Production of heterozygous alpha 1,3-galactosyltransferase (<Emphasis Type="Italic">GGTA1</Emphasis>) knock-out transgenic miniature pigs expressing human <Emphasis Type="Italic">CD39</Emphasis>

Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig’s cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig’s cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.     

利用ZFN删除转基因猪细胞中的多拷贝EGFP基因

锌指核酸酶（zinc finger nuclease, ZFN）是由特异性识别DNA的锌指结构域和Fok I切割结构域组成,能够在基因组特定位点上切割DNA,引起DNA双链断裂（double-strand break, DSB）. 通过DSB修复机制,可以使基因修饰的效率比传统方法提高102～104倍．目前,利用ZFN对动物内源基因进行敲除的研究较多,但对转基因动物中外源多拷贝基因进行敲除的报道较少．本研究首先利用荧光定量PCR法对本实验室培育的两头转基因猪中增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）基因的拷贝数进行鉴定,发现其拷贝数分别为11.95和17.36拷贝;然后将靶向EGFP的一对ZFN转染进拷贝数为1736的EGFP转基因猪的成纤维细胞中,并通过流式和CEL-1酶切方法检测敲除效率. 结果表明,转染400 ng、800 ng和1 200 ng ZFN的切割效率分别为0.97%、1.39%和1.76%,可见随着转染ZFN剂量的增加,ZFN的切割效率逐渐提高．但是,不发绿色荧光的细胞比例却没有明显提高,因此认为,ZFN敲除转基因动物中多拷贝基因的效率还是比较低．     

利用CRISPR/Cas9系统构建Tiki1基因修饰猪模型

Tiki1基因是哈佛大学儿童医学院贺熹教授实验室发现的一个对蛙头部的诱导起到决定性作用的新基因,但Tiki1基因在小鼠等啮齿类动物中缺失,因此无法利用小鼠等小动物来研究其在哺乳动物中的作用.本文利用CRISPR/Cas9系统结合体细胞克隆技术构建Tiki1基因修饰猪模型,研究Tiki1基因在猪发育中的作用.我们利用贺熹教授团队提供的人Tiki1基因序列,在猪的基因组数据库中比对出与其同源性最高的一段序列设计2个靶位点(g1和g2).以设计的靶位点构建打靶质粒转染猪胎儿成纤维细胞,经细胞筛选、PCR扩增及测序共鉴定了52个单细胞克隆株.最终选择靶位点g1为纯合双敲的5个单细胞克隆株和靶位点g2为纯合双敲的3个单细胞克隆株作为构建Tiki1基因敲除猪的核供体.我们共计构建了720个重组胚胎,分别植入3头代孕母猪,其中有1头经B超检测成功怀孕并妊娠到期产下13头发育正常的克隆猪,经测序鉴定其中12头为Tiki1基因双敲除猪模型,Tiki1基因敲除克隆猪健康存活至今.结果表明Tiki1基因对于猪早期发育的作用机理不同于蛙,其在猪早期发育的过程中的具体作用机理有待后续进一步的深入研究.     

Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases

Mammalian cells with multi‐gene knockouts could be of considerable utility in research, drug discovery, and cell‐based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events—a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc‐finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi‐locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97–105. © 2009 Wiley Periodicals, Inc.     

Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.     

Generation of GTKO <Emphasis Type="Italic">Diannan</Emphasis> Miniature Pig Expressing Human Complementary Regulator Proteins hCD55 and hCD59 via T2A Peptide-Based Bicistronic Vectors and SCNT

Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.     

Targeted disruption of the sheep MSTN gene by engineered zinc-finger nucleases

Prior to the development of zinc-finger nuclease technology, genetic manipulation by gene targeting achieved limited success in mammals, with the exception of mice and rat. Although ZFNs demonstrated highly effective gene targeted disruption in various model organisms, the activity of ZFNs in large domestic animals may be very low, and the probability of identifying ZFN-mediated positive targeted disruption events is small. In this paper, we used the context-dependent assembly method to synthesize two pairs of ZFNs targeted to the sheep MSTN gene. We verified the activity of these ZFNs using an mRFP-MBS-eGFP dual-fluorescence reporter system in HEK293T cells and, according to the expression level of eGFP, we obtained a pair of ZFNs that can recognize and cut the targeted MSTN site in the reporter vector. The activity of ZFN was increased by cold stimulation at 30 °C and by mutant the wildtype FokI in ZFN with its counterpart Sharkeys. Finally, the ZF-Sharkeys and reporter vector were cotransfected into sheep fetal fibroblasts and two MSTN mutant cell lines, identified by flow cytometry and sequencing, were obtained.     

Efficient Generation of Myostatin (MSTN) Biallelic Mutations in Cattle Using Zinc Finger Nucleases

Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle.     

Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration.     

Reduced sensitivity to human serum inactivation of enveloped viruses produced by pig cells transgenic for human CD55 or deficient for the galactosyl-alpha(1-3) galactosyl epitope

Complement activation mediated by the major xenogeneic epitope in the pig, galactosyl-alpha(1-3) galactosyl sugar structure (alpha-Gal), and human natural antibodies could cause hyperacute rejection (HAR) in pig-to-human xenotransplantation. The same reaction on viruses bearing alpha-Gal may serve as a barrier to zoonotic infection. Expressing human complement regulatory proteins or knocking out alpha-Gal epitopes in pig in order to overcome HAR may therefore pose an increased risk in xenotransplantation with regard to zoonosis. We investigated whether amphotropic murine leukemia virus, porcine endogenous retrovirus, and vesicular stomatitis virus (VSV) budding from primary transgenic pig aortic endothelial (TgPAE) cells expressing human CD55 (hCD55 or hDAF) was protected from human-complement-mediated inactivation. VSV propagated through the ST-IOWA pig cell line, in which alpha-galactosyl-transferase genes were disrupted (Gal null), was also tested for sensitivity to human complement. The TgPAE cells were positive for hCD55, and all pig cells except the Gal-null ST-IOWA expressed alpha-Gal epitopes. Through antibody binding, we were able to demonstrate the incorporation of hCD55 onto VSV particles. Viruses harvested from TgPAE cells were relatively resistant to complement-mediated inactivation by the three sources of human sera tested. Additionally, VSV from Gal-null pig cells was resistant to human complement inactivation. Such protection of enveloped viruses may increase the risk of zoonosis from pigs genetically modified for pig-to-human xenotransplantation.     

Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs

Galactose-alpha1,3-galactose (alpha1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig alpha1,3-galactosyltransferase (alpha1,3GT) by homologous recombination is a means to completely remove the alpha1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig alpha1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig alpha1,3GT allele.     

绿色荧光蛋白在α-1,3半乳糖基转移酶敲除猪组织器官的表达分析

猪是人类异种器官移植的理想供体,然而猪-人的异种器官移植会产生剧烈的排斥反应。虽然已制备的α-1,3半乳糖基转移酶基因敲除（Galactosyltransferase gene knockout, GTKO）猪可有效缓解猪-人异种器官移植引起的超急性免疫排斥,但缺少报告基因直观示踪移植后的细胞迁移及器官排斥状态。本文将CAG启动子驱动增强型绿色荧光蛋白（Enhanced green fluorescent protein, EGFP）的表达载体导入GTKO猪耳成纤维细胞,通过体细胞核移植技术制备了EGFP猪。利用双荧光蛋白观测镜、荧光显微镜及定量PCR扩增观察、检测和分析克隆猪各组织器官中EGFP蛋白和转录本的表达状况。结果显示,EGFP蛋白及转录本在克隆猪各组织器官中均有表达,但在肝脏和中枢神经系统中表达较弱。本文成功获得了各组织器官表达EGFP的GTKO猪,为EGFP示踪异种细胞组织移植奠定了基础。     

Targeted editing of goat genome with modular-assembly zinc finger nucleases based on activity prediction by computational molecular modeling

Zinc finger nuclease (ZFN) technology can mediate targeted genome modification to produce transgenic animals in a high-efficient and biological-safe way. Modular assembly is a rapid, convenient and open-source method for the synthesis of ZFNs. However, this biotechnology is hampered by multistep construction, low-efficiency editing and off-target cleavage. Here we synthesized and tested six pairs of three- or four-finger ZFNs to target one site in goat beta-lactoglobulin (BLG, a dominant allergen in goat milk) gene. Homology modeling was applied to build the structure model of ZFNs to predict their editing activities targeting at goat BLG gene. Goat fibroblast cells were transfected with plasmids that encoded ZFN pairs, and genomic DNA was isolated 72 h later for genome editing efficiency assay. The results of editing efficiency assay demonstrated that ZFNs with optimal interaction modes can edit goat BLG gene more efficiently, whereas ZFNs with unexpected interaction modes showed lower activities in editing BLG gene. We concluded that modular-assembly ZFNs can provide a rapid, public-available, and easy-to-practice platform for transgenic animal research and molecular modeling would help as a useful tool for ZFNs activity prediction.     

Generation of albino Xenopus tropicalis using zinc‐finger nucleases

To generate albino lines of Xenopus tropicalis, we injected fertilized eggs with mRNAs encoding zinc‐finger nucleases (ZFNs) targeting the tyrosinase coding region. Surprisingly, vitiligo was observed on the skin of F0 frogs that had been injected with ZFN mRNAs, indicating that both tyrosinase genes in the genome were disrupted in all melanocytes within the vitiligo patches. Mutation analysis using genomic DNA from the skin revealed that two mosaic F0 frogs underwent spatially complex tyrosinase gene mutations. The data implies that the ZFN‐induced tyrosinase gene ablations occurred randomly over space and time throughout the entire body, possibly until the young tadpole stage, and that melanocyte precursors lacking functional tyrosinase proliferated and formed vitiligo patches. Several albino X. tropicalis, which are compound heterozygotes for biallelic tyrosinase mutations, were obtained by mating the mosaic F0 frogs. To our knowledge, this is the first report of the albino vertebrates generated by the targeted gene knockout.     

Porcine endogenous retrovirus transmission characteristics of galactose alpha1-3 galactose-deficient pig cells

Galactose alpha1-3 galactose (Gal) trisaccharides are present on the surface of wild-type pig cells, as well as on viruses particles produced from such cells. The recognition of Gal sugars by natural anti-Gal antibodies (NAb) in human and Old World primate serum can cause the lysis of the particles via complement-dependent mechanisms and has therefore been proposed as an important antiviral mechanism. Recently, pigs have been generated that possess disrupted galactosyl-transferase (GGTA1) genes. The cells of these pigs do not express Gal sugars on their surface, i.e., are Gal null. Concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of the Gal sugars. We investigated the sensitivity of porcine endogenous retrovirus (PERV) produced from Gal-null and Gal-positive pig cells to inactivation by purified NAb and human serum. PERV produced in Gal-null pig cells was resistant to inactivation by either NAb or human serum. In contrast, although Gal-positive PERV particles were sensitive to inactivation by NAb and human serum, they required markedly higher concentrations of NAb for inactivation compared to the Gal-positive cells from which they were produced. Complete inactivation of Gal-positive PERV particles was not achievable despite the use of high levels of NAb, indicating that NAb-mediated inactivation of cell-free PERV particles is an inefficient process.     

The molecular basis for galalpha(1,3)gal expression in animals with a deletion of the alpha1,3galactosyltransferase gene

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes alpha1,3galactosyltransferase (alpha1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galalpha(1,3)Gal carbohydrate epitope was not observed as Galalpha(1,3)Gal staining was still present in tissues from GGTA1(-/-) animals. This shows that, contrary to previous dogma, alpha1,3GT is not the only enzyme able to synthesize Galalpha(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1(-/-) mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Galalpha(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galalpha(1,3)Gal epitope. Galalpha(1,3)Gal synthesized by iGb3S, in contrast to alpha1,3GT, was resistant to down-regulation by competition with alpha1,2fucosyltransferase. Moreover, Galalpha(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 (-/-) mice. Galalpha(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.     

应用SSA报告载体提高ZFN和CRISPR/Cas9对猪IGF2基因的打靶效率

IGF2(Insulin-like growth factor 2)基因作为最复杂多样的生长因子之一,对猪胎儿发育以及出生后生长发育和肌肉生成起着非常重要的作用。通过基因组编辑技术对我国本地猪种的IGF2基因作精确的遗传修饰,对于提高本地猪种的瘦肉率具有重要的育种意义。文章在蓝塘猪胎儿成纤维细胞(Porcine fetal fibroblasts, PEF)中检测了锌指核酸酶(Zinc finger nucleases, ZFN)和CRISPR/Cas9对IGF2基因的打靶效率,结果表明CRISPR/Cas9对IGF2基因的切割效率最高可达9.2%,显著高于ZFN的切割效率(<1%),但两者均未达到作为体细胞核移植(Somatic nuclear transfer, SCNT)供体细胞所需的打靶效率。应用SSA (Single-strand annealing)报告载体筛选技术来富集IGF2基因被ZFN和CRISPR/Cas9修饰过的PEF细胞,结果表明,该技术可使CRISPR/Cas9的打靶效率提高5倍左右,对ZFN的打靶效率具有更大的增强作用。