' UTR. Two boxes named Brd-Box and GY-Box located in the 3' UTR of the porcine gene containing two consensus polyadenylation signals (AATAAA) located upstream of the poly (A) stretch. And the Brd-Box plays an important role in negative regulation for the post- transcription via its influence on the stability and translation efficiency.3. Regulation factors in the promoter of human homologous geneA lot of binding site of transcription factors such as Sp1 and AP-1 were discovered nearby the CpG island after analysis of network regulation for the promoter of human clorf121 which is homologous to porcine PNAS-4, indicating the transcription efficiency will be higher than others. In addition, there are many binding sites of apoptosis repressor in the human promoter. This promoter is GC rich and lacks a TATA box as is typical for promoters of ubiquitously expressed "housekeeping genes".4. Characterization of predicted protein sequences
The conceptual translation product of the porcine PNAS-4 transcript encodes 194 amino acids residues, which is a non-secretary protein without signal peptide, or transmembrane regions. Three-dimensional simulation structure was carried out according to the Putative conserved domain related to Eukaryotie protein of unknown function (DUF862), which comprises three possible protein binding domains and four amino acids residues exposing outside. Comparison to seven amino acids sequences of different species reported in GenBank, the porcine putative protein has a high level identity (96-98%) in mammals, and furthermore, it shares 74% and 45% similarity with Xenopus laevis and Arabidopsis thaliana, respectively.
5. Detection of Polymorphisms and analysis of linkage disequilibria
The complete second intron comprising of 3.2kb in length (GenBank accession no. DQ406743) was obtained according to the human clorf121 genome structure. After analysis of four mutation site located on the second intron and 3'UTR respectively using PCR-RFLP and DHPLC methods, the allele frequency of the four SNPs displays variable in different pig breeds. And the four mutation sites appear linkage disequilibria in single population though they are approximately 21Kb apart from each other. There are significant associations between the genotype of PNAS-4 with phenotypes of Percentage of Leaf Fat, Internal Fat Rate, Carcass Length, Rib Numbers, Loin-muscle Area, Ratio of M vs S in Ham and Hemoglobin Concentration (P＜0.05).
6. Temporal and spatial express pattern
Real-time Q-PCR was performed to analyze tissue-specific expression of porcine PNAS-4 transcript using the housekeeping geneβ-actin as endogenous control. Relative mRNA expression of PNAS-4 was high in the lymph as well as in skeletal muscle, and with weakest signal observed in kidney and brain. In addition, the higher mRNA expression was found in cancerization cell than the normal kidney tissue. For temporal expression analysis, the porcine PNAS-4 appears down-regulated pattern during embryonic development while as up-regulated pattern after birth. And in the process of skeletal muscle development, there is no differential expression between indigenous and exotic pig breeds for this novel gone.
7. Expression P4-p28c in E. coli
A prokaryotic expression plasmids P4-pET28c was Constructed, and transferred into E. coli BL21 (DE3) expression strain. As a result from codon preference, no special signal peptide was found via conventional methods including SDS-PAGE.
8. The porcine CGI-146 intracellular distribution
The intracellular distribution of porcine CGI-146 (the product of PNAS-4) in PK-15 cells was analyzed by fluorescence and confocal analysis of transiently transfected with P4-pEGFP-C1. After stained with MitoTracker Red and Hoechst33342, CGI-146-pEGFP fusion proteins were predominantly detected in Golgi apparatus, which was identified using the homo-functional plasma P4-pEGFP-N3 as the complementation.
9. Overexpression of the porcine CGI-146
Transiently transfection of PK-15 cells with the overexressed plasmid P4-pcDNA3.1 which express 21.4kDa non-tagged protein, the cell shrinkage and the less well defined cell surface was observed in the normal view with normal optical microscope. After the transfected cells were stained with Annexin V and Propidium Iodide, the apoptosis (annexin V-positive/propidium iodide-negative) were observed by the fluorescence microscope with barely vector alone as controls.
As a new medical technology born in 20th century, clinical transplantation has become one of the important and powerful treatment methods for end-stage organ failure. There are tens of thousands patients coming to a new life by this medical technology every year in all over the world. However many patients die in the course of waiting because the donor organs are serious insufficiency. Therefore the Xenotransplantation has become to the most appropriate method to solve the urgently problem of medicine.
The major obstacle to success is the hyperacute rejection (HAR) in pig-to-human discordant xenotransplantation, which attribute to the presence of the Galactose-α1,3-galactose (αGal) epitopes on the surface of pig cells and tissues. And the Synthesis of theα1,3-Gal epitope is catalyzed by the enzymeα1,3-galactosyltransferase (α1, 3-GT, GGAT1), but humans, apes, and Old World monkeys have lost the corresponding galactosyltransferase activity because only with a non-functional copy of theα1,3-GT gene in the course of evolution and therefore produce a large quantity of natural antibodies to the epitope, which are responsible for hyperacute rejection of porcine organs. Consequently, the genetic knockout of the a1,3-galactosyltransferase locus in pigs would provide permanent and complete protection against the HAR at extant range using gene engineering technology.
Gene targeting is an important genetic manipulation technology, which defined as the introduction of site-specific modifications into the genome by homologous recombination, has revolutionarized the field of mouse genetics and allowed the analysis of diverse aspects of gene function in vivo. In present, the cloning of sheep, goat, cattle, and pigs by somatic cell nuclear transfer provides a feasible means of disrupting or deleting theα1,3-GT in mammals other than mice.
According to the porcineα1,3-GT fragment obtained from the NCBI, a gene targeting vector, which comprise of positive and negative selection, was used to establish the highly inbred Wuzhishan miniature pig (WZSP) line knocked out theα1,3-GT gene by the method of gene targeting in somatic cells. At the same time, to offer the possible experimental animals used for Xenotransplantation in our country.
Using the long-range (LR) PCR method, we have cloned two DNA fragments which are 5.4Kb and 1.6Kb respectively from the highly inbred WZSP genome. The 5.4Kb DNA fragment includes the whole eighth intron of porcineα1,3-GT gene, and the other spans the most part of exon 9, which are cloned to vector for sequencing. And sequencing analysis showed that the two fragment are genomic DNA of the porcineα1,3-GT gene.
Because there is a restriction digestion enzyme SalⅠlocated in the former of 5.4Kb DNA fragment, 5.1Kb was amplified for 5'arm of targeting vector using conventional PCR method while the 1.6Kb DNA fragment as the 3'arm. And then, the construction taregeting vector containing 15.2 Kb used for knock-out porcineα1,3-GT gene was completed and conformed by sequencing, in which the neomycin phosphotransferase (neo') and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selection markers.
引文
1．陈立栋：牛α 1，3-半乳糖基转移酶基因敲除的研究．博士研究生学位论文 2003．
2．韩颖：异种器官移植的发展与医学伦理学思考．中国医学伦理学 2005，18：27-29．
3．李雪辉：猪体细胞敲除α 1，3-半乳糖基转移酶基因的研究．硕士研究生学位论文 2003．
4．林丛，许克锋，张文秀，侯宽永，毛泽斌：反义RNA对猪α．1，3-半乳糖苷转移酶活性的影响．中国生物化学与分子生物学报 2001，17：515-518．
5．马志方：异种器官移植早期捧斥及病理改变研究进展．国外医学泌尿系统分册 2004，24：485-488．
6．潘佩文：猪不同品种或发育阶段肌肉组织中差异表达EST的分离、鉴定和定位．博士研究生学位论文 2002．
7．魏庆信，郑新民，肖作焕：异种器官移植用转基因猪研究进展．湖北农业科学 2002：62-64．
8．俞远京：猪异种器官移植的人源化修饰．遗传 2003，25：596-600．
9．周健，林爱星，陈永福：敲除猪α 1，3-半乳糖基转移酶基因并敲入HLA-G1基因的打靶载体构建．中国生物医学工程学报 2005，24：498-502．
10．朱正茂：应用cDNA宏阵列技术发现猪某些产肉性状的候选基因．博士研究生学位论文 2003．
11．左波：猪3、4和7号染色体QTL定位及六个候选基因的分离、鉴定．博士研究生学位论文 2004
12. Bellas RE, FitzGerald MJ, Fausto N, Sonenshein GE: Inhibition of NF-kappa B activity induces apoptosis in murine hepatocytes. Am J Pathol 1997, 151: 891-896.
13. Belusa R, Wang ZM, Matsubara T, Sahlgren B, Dulubova I, Nairn AC, Ruoslahti E, Greengard P, Aperia A: Mutation of the protein kinase C phosphorylation site on rat alphal Na+, K+-ATPase alters regulation of intracellular Na+ and pH and influences cell shape and adhesiveness. J Biol Chem 1997, 272: 20179-20184.
14. Bennett M, Macdonald K, Chan SW, Luzio JP, Simari R, Weissberg P: Cell surface trafficking of Fas: a rapid mechanism of p53-mediated apoptosis. Science 1998, 282: 290-293.
15. Blanken WM, Van den Eijnden DH: Biosynthesis of terminal Gal alpha 1—3Gal beta 1—4GIcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal: N-acetyllactosaminide alpha 1—3-galactosyltransferase from calf thymus. J Biol Chem 1985, 260: 12927-12934.
16. Braun RE, Lo D, Pinkert CA, Widera G, Flavell RA, Palmiter RD, Brinster RL: Infertility in male transgenic mice: disruption of sperm development by HSV-tk expression in postmeiotic germ cells. Biol Reprod 1990, 43: 684-693.
17. Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M, Roth R, George D, Eletr S, Albrecht G, Vermaas E, Williams SR, Moon K, Burcham T, Pallas M, DuBridge RB, et al.: Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol 2000, 18: 630-634.
18. Brown EA, Zajac AJ, Lemon SM: In vitro characterization of an internal ribosomal entry site (IRES) present within the 5' nontranslated region of hepatitis A virus RNA: comparison with the IRES of encephalomyocarditis virus. J Virol 1994, 68: 1066-1074.
19. Bucher P, Morel P, Buhler LH: Xenotransplantation: an update on recent progress and future perspectives. Transpl Int 2005, 18: 894-901.
20. Calne RY: Organ transplantation between widely disparate species. Transplant Proc 1970, 2: 550-556.
21. Charlotte Rehfeldt NCS, Ilse Fiedler and Jochen Wegner: Environmental and Genetic Factors as Sources of Variation in Skeletal Muscle Fibre Number. Basic Appl. Myol. 1999, 9: 235-253.
22. Cho SK, Yeh J, Cho M, Cummings RD: Transcriptional regulation of alpha 1, 3-galactosyltransferase in embryonal carcinoma cells by retinoic acid. Masking of Lewis X antigens by alpha-galactosylation. J Biol Chem 1996, 271: 3238-3246.
23. Cobb JP, Mindrinos MN, Miller-Graziano C, Calvano SE, Baker HV, Xiao W, Laudanski K, Brownstein BH, Elson CM, Hayden DL, Herndon DN, Lowry SF, Maier RV, Schoenfeld DA, Moldawer LL, Davis RW, Tompkins RG, Baker HV, Bankey P, Billiar T, et al.: Application of genome-wide expression analysis to human health and disease. Proc Natl Acad Sci U S A 2005, 102: 4801-4806.
24. Cooper DK: Is xenotransplantation a realistic clinical option? Transplant Proc 1992, 24: 2393-2396.
25. Coulson AS: The Golgi apparatus in small lymphocytes and in transformed blast cells. Q J Exp Physiol Cogn Med Sci 1965, 50: 271-276.
26. Cui Q, Jiang T, Lin B, Ma S: Esuh8: a novel tool to predict protein subcellular localizations in eukaryotic organisms. BMC Bioinformatics 2004, 5: 66.
27. Curtin JF, Cotter TG: Live and let die: regulatory mechanisms in Fas-mediated apoptosis. Cell Signal 2003, 15: 983-992.
28. Cutolo M, Sulli A, Barone A, Seriolo B, Accardo S: Sex hormones, proto-oncogene expression and apoptosis: their effects on rheumatoid synovial tissue. Clin Exp Rheumatol 1996, 14: 87-94.
29. Czernik PJ, Peterson CA, Hurlburt BK: Preferential binding of MyoD-E12 versus myogenin-E12 to the murine sarcoma virus enhancer in vitro. J Biol Chem 1996, 271: 9141-9149.
30. Dabkowski PL, Vaughan HA, McKenzie IF, Sandrin MS: Characterisation of a cDNA clone encoding the pig alpha 1, 3 galactosyltransferase: implications for xenotransplantation. Transplant Proc 1993, 25: 2921.
31. Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL: Targeted disruption of the alpha1, 3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002, 20: 251-255.
32. Dang CV: c-Myc target genes involved in cell growth, apoptosis, and metabolism. Mol Cell Biol 1999, 19: 1-11.
33. Daniotti JL, Martina JA, Giraudo CG, Zurita AR, Maccioni HJ: GM3 alpha2, 8-sialyltransferase (GD3 synthase): protein characterization and sub-golgi location in CHO-K1 cells. J Neurochem 2000, 74: 1711-1720.
34. Davies MV, Kaufman RJ: The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation. J Virol 1992, 66: 1924-1932.
35. de Hoop MJ, Ab G: Import of proteins into peroxisomes and other microbodies. Biochem J 1992, 286 (Pt 3): 657-669.
36. Dehoux JP, de la Parra B, Latinne D, Bazin H, Gianello P: Characterization of baboon anti-porcine IgG antibodies during acute vascular rejection of porcine kidney xenograft. Xenotransplantation 2002, 9: 338-349.
37. Deng C, Capecchi MR: Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol Cell Biol 1992, 12: 3365-3371.
38. Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, Martinou JC: Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 1999, 144: 891-901.
39. Deschamps JY, Roux FA, Sai P, Gouin E: History of xenotransplantation. Xenotransplantation 2005, 12: 91-109.
40. Dorling A: Are anti-endothelial cell antibodies a pre-requisite for the acute vascular rejection of xenografts? Xenotransplantation 2003, 10: 16-23.
41. Dunkov BC, Georgieva T, Yoshiga T, Hall M, Law JH: Aedes aegypti ferritin heavy chain homologue: feeding of iron or blood influences message levels, lengths and subunit abundance. J Insect Sci 2002, 2: 7.
42. Finucane DM, Waterhouse NJ, Amarante-Mendes GP, Cotter TG, Green DR: Collapse of the inner mitochondrial transmembrane potential is not required for apoptosis of HL60 cells. Exp Cell Res 1999, 251: 166-174.
43. Forrest MS, Lan Q, Hubbard AE, Zhang L, Vermeulen R, Zhao X, Li G, Wu YY, Shen M, Yin S, Chanock SJ, Rothman N, Smith MT: Discovery of novel biomarkers by microarray analysis of peripheral blood mononuclear cell gene expression in benzene-exposed workers. Environ Health Perspect 2005, 113: 801-807.
44. Fujiwara Y, Asogawa M: Prediction of subcellular localizations using amino acid composition and order. Genome Inform Ser Workshop Genome Inform 2001, 12: 103-112.
45. Galili U, Rachmilewitz EA, Peleg A, Flechner I: A unique natural human IgG antibody with anfi-alpha-galactosyl specificity. J Exp Med 1984, 160: 1519-1531.
46. Galili U, Macher BA, Buehler J, Shohet SB: Human natural anti-alpha-galactosyl IgG Ⅱ. The specific recognition of alpha (1—3)-iinked galactose residues. J Exp Med 1985, 162: 573-582.
47. Galili U, Clark MR, Shohet SB, Buehler I, Macher BA: Evolutionary relationship between the natural anti-Gal antibody and the Gal alpha 1—3Gal epitope in primates. Proc Natl Acad Sci U S A 1987, 84: 1369-1373.
48. Gamen S, Anel A, Pineiro A, Naval J: Caspases are the main executioners of Fas-mediated apoptosls, irrespective of the ceramide signalling pathway. Cell Death Differ 1998, 5: 241-249.
49. Gehnini S, Orlando C, Sestini R, Vona G, Pinzani P, Ruocco L, Pazzagli M: Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification. Clin Chem 1997, 43: 752-758.
50. Geuze HJ, Slot JW, van der Ley PA, Scbeffer PC: Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections. J Cell Biol 1981, 89: 653-665.
51. Glaumann H, Ericsson JL: Evidence for the participation of the Golgi apparatus in the intracellular transport of nascent albumin in the liver cell. J Cell Biol 1970, 47: 555-567.
52. Gould SJ, Keller GA, Subramani S: Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. J Cell Biol 1988, 107: 897-905.
53. Gould SJ, Keller GA, Hosken N, Wilkinson J, Subramani S: A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol 1989, 108: 1657-1664.
54. Gould S J, Keller GA, Schneider M, Howell SH, Garrard LJ, Goodman JM, Distel B, Tabak H, Subramani S: Peroxisomal protein import is conserved between yeast, plants, insects and mammals. Embo J 1990, 9: 85-90.
55. Gravotta D, Adesnik M, Sabatini DD: Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins. J Cell Biol 1990, 111: 2893-2908.
56. Gregoli PA, Bondurant MC: The roles of Bcl-X(L) and apopain in the control of erythropoiesis by erythropoietin. Blood 1997, 90: 630-640.
57. Grosjean H, Fiers W: Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene 1982, 18: 199-209.
58. Hanzlik TN, Abdel-Aal YA, Harshman LG, Hammock BD: Isolation and sequencing of cDNA clones coding for juvenile hormone esterase from Heliothis virescens. Evidence for a catalytic mechanism for the serine carboxylesterases different from that of the serine proteases. J Biol Chem 1989, 264: 12419-12425.
59. Haynes AP, Daniels I, Abhulayha AM, Carter GI, Metheringham R, Gregory CD, Thomson BJ: CD95 (Fas) expression is regulated by sequestration in the Golgi complex in B-cell lymphoma. Br J Haematol 2002, 118: 488-494.
60. Hecker M, Mulsch A, Busse R: Subcellular localization and characterization of neuronal nitric oxide synthase. J Neurochem 1994, 62: 1524-1529.
61. Heid CA, Stevens J, Livak KJ, Williams PM: Real time quantitative PCR. Genome Res 1996, 6: 986-994.
62. Heinrich UR, Liondyno M, Maurer J, Mann W, Guth PS, Forstermann U: Localization of the two constitutively expressed nitric oxide synthase isoforms (nNOS and eNOS) in the same cell types in the saccule maculae of the frog Rana pipiens by immunoelectron microscopy: evidence for a back-up system? J Electron Microsc (Tokyo) 2003, 52: 197-206.
63. Helps C, Lait P, Tasker S, Harbour D: Melting curve analysis of feline calicivirus isolates detected by real-time reverse transcription PCR. J Virol Methods 2002, 106: 241-244.
64. Henion TR, Macher BA, Anaraki F, Galili U: Defining the minimal size of catalytically active primate alpha 1, 3 galactosyltransferase: structure-function studies on the recombinant truncated enzyme. Glycobiology 1994, 4: 193-201.
65. Iakovlev AI, Ts KT, Klepukov AA: [Micro-agglutination reaction of Rickettsia and viruses with the aid of luminescence microscopy.]. Vopr Virusol 1958, 3: 369-372.
66. Ikebukuro K, Adachi Y, Toki J, Taketani S, Tokunaga R, Hioki K, Ikehara S: Morphological change, loss of deltapsi(m) and activation of caspases upon apoptosis of colorectal adenocarcinoma induced by 5-FU. Cancer Lett 2000, 153: 101-108.
67. Ikematsu S, Kaname T, Ozawa M, Yonezawa S, Sate E, Uehara F, Obama H, Yamamura K, Muramatsu T: Transgenic mouse lines with ectopic expression of alpha-1, 3-galactosyltransferase: production and characteristics. Glycobiology 1993, 3: 575-580.
68. Jin DI, Lee SH, Choi JH, Lee JS, Lee JE, Park KW, Seo JS: Targeting efficiency of a-1, 3-galactosyl transferase gene in pig fetal fibroblast cells. Exp Mol Med 2003, 35: 572-577.
69. Joe Sambrook DR: Molecular Cloning: A Laboratoty Mannual. 2001: 68-86.
70. Joziasse DH, Oriel R: Xenotransplantation: the importance of the Galalpha1, 3Ga1 epitope in hyperacute vascular rejection. Biochim Biophys Acta 1999, 1455: 403-418.
71. Joziasse DH, Shaper JH, Van den Eijnden DH, Van Tunen AJ, Shaper NL: Bovine alpha 1—3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA. J Biol Chem 1989, 264: 14290-14297.
72. Juin P, Hueber AO, Littiewood T, Evan G: c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release. Genes Dev 1999, 13: 1367-1381.
73. Kasibhatla S, Beere HM, Brunner T, Echeverri F, Green DR: A 'non-canonical' DNA-binding element mediates the response of the Fas-ligand promoter to c-Myc. Curr Biol 2000, 10: 1205-1208.
74. Kong Y, Flick MJ, Kudla AJ, Konieczny SF: Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD. Mol Cell Biol 1997, 17: 4750-4760.
75. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH: Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994, 84: 1415-1420.
76. Kozak M: An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 1987, 15: 8125-8148.
77. Krajewski S, Krajewska M, Reed JC: Immunohistochemical analysis of in vivo patterns of Bak expression, a proapoptotic member of the Bcl-2 protein family. Cancer Res 1996, 56: 2849-2855.
78. Kumar R, Yang J, Larsen RD, Stanley P: Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation. Proc Natl Acad Sci U S A 1990, 87: 9948-9952.
79. Lai EC: Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet 2002, 30: 363-364.
80. Lai EC, Burks C, Posakony JW: The K box, a conserved 3' UTR sequence motif, negatively regulates accumulation of enhancer of split complex transcripts. Development 1998, 125: 4077-4088.
81. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS: Production of alpha-1, 3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002, 295: 1089-1092.
82. Lakso M, Saner B, Mosinger B, Jr., Lee EJ, Manning RW, Yu SH, Mulder KL, Westphal H: Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci U S A 1992, 89: 6232-6236.
83. Lambert DL, Malik N, Shepherd L, Gunn J, Francis SE, King A, Crossman DC, Cumberland DC, Holt CM: Localization of c-Myb and induction of apoptosis by antisense oligonucleotide c-Myb after angioplasty of porcine coronary arteries. Arterioscler Thromb Vasc Biol 2001, 21: 1727-1732.
84. Lanza RP, Cibelli JB, West MD: Prospects for the use of nuclear transfer in human transplantation. Nat Biotechnol 1999, 17: 1171-1174.
85. Larsen RD, Rajan VP, Ruff MM, Kukowska-Latallo J, Cummings RD, Lowe JB: Isolation of a cDNA encoding a murine UDPgalactose: beta-D-galactosyl- 1, 4-N-acetyl-D-glucosaminide alpha-1, 3-galactosyltransferase: expression cloning by gene transfer. Proc Natl Acad Sci U S A 1989, 86: 8227-8231.
86. Le SY, Maizel JV, Jr.: A common RNA structural motif involved in the internal initiation of translation of cellular mRNAs. Nucleic Acids Res 1997, 25: 362-369.
87. Lenato GM, Lastella P, Di Giacomo MC, Resta N, Suppressa P, Pasculli G, Sabba C, Guanti G: DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population. Hum Mutat 2006, 27: 213-214.
88. Levrat C, Ardail D, Louisot P: Comparative Study of the N-glycoprotein synthesis through dolichol intermediates in mitochondria, Golgi apparatus-rich fraction and endoplasmic reticulum-rich fraction. Int J Biochem 1990, 22: 287-293.
89. Li K, Li Y, Shelton JM, Richardson JA, Spencer E, Chen 73, Wang X, Williams RS: Cytochrome c deficiency causes embryonic lethality and attenuates stress-induced apoptosis. Cell 2000, 101: 389-399.
90. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X: Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997, 91: 479-489.
91. Lippincott-Schwartz J, Roberts TH, Hirschberg K: Secretory protein trafficking and organelle dynamics in living cells. Annu Rev Cell Dev Biol 2000, 16: 557-589.
92. Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN, James CD: Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Res 1998, 26: 1396-1400.
93. Livak K J, Flood SJ, Marmaro J, Giusti W, Deetz K: Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 1995, 4: 357-362.
94. Long M, Rosenberg C, Gilbert W: Intron phase correlations and the evolution of the intron/exon structure of genes. Proc Natl Acad Sci U S A 1995, 92: 12495-12499.
95. Ly JD, Grubb DR, Lawen A: The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis 2003, 8: 115-128.
96. Mackay IM, Arden KE, Nitsche A: Real-time PCR in virology. Nucleic Acids Res 2002, 30: 1292-1305.
97. Maleki SJ, Royer CA, Hurlburt BK: Analysis of the DNA-binding properties of MyoD, myogenin, and E12 by fluorescence anisotropy. Biochemistry 2002, 41: 10888-10894.
98. Martin S J, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR: Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Ab1. J Exp Med 1995, 182: 1545-1556.
99. McDonnell TJ, Beham A, Sarkiss M, Andersen MM, Lo P: Importance of the Bcl-2 family in cell death regulation. Experientia 1996, 52: 1008-1017.
100. McKnight S, Tjian R: Transcriptional selectivity of viral genes in mammalian cells. Cell 1986, 46: 795-805.
101. Metcalfe AD, Hunter HR, Bloor D J, Lieberman BA, Picton HM, Leese HJ, Kimber SJ, Brison DR: Expression of 11 members of the BCL-2 family of apoptosis regulatory molecules during human preimplantation embryo development and fragmentation. Mol Reprod Dev 2004, 68: 35-50.
102. Michaels MG: Xenotransplant-associated infections. Lab Anita Sci 1998, 48: 228-233.
103. Minegishi N, Suzuki N, Kawatani Y, Shimizu R, Yamamoto M: Rapid turnover of GATA-2 via ubiquitin-proteasome protein degradation pathway. Genes Cells 2005, 10: 693-704.
104. Moreadith RW, Radford NB: Gene targeting in embryonic stem cells: the new physiology and metabolism. J Mol Med 1997, 75: 208-216.
105. Morris T, Robertson B, Gallagher M: Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system. J Clin Microbiol 1996, 34: 2933-2936.
106. Mountz JD, Zhou T, Wu J, Wang W, Su X, Cheng J: Regulation of apoptosis in immune cells. J Clin Immunol 1995, 15: 1-16.
107. Muller U: Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech Dev 1999, 82: 3-21.
108. Nagase M, Hirose S, Fujita T: Unique repetitive sequence and unexpected regulation of expression of rat endothelial receptor for oxidized low-density lipoprotein (LOX-1). Biochem J 1998, 330 (Pt 3): 1417-1422.
109. Nakamura K, Bossy-Wetzel E, Burns K, Fadel MP, Lozyk M, Goping IS, Opas M, Bleackley RC, Green DR, Michalak M: Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis. J Cell Biol 2000, 150: 731-740.
110. Numata M, Orlowski J: Molecular cloning and characterization of a novel (Na+, K+)/H+ exchanger localized to the trans-Golgi network. J Biol Chem 2001, 276: 17387-17394.
111. Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, Tokino T, Taniguchi T, Tanaka N: Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 2000, 288: 1053-1058.
112. Ogle BM, Platt JL: Genetic therapies and xenotransplantation. Expert Opin Biol Ther 2002, 2: 299-310.
113. Oltean S, Banerjee R: A B12-responsive internal ribosome entry site (IRES) element in human methionine synthase. J Biol Chem 2005, 280: 32662-32668.
114. Orkin SH, Weiss MJ: Apoptosis. Cutting red-cell production. Nature 1999, 401: 433, 435-436.
115. Ossina NK, Cannas A, Powers VC, Fitzpatrick PA, Knight JD, Gilbert JR, Shekhtman EM, Tomei LD, Umansky SR, Kiefer MC: Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem 1997, 272: 16351-16357.
116. Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM: Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 1982, 300: 611-615.
117. Panepinto LM, Phillips RW: The Yucatan miniature pig: characterization and utilization in biomedical research. Lab Anim Sci 1986, 36: 344-347.
118. Parcharidou A, Raza A, Economopoulos T, Papageorgiou E, Anagnostou D, Papadaki T, Raptis S: Extensive apoptosis of bone marrow cells as evaluated by the in situ end-labelling (ISEL) technique may be the basis for ineffective haematopoiesis in patients with myelodysplastic syndromes. Eur J Haematol 1999, 62: 19-26.
119. Patience C, Takeuchi Y, Weiss RA: Infection of human cells by an endogenous retrovirus of pigs. Nat Med 1997, 3: 282-286.
120. Phelps CJ, Koike C, Vanght TD, Boone J, Wells KD, Chen SH, Ball S, Specht SM, Polejaeva IA, Monahan JA, Jobst PM, Sharma SB, Lamborn AE, Garst AS, Moore M, Demetris A J, Rudert WA, Bottino R, Bertera S, Trucco M, et al.: Production of alpha 1, 3-galactosyltransferase-deficient pigs. Science 2003, 299: 411-414.
121. Platt JL, Vercellotti GM, Dalmasso AP, Matas AJ, Bolman RM, Najarian JS, Bach FH: Transplantation of discordant xenografts: a review of progress. Immunol Today 1990, 11: 450-456; discussion 456-457.
122. Posner BI, Gonzalez RM, Guyda HJ: Intracellular (Golgi) receptors for insulinlike peptides in rat liver. Can J Biochem 1980, 58: 1075-1081.
123. Raff M: Cell suicide for beginners. Nature 1998, 396: 119-122.
124. Rajewsky K, Gu H, Kuhn R, Betz UA, Muller W, Roes J, Schwenk F: Conditional gene targeting. J Clin Invest 1996, 98: 600-603.
125. Ravnik-Glavac M, Atkinson A, Glavac D, Dean M: DHPLC screening of cystic fibrosis gone mutations. Hum Mutat 2002, 19: 374-383.
126. Rehfeldt C. FI, Dietl G., Ender K.: Myogenesis and postnatal skeletal muscle cell growth as influenced by selection. Livestock Production Science 2000, 66: 177-188.
127. Roberts PS, Jozwiak S, Kwiatkowski DJ, Dabora SL: Denaturing high-performance liquid chromatography (DHPLC) is a highly sensitive, semi-automated method for identifying mutations in the TSC1 gene. J Biochem Biophys Methods 2001, 47: 33-37.
128. Sachs DH: The pig as a potential xenograft donor. Vet Immunol Immunopathol 1994, 43: 185-191.
129. Sate S, Hughes RC: Regulation of secretion and surface expression of Mac-2, a galactoside-binding protein of macrophages. J Biol Chem 1994, 269: 4424-4430.
130. Schulz I, Zeitschel U, Rudolph T, Ruiz-Carrillo D, Rahfeld JU, Gerhartz B, Bigl V, Demuth HU, Rossner S: Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion. J Neurochem 2005, 94: 970-979.
131. Sendal Y, Sawada T, Urakawa M, Shinkai Y, Kubota K, Teraoka S, Hoshi H, Aoyagi Y: Heterozygous disruption of the alpha1, 3-galactosyltransferase gene in cattle. Transplantation 2003, 76: 900-902.
132. Sessa WC, Barber CM, Lynch KR: Mutation of N-myristoylation site converts endothelial cell nitric oxide synthase from a membrane to a cytosolic protein. Circ Res 1993, 72: 921-924.
133. Shi B, Hsu HL, Evens AM, Gordon LI, Gartenhaus RB: Expression of the candidate MCT-1 oncogene in B- and T-cell lymphoid malignancies. Blood 2003, 102: 297-302.
134. Shorter J, Warren G: Golgi architecture and inheritance. Annu Rev Cell Dev Biol 2002, 18: 379-420.
135. Shulman MJ, Nissen L, Collins C: Homologous recombination in hybridoma cells: dependence on time and fragment length. Mol Cell Biol 1990, 10: 4466-4472.
136. Slot JW, Geuze HJ: Sizing of protein A-colloidal gold probes for immunoelectron microscopy. J Cell Biol 1981, 90: 533-536.
137. Steller H: Mechanisms and genes of cellular suicide. Science 1995, 267: 1445-1449.
138. Stolle K, Schnoor M, Fuellen G, Spitzer M, Engel T, Spener F, Cullen P, Lorkowski S: Cloning, cellular localization, genomic organization, and tissue-specific expression of the TGFbetal-inducible SMAP-5 gene. Gene 2005, 351: 119-130.
139. Storrie B, Nilsson T: The Golgi apparatus: balancing new with old. Traffic 2002, 3:521-529.
140. Strahan KM, Gu F, Preece AF, Gustavsson I, Andersson L, Gustafsson K: cDNA sequence and chromosome localization of pig alpha 1, 3 galactosyltransferase. Immunogenetics 1995, 41: 101-105.
141. Swan DC, Tucker RA, Holloway BP, Icenogle JP: A sensitive, type-specific, fluorogenic probe assay for detection of human papillomavirus DNA. J Clin Microbiol 1997, 35: 886-891.
142. Tearle RG, Tango MJ, Zannettino ZL, Katerelos M, Shinkel TA, Van Denderen BJ, Lonie AJ, Lyons I, Nettle MB, Cox T, Becker C, Peura AM, Wigley PL, Crawford R J, Robins A J, Pearse MJ, d'Apice AJ: The alpha-1, 3-galactosyltransferase knockout mouse. Implications for xenotransplantation. Transplantation 1996, 61: 13-19.
143. Teasdale RD, Jackson MR: Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the golgi apparatus. Annu Rev Cell Dev Biol 1996, 12: 27-54.
144. Thall AD, Maly P, Lowe JB: Oocyte Gal alpha 1, 3 Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse. J Biol Chem 1995, 270: 21437-21440.
145. Thompson EB: The many roles of c-Myc in apoptosis. Annu Rev Physiol 1998, 60: 575-600.
146. Tinglu G, Ghosh A, Ghosh BK: Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold. J Bacteriol 1984, 159: 668-677.
147. van Heerde WL, Robert-Offerman S, Dumont E, Hofstra L, Doevendans PA, Smits JF, Daemen MJ, Reutelingsperger CP: Markers of apoptosis in cardiovascular tissues: focus on Annexin V. Cardiovasc Res 2000, 45: 549-559.
148. Vaux DL, Korsmeyer SJ: Cell death in development. Cell 1999, 96: 245-254.
149. Velculescu VE, Zhang L, Vogelstein B, Kinzler KW: Serial analysis of gene expression. Science 1995, 270: 484-487.
150. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C: A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995, 184: 39-51.
151. Vignal A, Milan D, SanCristobal M, Eggen A: A review on SNP and other types of molecular markers and their use in animal genetics. Genet Sel Evol 2002, 34: 275-305.
152. Wang CY, Mayo MW, Komeluk RG, Goeddel DV, Baldwin AS, Jr.: NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998, 281: 1680-1683.
153. Wolf BB, Green DR: Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 1999, 274: 20049-20052.
154. Wolford JK, Blunt D, Ballecer C, Prochazka M: High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC). Hum Genet 2000, 107: 483-487.
155. Wylie FG, Lock JG, Jamriska L, Khromykh T, Brown DL, Stow JL: GAIP participates in budding of membrane carriers at the trans-Golgi network. Traffic 2003, 4: 175-189.
156. Xiao W, Oefner PJ: Denaturing high-performance liquid chromatography: A review. Hum Mutat 2001, 17: 439-474.
157. Ye SQ, Zhang LQ, Zheng F, Virgil D, Kwiterovich PO: miniSAGE: gene expression profiling using serial analysis of gene expression from 1 microg total RNA. Anal Biochem 2000, 287: 144-152.
158. Yin C, Knudson CM, Korsmeyer SJ, Van Dyke T: Bax suppresses tumorigenesis and stimulates apoptosis in vivo. Nature 1997, 385: 637-640.
159. Yu A, McMaster CR, Byers DM, Ridgway ND, Cook HW: Resistance to UV-induced apoptosis in Chinese-hamster ovary cells overexpressing phosphatidylserine synthases. Biochem J 2004, 381: 609-618.
160. Zhang L, Yoshimura Y, Hatta T, Otani H: Myogenic determination and differentiation of the mouse palatal muscle in relation to the developing mandibular nerve. J Dent Res 1999, 78: 1417-1425.
161. Zhang MQ: Identification of Human Gene Core-promoters In Silico. Genome Research 1998.
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12. Bellas RE, FitzGerald MJ, Fausto N, Sonenshein GE: Inhibition of NF-kappa B activity induces apoptosis in murine hepatocytes. Am J Pathol 1997, 151: 891-896.
13. Belusa R, Wang ZM, Matsubara T, Sahlgren B, Dulubova I, Nairn AC, Ruoslahti E, Greengard P, Aperia A: Mutation of the protein kinase C phosphorylation site on rat alphal Na+, K+-ATPase alters regulation of intracellular Na+ and pH and influences cell shape and adhesiveness. J Biol Chem 1997, 272: 20179-20184.
14. Bennett M, Macdonald K, Chan SW, Luzio JP, Simari R, Weissberg P: Cell surface trafficking of Fas: a rapid mechanism of p53-mediated apoptosis. Science 1998, 282: 290-293.
15. Blanken WM, Van den Eijnden DH: Biosynthesis of terminal Gal alpha 1—3Gal beta 1—4GIcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal: N-acetyllactosaminide alpha 1—3-galactosyltransferase from calf thymus. J Biol Chem 1985, 260: 12927-12934.
16. Braun RE, Lo D, Pinkert CA, Widera G, Flavell RA, Palmiter RD, Brinster RL: Infertility in male transgenic mice: disruption of sperm development by HSV-tk expression in postmeiotic germ cells. Biol Reprod 1990, 43: 684-693.
17. Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M, Roth R, George D, Eletr S, Albrecht G, Vermaas E, Williams SR, Moon K, Burcham T, Pallas M, DuBridge RB, et al.: Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol 2000, 18: 630-634.
18. Brown EA, Zajac AJ, Lemon SM: In vitro characterization of an internal ribosomal entry site (IRES) present within the 5' nontranslated region of hepatitis A virus RNA: comparison with the IRES of encephalomyocarditis virus. J Virol 1994, 68: 1066-1074.
19. Bucher P, Morel P, Buhler LH: Xenotransplantation: an update on recent progress and future perspectives. Transpl Int 2005, 18: 894-901.
20. Calne RY: Organ transplantation between widely disparate species. Transplant Proc 1970, 2: 550-556.
21. Charlotte Rehfeldt NCS, Ilse Fiedler and Jochen Wegner: Environmental and Genetic Factors as Sources of Variation in Skeletal Muscle Fibre Number. Basic Appl. Myol. 1999, 9: 235-253.
22. Cho SK, Yeh J, Cho M, Cummings RD: Transcriptional regulation of alpha 1, 3-galactosyltransferase in embryonal carcinoma cells by retinoic acid. Masking of Lewis X antigens by alpha-galactosylation. J Biol Chem 1996, 271: 3238-3246.
23. Cobb JP, Mindrinos MN, Miller-Graziano C, Calvano SE, Baker HV, Xiao W, Laudanski K, Brownstein BH, Elson CM, Hayden DL, Herndon DN, Lowry SF, Maier RV, Schoenfeld DA, Moldawer LL, Davis RW, Tompkins RG, Baker HV, Bankey P, Billiar T, et al.: Application of genome-wide expression analysis to human health and disease. Proc Natl Acad Sci U S A 2005, 102: 4801-4806.
24. Cooper DK: Is xenotransplantation a realistic clinical option? Transplant Proc 1992, 24: 2393-2396.
25. Coulson AS: The Golgi apparatus in small lymphocytes and in transformed blast cells. Q J Exp Physiol Cogn Med Sci 1965, 50: 271-276.
26. Cui Q, Jiang T, Lin B, Ma S: Esuh8: a novel tool to predict protein subcellular localizations in eukaryotic organisms. BMC Bioinformatics 2004, 5: 66.
27. Curtin JF, Cotter TG: Live and let die: regulatory mechanisms in Fas-mediated apoptosis. Cell Signal 2003, 15: 983-992.
28. Cutolo M, Sulli A, Barone A, Seriolo B, Accardo S: Sex hormones, proto-oncogene expression and apoptosis: their effects on rheumatoid synovial tissue. Clin Exp Rheumatol 1996, 14: 87-94.
29. Czernik PJ, Peterson CA, Hurlburt BK: Preferential binding of MyoD-E12 versus myogenin-E12 to the murine sarcoma virus enhancer in vitro. J Biol Chem 1996, 271: 9141-9149.
30. Dabkowski PL, Vaughan HA, McKenzie IF, Sandrin MS: Characterisation of a cDNA clone encoding the pig alpha 1, 3 galactosyltransferase: implications for xenotransplantation. Transplant Proc 1993, 25: 2921.
31. Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL: Targeted disruption of the alpha1, 3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002, 20: 251-255.
32. Dang CV: c-Myc target genes involved in cell growth, apoptosis, and metabolism. Mol Cell Biol 1999, 19: 1-11.
33. Daniotti JL, Martina JA, Giraudo CG, Zurita AR, Maccioni HJ: GM3 alpha2, 8-sialyltransferase (GD3 synthase): protein characterization and sub-golgi location in CHO-K1 cells. J Neurochem 2000, 74: 1711-1720.
34. Davies MV, Kaufman RJ: The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation. J Virol 1992, 66: 1924-1932.
35. de Hoop MJ, Ab G: Import of proteins into peroxisomes and other microbodies. Biochem J 1992, 286 (Pt 3): 657-669.
36. Dehoux JP, de la Parra B, Latinne D, Bazin H, Gianello P: Characterization of baboon anti-porcine IgG antibodies during acute vascular rejection of porcine kidney xenograft. Xenotransplantation 2002, 9: 338-349.
37. Deng C, Capecchi MR: Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol Cell Biol 1992, 12: 3365-3371.
38. Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, Martinou JC: Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 1999, 144: 891-901.
39. Deschamps JY, Roux FA, Sai P, Gouin E: History of xenotransplantation. Xenotransplantation 2005, 12: 91-109.
40. Dorling A: Are anti-endothelial cell antibodies a pre-requisite for the acute vascular rejection of xenografts? Xenotransplantation 2003, 10: 16-23.
41. Dunkov BC, Georgieva T, Yoshiga T, Hall M, Law JH: Aedes aegypti ferritin heavy chain homologue: feeding of iron or blood influences message levels, lengths and subunit abundance. J Insect Sci 2002, 2: 7.
42. Finucane DM, Waterhouse NJ, Amarante-Mendes GP, Cotter TG, Green DR: Collapse of the inner mitochondrial transmembrane potential is not required for apoptosis of HL60 cells. Exp Cell Res 1999, 251: 166-174.
43. Forrest MS, Lan Q, Hubbard AE, Zhang L, Vermeulen R, Zhao X, Li G, Wu YY, Shen M, Yin S, Chanock SJ, Rothman N, Smith MT: Discovery of novel biomarkers by microarray analysis of peripheral blood mononuclear cell gene expression in benzene-exposed workers. Environ Health Perspect 2005, 113: 801-807.
44. Fujiwara Y, Asogawa M: Prediction of subcellular localizations using amino acid composition and order. Genome Inform Ser Workshop Genome Inform 2001, 12: 103-112.
45. Galili U, Rachmilewitz EA, Peleg A, Flechner I: A unique natural human IgG antibody with anfi-alpha-galactosyl specificity. J Exp Med 1984, 160: 1519-1531.
46. Galili U, Macher BA, Buehler J, Shohet SB: Human natural anti-alpha-galactosyl IgG Ⅱ. The specific recognition of alpha (1—3)-iinked galactose residues. J Exp Med 1985, 162: 573-582.
47. Galili U, Clark MR, Shohet SB, Buehler I, Macher BA: Evolutionary relationship between the natural anti-Gal antibody and the Gal alpha 1—3Gal epitope in primates. Proc Natl Acad Sci U S A 1987, 84: 1369-1373.
48. Gamen S, Anel A, Pineiro A, Naval J: Caspases are the main executioners of Fas-mediated apoptosls, irrespective of the ceramide signalling pathway. Cell Death Differ 1998, 5: 241-249.
49. Gehnini S, Orlando C, Sestini R, Vona G, Pinzani P, Ruocco L, Pazzagli M: Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification. Clin Chem 1997, 43: 752-758.
50. Geuze HJ, Slot JW, van der Ley PA, Scbeffer PC: Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections. J Cell Biol 1981, 89: 653-665.
51. Glaumann H, Ericsson JL: Evidence for the participation of the Golgi apparatus in the intracellular transport of nascent albumin in the liver cell. J Cell Biol 1970, 47: 555-567.
52. Gould SJ, Keller GA, Subramani S: Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. J Cell Biol 1988, 107: 897-905.
53. Gould SJ, Keller GA, Hosken N, Wilkinson J, Subramani S: A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol 1989, 108: 1657-1664.
54. Gould S J, Keller GA, Schneider M, Howell SH, Garrard LJ, Goodman JM, Distel B, Tabak H, Subramani S: Peroxisomal protein import is conserved between yeast, plants, insects and mammals. Embo J 1990, 9: 85-90.
55. Gravotta D, Adesnik M, Sabatini DD: Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins. J Cell Biol 1990, 111: 2893-2908.
56. Gregoli PA, Bondurant MC: The roles of Bcl-X(L) and apopain in the control of erythropoiesis by erythropoietin. Blood 1997, 90: 630-640.
57. Grosjean H, Fiers W: Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene 1982, 18: 199-209.
58. Hanzlik TN, Abdel-Aal YA, Harshman LG, Hammock BD: Isolation and sequencing of cDNA clones coding for juvenile hormone esterase from Heliothis virescens. Evidence for a catalytic mechanism for the serine carboxylesterases different from that of the serine proteases. J Biol Chem 1989, 264: 12419-12425.
59. Haynes AP, Daniels I, Abhulayha AM, Carter GI, Metheringham R, Gregory CD, Thomson BJ: CD95 (Fas) expression is regulated by sequestration in the Golgi complex in B-cell lymphoma. Br J Haematol 2002, 118: 488-494.
60. Hecker M, Mulsch A, Busse R: Subcellular localization and characterization of neuronal nitric oxide synthase. J Neurochem 1994, 62: 1524-1529.
61. Heid CA, Stevens J, Livak KJ, Williams PM: Real time quantitative PCR. Genome Res 1996, 6: 986-994.
62. Heinrich UR, Liondyno M, Maurer J, Mann W, Guth PS, Forstermann U: Localization of the two constitutively expressed nitric oxide synthase isoforms (nNOS and eNOS) in the same cell types in the saccule maculae of the frog Rana pipiens by immunoelectron microscopy: evidence for a back-up system? J Electron Microsc (Tokyo) 2003, 52: 197-206.
63. Helps C, Lait P, Tasker S, Harbour D: Melting curve analysis of feline calicivirus isolates detected by real-time reverse transcription PCR. J Virol Methods 2002, 106: 241-244.
64. Henion TR, Macher BA, Anaraki F, Galili U: Defining the minimal size of catalytically active primate alpha 1, 3 galactosyltransferase: structure-function studies on the recombinant truncated enzyme. Glycobiology 1994, 4: 193-201.
65. Iakovlev AI, Ts KT, Klepukov AA: [Micro-agglutination reaction of Rickettsia and viruses with the aid of luminescence microscopy.]. Vopr Virusol 1958, 3: 369-372.
66. Ikebukuro K, Adachi Y, Toki J, Taketani S, Tokunaga R, Hioki K, Ikehara S: Morphological change, loss of deltapsi(m) and activation of caspases upon apoptosis of colorectal adenocarcinoma induced by 5-FU. Cancer Lett 2000, 153: 101-108.
67. Ikematsu S, Kaname T, Ozawa M, Yonezawa S, Sate E, Uehara F, Obama H, Yamamura K, Muramatsu T: Transgenic mouse lines with ectopic expression of alpha-1, 3-galactosyltransferase: production and characteristics. Glycobiology 1993, 3: 575-580.
68. Jin DI, Lee SH, Choi JH, Lee JS, Lee JE, Park KW, Seo JS: Targeting efficiency of a-1, 3-galactosyl transferase gene in pig fetal fibroblast cells. Exp Mol Med 2003, 35: 572-577.
69. Joe Sambrook DR: Molecular Cloning: A Laboratoty Mannual. 2001: 68-86.
70. Joziasse DH, Oriel R: Xenotransplantation: the importance of the Galalpha1, 3Ga1 epitope in hyperacute vascular rejection. Biochim Biophys Acta 1999, 1455: 403-418.
71. Joziasse DH, Shaper JH, Van den Eijnden DH, Van Tunen AJ, Shaper NL: Bovine alpha 1—3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA. J Biol Chem 1989, 264: 14290-14297.
72. Juin P, Hueber AO, Littiewood T, Evan G: c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release. Genes Dev 1999, 13: 1367-1381.
73. Kasibhatla S, Beere HM, Brunner T, Echeverri F, Green DR: A 'non-canonical' DNA-binding element mediates the response of the Fas-ligand promoter to c-Myc. Curr Biol 2000, 10: 1205-1208.
74. Kong Y, Flick MJ, Kudla AJ, Konieczny SF: Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD. Mol Cell Biol 1997, 17: 4750-4760.
75. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH: Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994, 84: 1415-1420.
76. Kozak M: An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 1987, 15: 8125-8148.
77. Krajewski S, Krajewska M, Reed JC: Immunohistochemical analysis of in vivo patterns of Bak expression, a proapoptotic member of the Bcl-2 protein family. Cancer Res 1996, 56: 2849-2855.
78. Kumar R, Yang J, Larsen RD, Stanley P: Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation. Proc Natl Acad Sci U S A 1990, 87: 9948-9952.
79. Lai EC: Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet 2002, 30: 363-364.
80. Lai EC, Burks C, Posakony JW: The K box, a conserved 3' UTR sequence motif, negatively regulates accumulation of enhancer of split complex transcripts. Development 1998, 125: 4077-4088.
81. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS: Production of alpha-1, 3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002, 295: 1089-1092.
82. Lakso M, Saner B, Mosinger B, Jr., Lee EJ, Manning RW, Yu SH, Mulder KL, Westphal H: Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci U S A 1992, 89: 6232-6236.
83. Lambert DL, Malik N, Shepherd L, Gunn J, Francis SE, King A, Crossman DC, Cumberland DC, Holt CM: Localization of c-Myb and induction of apoptosis by antisense oligonucleotide c-Myb after angioplasty of porcine coronary arteries. Arterioscler Thromb Vasc Biol 2001, 21: 1727-1732.
84. Lanza RP, Cibelli JB, West MD: Prospects for the use of nuclear transfer in human transplantation. Nat Biotechnol 1999, 17: 1171-1174.
85. Larsen RD, Rajan VP, Ruff MM, Kukowska-Latallo J, Cummings RD, Lowe JB: Isolation of a cDNA encoding a murine UDPgalactose: beta-D-galactosyl- 1, 4-N-acetyl-D-glucosaminide alpha-1, 3-galactosyltransferase: expression cloning by gene transfer. Proc Natl Acad Sci U S A 1989, 86: 8227-8231.
86. Le SY, Maizel JV, Jr.: A common RNA structural motif involved in the internal initiation of translation of cellular mRNAs. Nucleic Acids Res 1997, 25: 362-369.
87. Lenato GM, Lastella P, Di Giacomo MC, Resta N, Suppressa P, Pasculli G, Sabba C, Guanti G: DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population. Hum Mutat 2006, 27: 213-214.
88. Levrat C, Ardail D, Louisot P: Comparative Study of the N-glycoprotein synthesis through dolichol intermediates in mitochondria, Golgi apparatus-rich fraction and endoplasmic reticulum-rich fraction. Int J Biochem 1990, 22: 287-293.
89. Li K, Li Y, Shelton JM, Richardson JA, Spencer E, Chen 73, Wang X, Williams RS: Cytochrome c deficiency causes embryonic lethality and attenuates stress-induced apoptosis. Cell 2000, 101: 389-399.
90. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X: Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997, 91: 479-489.
91. Lippincott-Schwartz J, Roberts TH, Hirschberg K: Secretory protein trafficking and organelle dynamics in living cells. Annu Rev Cell Dev Biol 2000, 16: 557-589.
92. Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN, James CD: Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Res 1998, 26: 1396-1400.
93. Livak K J, Flood SJ, Marmaro J, Giusti W, Deetz K: Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 1995, 4: 357-362.
94. Long M, Rosenberg C, Gilbert W: Intron phase correlations and the evolution of the intron/exon structure of genes. Proc Natl Acad Sci U S A 1995, 92: 12495-12499.
95. Ly JD, Grubb DR, Lawen A: The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis 2003, 8: 115-128.
96. Mackay IM, Arden KE, Nitsche A: Real-time PCR in virology. Nucleic Acids Res 2002, 30: 1292-1305.
97. Maleki SJ, Royer CA, Hurlburt BK: Analysis of the DNA-binding properties of MyoD, myogenin, and E12 by fluorescence anisotropy. Biochemistry 2002, 41: 10888-10894.
98. Martin S J, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR: Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Ab1. J Exp Med 1995, 182: 1545-1556.
99. McDonnell TJ, Beham A, Sarkiss M, Andersen MM, Lo P: Importance of the Bcl-2 family in cell death regulation. Experientia 1996, 52: 1008-1017.
100. McKnight S, Tjian R: Transcriptional selectivity of viral genes in mammalian cells. Cell 1986, 46: 795-805.
101. Metcalfe AD, Hunter HR, Bloor D J, Lieberman BA, Picton HM, Leese HJ, Kimber SJ, Brison DR: Expression of 11 members of the BCL-2 family of apoptosis regulatory molecules during human preimplantation embryo development and fragmentation. Mol Reprod Dev 2004, 68: 35-50.
102. Michaels MG: Xenotransplant-associated infections. Lab Anita Sci 1998, 48: 228-233.
103. Minegishi N, Suzuki N, Kawatani Y, Shimizu R, Yamamoto M: Rapid turnover of GATA-2 via ubiquitin-proteasome protein degradation pathway. Genes Cells 2005, 10: 693-704.
104. Moreadith RW, Radford NB: Gene targeting in embryonic stem cells: the new physiology and metabolism. J Mol Med 1997, 75: 208-216.
105. Morris T, Robertson B, Gallagher M: Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system. J Clin Microbiol 1996, 34: 2933-2936.
106. Mountz JD, Zhou T, Wu J, Wang W, Su X, Cheng J: Regulation of apoptosis in immune cells. J Clin Immunol 1995, 15: 1-16.
107. Muller U: Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech Dev 1999, 82: 3-21.
108. Nagase M, Hirose S, Fujita T: Unique repetitive sequence and unexpected regulation of expression of rat endothelial receptor for oxidized low-density lipoprotein (LOX-1). Biochem J 1998, 330 (Pt 3): 1417-1422.
109. Nakamura K, Bossy-Wetzel E, Burns K, Fadel MP, Lozyk M, Goping IS, Opas M, Bleackley RC, Green DR, Michalak M: Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis. J Cell Biol 2000, 150: 731-740.
110. Numata M, Orlowski J: Molecular cloning and characterization of a novel (Na+, K+)/H+ exchanger localized to the trans-Golgi network. J Biol Chem 2001, 276: 17387-17394.
111. Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, Tokino T, Taniguchi T, Tanaka N: Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 2000, 288: 1053-1058.
112. Ogle BM, Platt JL: Genetic therapies and xenotransplantation. Expert Opin Biol Ther 2002, 2: 299-310.
113. Oltean S, Banerjee R: A B12-responsive internal ribosome entry site (IRES) element in human methionine synthase. J Biol Chem 2005, 280: 32662-32668.
114. Orkin SH, Weiss MJ: Apoptosis. Cutting red-cell production. Nature 1999, 401: 433, 435-436.
115. Ossina NK, Cannas A, Powers VC, Fitzpatrick PA, Knight JD, Gilbert JR, Shekhtman EM, Tomei LD, Umansky SR, Kiefer MC: Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem 1997, 272: 16351-16357.
116. Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM: Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 1982, 300: 611-615.
117. Panepinto LM, Phillips RW: The Yucatan miniature pig: characterization and utilization in biomedical research. Lab Anim Sci 1986, 36: 344-347.
118. Parcharidou A, Raza A, Economopoulos T, Papageorgiou E, Anagnostou D, Papadaki T, Raptis S: Extensive apoptosis of bone marrow cells as evaluated by the in situ end-labelling (ISEL) technique may be the basis for ineffective haematopoiesis in patients with myelodysplastic syndromes. Eur J Haematol 1999, 62: 19-26.
119. Patience C, Takeuchi Y, Weiss RA: Infection of human cells by an endogenous retrovirus of pigs. Nat Med 1997, 3: 282-286.
120. Phelps CJ, Koike C, Vanght TD, Boone J, Wells KD, Chen SH, Ball S, Specht SM, Polejaeva IA, Monahan JA, Jobst PM, Sharma SB, Lamborn AE, Garst AS, Moore M, Demetris A J, Rudert WA, Bottino R, Bertera S, Trucco M, et al.: Production of alpha 1, 3-galactosyltransferase-deficient pigs. Science 2003, 299: 411-414.
121. Platt JL, Vercellotti GM, Dalmasso AP, Matas AJ, Bolman RM, Najarian JS, Bach FH: Transplantation of discordant xenografts: a review of progress. Immunol Today 1990, 11: 450-456; discussion 456-457.
122. Posner BI, Gonzalez RM, Guyda HJ: Intracellular (Golgi) receptors for insulinlike peptides in rat liver. Can J Biochem 1980, 58: 1075-1081.
123. Raff M: Cell suicide for beginners. Nature 1998, 396: 119-122.
124. Rajewsky K, Gu H, Kuhn R, Betz UA, Muller W, Roes J, Schwenk F: Conditional gene targeting. J Clin Invest 1996, 98: 600-603.
125. Ravnik-Glavac M, Atkinson A, Glavac D, Dean M: DHPLC screening of cystic fibrosis gone mutations. Hum Mutat 2002, 19: 374-383.
126. Rehfeldt C. FI, Dietl G., Ender K.: Myogenesis and postnatal skeletal muscle cell growth as influenced by selection. Livestock Production Science 2000, 66: 177-188.
127. Roberts PS, Jozwiak S, Kwiatkowski DJ, Dabora SL: Denaturing high-performance liquid chromatography (DHPLC) is a highly sensitive, semi-automated method for identifying mutations in the TSC1 gene. J Biochem Biophys Methods 2001, 47: 33-37.
128. Sachs DH: The pig as a potential xenograft donor. Vet Immunol Immunopathol 1994, 43: 185-191.
129. Sate S, Hughes RC: Regulation of secretion and surface expression of Mac-2, a galactoside-binding protein of macrophages. J Biol Chem 1994, 269: 4424-4430.
130. Schulz I, Zeitschel U, Rudolph T, Ruiz-Carrillo D, Rahfeld JU, Gerhartz B, Bigl V, Demuth HU, Rossner S: Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion. J Neurochem 2005, 94: 970-979.
131. Sendal Y, Sawada T, Urakawa M, Shinkai Y, Kubota K, Teraoka S, Hoshi H, Aoyagi Y: Heterozygous disruption of the alpha1, 3-galactosyltransferase gene in cattle. Transplantation 2003, 76: 900-902.
132. Sessa WC, Barber CM, Lynch KR: Mutation of N-myristoylation site converts endothelial cell nitric oxide synthase from a membrane to a cytosolic protein. Circ Res 1993, 72: 921-924.
133. Shi B, Hsu HL, Evens AM, Gordon LI, Gartenhaus RB: Expression of the candidate MCT-1 oncogene in B- and T-cell lymphoid malignancies. Blood 2003, 102: 297-302.
134. Shorter J, Warren G: Golgi architecture and inheritance. Annu Rev Cell Dev Biol 2002, 18: 379-420.
135. Shulman MJ, Nissen L, Collins C: Homologous recombination in hybridoma cells: dependence on time and fragment length. Mol Cell Biol 1990, 10: 4466-4472.
136. Slot JW, Geuze HJ: Sizing of protein A-colloidal gold probes for immunoelectron microscopy. J Cell Biol 1981, 90: 533-536.
137. Steller H: Mechanisms and genes of cellular suicide. Science 1995, 267: 1445-1449.
138. Stolle K, Schnoor M, Fuellen G, Spitzer M, Engel T, Spener F, Cullen P, Lorkowski S: Cloning, cellular localization, genomic organization, and tissue-specific expression of the TGFbetal-inducible SMAP-5 gene. Gene 2005, 351: 119-130.
139. Storrie B, Nilsson T: The Golgi apparatus: balancing new with old. Traffic 2002, 3:521-529.
140. Strahan KM, Gu F, Preece AF, Gustavsson I, Andersson L, Gustafsson K: cDNA sequence and chromosome localization of pig alpha 1, 3 galactosyltransferase. Immunogenetics 1995, 41: 101-105.
141. Swan DC, Tucker RA, Holloway BP, Icenogle JP: A sensitive, type-specific, fluorogenic probe assay for detection of human papillomavirus DNA. J Clin Microbiol 1997, 35: 886-891.
142. Tearle RG, Tango MJ, Zannettino ZL, Katerelos M, Shinkel TA, Van Denderen BJ, Lonie AJ, Lyons I, Nettle MB, Cox T, Becker C, Peura AM, Wigley PL, Crawford R J, Robins A J, Pearse MJ, d'Apice AJ: The alpha-1, 3-galactosyltransferase knockout mouse. Implications for xenotransplantation. Transplantation 1996, 61: 13-19.
143. Teasdale RD, Jackson MR: Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the golgi apparatus. Annu Rev Cell Dev Biol 1996, 12: 27-54.
144. Thall AD, Maly P, Lowe JB: Oocyte Gal alpha 1, 3 Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse. J Biol Chem 1995, 270: 21437-21440.
145. Thompson EB: The many roles of c-Myc in apoptosis. Annu Rev Physiol 1998, 60: 575-600.
146. Tinglu G, Ghosh A, Ghosh BK: Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold. J Bacteriol 1984, 159: 668-677.
147. van Heerde WL, Robert-Offerman S, Dumont E, Hofstra L, Doevendans PA, Smits JF, Daemen MJ, Reutelingsperger CP: Markers of apoptosis in cardiovascular tissues: focus on Annexin V. Cardiovasc Res 2000, 45: 549-559.
148. Vaux DL, Korsmeyer SJ: Cell death in development. Cell 1999, 96: 245-254.
149. Velculescu VE, Zhang L, Vogelstein B, Kinzler KW: Serial analysis of gene expression. Science 1995, 270: 484-487.
150. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C: A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995, 184: 39-51.
151. Vignal A, Milan D, SanCristobal M, Eggen A: A review on SNP and other types of molecular markers and their use in animal genetics. Genet Sel Evol 2002, 34: 275-305.
152. Wang CY, Mayo MW, Komeluk RG, Goeddel DV, Baldwin AS, Jr.: NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998, 281: 1680-1683.
153. Wolf BB, Green DR: Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 1999, 274: 20049-20052.
154. Wolford JK, Blunt D, Ballecer C, Prochazka M: High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC). Hum Genet 2000, 107: 483-487.
155. Wylie FG, Lock JG, Jamriska L, Khromykh T, Brown DL, Stow JL: GAIP participates in budding of membrane carriers at the trans-Golgi network. Traffic 2003, 4: 175-189.
156. Xiao W, Oefner PJ: Denaturing high-performance liquid chromatography: A review. Hum Mutat 2001, 17: 439-474.
157. Ye SQ, Zhang LQ, Zheng F, Virgil D, Kwiterovich PO: miniSAGE: gene expression profiling using serial analysis of gene expression from 1 microg total RNA. Anal Biochem 2000, 287: 144-152.
158. Yin C, Knudson CM, Korsmeyer SJ, Van Dyke T: Bax suppresses tumorigenesis and stimulates apoptosis in vivo. Nature 1997, 385: 637-640.
159. Yu A, McMaster CR, Byers DM, Ridgway ND, Cook HW: Resistance to UV-induced apoptosis in Chinese-hamster ovary cells overexpressing phosphatidylserine synthases. Biochem J 2004, 381: 609-618.
160. Zhang L, Yoshimura Y, Hatta T, Otani H: Myogenic determination and differentiation of the mouse palatal muscle in relation to the developing mandibular nerve. J Dent Res 1999, 78: 1417-1425.
161. Zhang MQ: Identification of Human Gene Core-promoters In Silico. Genome Research 1998.