转基因克隆猪的研制与ZFNs介导基因敲除的初步研究
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摘要
转基因体细胞克隆猪在基础科学研究，人类医学，生物产品生产以及畜牧业生产等领域都具有非常重要的应用价值。目前，得益于新的基因打靶工具—锌指核酸酶（Znic finger nucleases, ZFNs）的出现，基因敲除效率得到大大提高，世界范围内的转基因体细胞克隆猪的研究也迈上了新的台阶。本研究以建立转基因体细胞克隆猪技术平台，研制转绿色荧光蛋白和转纤维素酶转基因猪为主要目的，系统地研究了源于细菌以及低等真核生物的纤维素酶在高等哺乳动物细胞中的密码子优化与表达，提高了纤维素酶在猪细胞中的表达水平。成功制作了转绿色荧光蛋白的转基因猪。最后对ZFNs介导的猪IGF2和Kit基因敲除进行了初步探索和分析。
本研究首先对源于细菌和低等真核生物的三种糖苷水解酶bgl4，xynB和egxA基因进行了两种方式的密码子优化，并比较了它们在四种不同种属的哺乳动物细胞中的表达水平，发现在PK15细胞系中，使用“一个密码子对应一种氨基酸”方式进行密码子优化后，其表达水平显著高于原始基因以及使用“人源化密码子”优化的基因。根据这一发现，本研究建立了稳定表达“一个密码子对应一种氨基酸”优化后的bgl4和xynB基因的PK15细胞系，并对表达产物的酶活力进行了检测。
其次建立了蓝塘猪胎儿成纤维细胞系（Pig embryonic fibroblasts, PEF）5个，长白猪胎儿成纤维细胞系10个以及1个出生后30天长白猪耳皮肤成纤维细胞系。对蓝塘猪、长白猪PEF细胞系进行了绿色荧光蛋白GFP、葡糖苷酶bgl4、木聚糖酶xynB的电击转染，成功获得了含有以上基因的转基因PEF细胞系。以获得的转基因细胞系为核供体，进行体细胞核移植，得到重构胚胎后进行胚胎移植实验，最后获得了转绿色荧光蛋白转基因猪两批共7头，健康状况良好。
为了设计靶向猪IGF2基因和Kit基因的ZFNs，本研究中通过PCR克隆方法，获得了蓝塘猪Kit基因内含子8到内含子11的基因组序列，并发现该序列与已报道的大白猪序列相比，有一段长为10bp的插入序列。同时验证了长白猪和蓝塘猪IGF2基因内含子3上第3072的QTN的类型：长白猪为A，蓝塘猪为G。
最后，通过T-A克隆测序法以及CEL-I酶切法，验证了由Sigma公司设计的3对靶向IGF2基因和6对靶向Kit基因的ZFNs在PK15细胞以及蓝塘猪PEF细胞中的活性。结果显示在蓝塘猪PEF细胞中，靶向IGF2基因的第3对ZFNs具有最高的活性，为8.91%，靶向Kit基因的第4对ZFNs活性最高，为7.14%。同时通过比较ZFNs在PK15和PEF细胞中的活性，发现两种细胞中ZFNs的活性并不一致，说明细胞类型对ZFNs的活性有一定影响。通过以上的研究，我们对ZFNs的活性有了较深入的了解，为制定合适的富集中靶细胞的策略奠定了基础，同时为制作IGF2和Kit基因敲除猪奠定了基础。
Transgenic somatic cell nuclear transfer in pigs has wide applications in basicresearch areas, human medicine and agricultural production. And the new geneticengineering tools, Znic finger nucleases, have largely improved the efficiency oftransgenic pig production. Compared to traditional gene targeting method, zinc fingernuclease is a very powerful gene targeting tool. The application of zinc fingernuclease technology to produce transgenic animals, especially transgenic farmanimals will be the main direction for future development. In this study, we developeda technical platform for transgenic pig production and produced7green fluorescenceprotein transgenic pigs, and we also primarily explored the application of Zinc fingernuclease in gene knock-out pig production.
First of all, we optimized three fibrolytic enzyme genes: bgl4, xynB and egxA intwo different codon optimization methods and compared the expression level in fourdifferent mammalian cell lines. We found that “one codon-one amino acid” optimizedsequences had higher expression level than the original ones and the “Humanized”ones. We established two PK15cell lines, which can stable express BGL4and XYNB separately, and the enzyme activity was measured
Five embryo fibroblast cell lines from Lantang pig, ten embryo fibroblast celllines from Landrace pig and one skin fibroblast cell line from a30days old Landracepiglet were established. Embryo fibroblast cell lines from Lantang and Landrace pigwere transfected with pEGFP-N1and pEGFP-BGL4, pEGFP-XYNB plasmids DNAby NeonTMtransfection system, and six transgenic cell lines were gained. We didnuclear transfer experiments using the transgenic cell lines as donor cells and thereconstructed embryos were transplanted into female pigs. At last, we obtained7liveGFP transgenic piglets.
In order to design ZFNs which targeting the IGF2and Kit gene of pig, weamplified and sequenced the genomic fragment of Kit gene from intron8to intron11,we also found that this sequence has a10bp insertion differs from the largewhite Kitgene in NCBI. We also sequenced partial fragment of IGF2intron3, verified the QTNtype of IGF2gene intron3of the Landrace and Langtang pig. Landrace is A type andLantang pig is G type. Based on these sequencing results, we custom made zinc fingernucleases (ZFNs) from Sigma Corporation. By T-A cloning sequencing method andCEL-I method, we measured the cleavage activity of ZFNs in PK15cells and PEFcells. Results shows that in Langtang Pig PEF cells, IGF2ZFN set3has the highestactivity,8.91%cells were gene knock-out. And the highest sctivity of Kit ZFNs isfrom set1, which is7.14%. At the same time, by comparing ZFN activity in the PK15and PEF cells, we found that ZFN activities are not consistent in the two types of cells,indicated that cell type has impact on ZFN activity. Through this study, we have amore in-depth understanding of ZFN activity, and most importantly, laid thefoundation for the production of IGF2and the Kit gene knockout pigs.
本研究首先对源于细菌和低等真核生物的三种糖苷水解酶bgl4，xynB和egxA基因进行了两种方式的密码子优化，并比较了它们在四种不同种属的哺乳动物细胞中的表达水平，发现在PK15细胞系中，使用“一个密码子对应一种氨基酸”方式进行密码子优化后，其表达水平显著高于原始基因以及使用“人源化密码子”优化的基因。根据这一发现，本研究建立了稳定表达“一个密码子对应一种氨基酸”优化后的bgl4和xynB基因的PK15细胞系，并对表达产物的酶活力进行了检测。
其次建立了蓝塘猪胎儿成纤维细胞系（Pig embryonic fibroblasts, PEF）5个，长白猪胎儿成纤维细胞系10个以及1个出生后30天长白猪耳皮肤成纤维细胞系。对蓝塘猪、长白猪PEF细胞系进行了绿色荧光蛋白GFP、葡糖苷酶bgl4、木聚糖酶xynB的电击转染，成功获得了含有以上基因的转基因PEF细胞系。以获得的转基因细胞系为核供体，进行体细胞核移植，得到重构胚胎后进行胚胎移植实验，最后获得了转绿色荧光蛋白转基因猪两批共7头，健康状况良好。
为了设计靶向猪IGF2基因和Kit基因的ZFNs，本研究中通过PCR克隆方法，获得了蓝塘猪Kit基因内含子8到内含子11的基因组序列，并发现该序列与已报道的大白猪序列相比，有一段长为10bp的插入序列。同时验证了长白猪和蓝塘猪IGF2基因内含子3上第3072的QTN的类型：长白猪为A，蓝塘猪为G。
最后，通过T-A克隆测序法以及CEL-I酶切法，验证了由Sigma公司设计的3对靶向IGF2基因和6对靶向Kit基因的ZFNs在PK15细胞以及蓝塘猪PEF细胞中的活性。结果显示在蓝塘猪PEF细胞中，靶向IGF2基因的第3对ZFNs具有最高的活性，为8.91%，靶向Kit基因的第4对ZFNs活性最高，为7.14%。同时通过比较ZFNs在PK15和PEF细胞中的活性，发现两种细胞中ZFNs的活性并不一致，说明细胞类型对ZFNs的活性有一定影响。通过以上的研究，我们对ZFNs的活性有了较深入的了解，为制定合适的富集中靶细胞的策略奠定了基础，同时为制作IGF2和Kit基因敲除猪奠定了基础。
Transgenic somatic cell nuclear transfer in pigs has wide applications in basicresearch areas, human medicine and agricultural production. And the new geneticengineering tools, Znic finger nucleases, have largely improved the efficiency oftransgenic pig production. Compared to traditional gene targeting method, zinc fingernuclease is a very powerful gene targeting tool. The application of zinc fingernuclease technology to produce transgenic animals, especially transgenic farmanimals will be the main direction for future development. In this study, we developeda technical platform for transgenic pig production and produced7green fluorescenceprotein transgenic pigs, and we also primarily explored the application of Zinc fingernuclease in gene knock-out pig production.
First of all, we optimized three fibrolytic enzyme genes: bgl4, xynB and egxA intwo different codon optimization methods and compared the expression level in fourdifferent mammalian cell lines. We found that “one codon-one amino acid” optimizedsequences had higher expression level than the original ones and the “Humanized”ones. We established two PK15cell lines, which can stable express BGL4and XYNB separately, and the enzyme activity was measured
Five embryo fibroblast cell lines from Lantang pig, ten embryo fibroblast celllines from Landrace pig and one skin fibroblast cell line from a30days old Landracepiglet were established. Embryo fibroblast cell lines from Lantang and Landrace pigwere transfected with pEGFP-N1and pEGFP-BGL4, pEGFP-XYNB plasmids DNAby NeonTMtransfection system, and six transgenic cell lines were gained. We didnuclear transfer experiments using the transgenic cell lines as donor cells and thereconstructed embryos were transplanted into female pigs. At last, we obtained7liveGFP transgenic piglets.
In order to design ZFNs which targeting the IGF2and Kit gene of pig, weamplified and sequenced the genomic fragment of Kit gene from intron8to intron11,we also found that this sequence has a10bp insertion differs from the largewhite Kitgene in NCBI. We also sequenced partial fragment of IGF2intron3, verified the QTNtype of IGF2gene intron3of the Landrace and Langtang pig. Landrace is A type andLantang pig is G type. Based on these sequencing results, we custom made zinc fingernucleases (ZFNs) from Sigma Corporation. By T-A cloning sequencing method andCEL-I method, we measured the cleavage activity of ZFNs in PK15cells and PEFcells. Results shows that in Langtang Pig PEF cells, IGF2ZFN set3has the highestactivity,8.91%cells were gene knock-out. And the highest sctivity of Kit ZFNs isfrom set1, which is7.14%. At the same time, by comparing ZFN activity in the PK15and PEF cells, we found that ZFN activities are not consistent in the two types of cells,indicated that cell type has impact on ZFN activity. Through this study, we have amore in-depth understanding of ZFN activity, and most importantly, laid thefoundation for the production of IGF2and the Kit gene knockout pigs.
引文
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