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

doi:10.16288/j.yczz.15-160

遗传 ›› 2015, Vol. 37 ›› Issue (12): 1211-1217.doi: 10.16288/j.yczz.15-160

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

李智方^{1, 2}, 冯冲², 纪慧丽^{1, 2}, 石宁宁², 宋小凤², 赵勤丽², 龙川², 潘登科², 杨小淦¹

1. 广西大学,亚热带农业生物资源保护与利用国家重点实验室,南宁 530004;
2. 中国农业科学院北京畜牧兽医研究所,农业部畜禽遗传资源与种质创新重点实验室,北京 100193

收稿日期:2015-04-15 出版日期:2015-12-20 发布日期:2015-08-12
通讯作者: 杨小淦,博士,副研究员;研究方向：动物繁殖生物技术。E-mail:xgyang@gxu.edu.cn;潘登科,博士,副研究员;研究方向：转基因猪模型。E-mail:pandengke2002@163.com E-mail:LIZHIFANG31@163.com
作者简介:李智方,硕士研究生;专业方向：动物遗传育种与繁殖。E-mail：LIZHIFANG31@163.com
基金资助:
亚热带农业生物资源保护利用国家重点实验室开放课题（编号：SKL201404）和国家高技术研究发展计划（863计划）项目（编号：2012AA020601）资助

Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs

Zhifang Li^{1, 2}, Chong Feng², Huili Ji^{1, 2}, Ningning Shi², Xiaofeng Song², Qinli Zhao², Chuan Long², Dengke Pan², Xiaogan Yang¹

1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Guangxi University, Nanning 530004,China;
2. Key Laboratory of Farm Animal Genetic Resource and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences,Beijing 100193, China

Received:2015-04-15 Online:2015-12-20 Published:2015-08-12

摘要/Abstract

摘要： 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。

关键词: GTKO, 增强型绿色荧光蛋白, 猪, 异种移植

Abstract: The pig is an ideal source to provide organs because its organ size and physiology are similar to humans. However, an acute rejection will ensue after pig-to-human xenotransplantation. The α-1,3 galactosyltransferase gene knockout (GTKO) pigs were generated in recent years, and could solve the problem of hyperacute rejection. But due to lack of reporting genes, the rejection status of cells and organs post pig-to-human xenotransplantation cannot be visualized. In this study, we introduced the enhanced green fluorescent protein (EGFP) gene driven by the CAG promoter into GTKO porcine ear fibroblasts. Then we produced transgenic pigs expressing the EGFP gene by nuclear transfer technology. Expression levels of EGFP in different tissues and organs of the cloned pig were investigated by Nightsea DFP-1 Fluorescent Protein Flashlight, fluorescence microscope and quantitative PCR assays. The results showed that the protein and transcript of EGFP were expressed in all tissues and organs of the GTKO pig, but the expression was weak in the liver and central nervous system. In conclusion, we have successfully produced the transgenic GTKO pigs expressing EGFP in all tested tissues and organs, which builds up a good basis to track transplanted cells or tissues.

Key words: GTKO, EGFP, pig, xenotransplation

李智方, 冯冲, 纪慧丽, 石宁宁, 宋小凤, 赵勤丽, 龙川, 潘登科, 杨小淦. 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[J]. 遗传, 2015, 37(12): 1211-1217.

Zhifang Li, Chong Feng, Huili Ji, Ningning Shi, Xiaofeng Song, Qinli Zhao, Chuan Long, Dengke Pan, Xiaogan Yang. Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs[J]. HEREDITAS(Beijing), 2015, 37(12): 1211-1217.

参考文献

[1] Pierson RN III, Dorling A, Ayares D, Rees MA, Seebach JD, Fishman JA, Hering BJ, Cooper DKC. Current status of xenotransplantation and prospects for clinical application. Xenotransplantation , 2009, 16(5): 263-280.
[2] Lai LX, Kolber-Simonds D, Park K-W, Cheong H-T, Greenstein JL, Im G-S, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS. Production of α-1, 3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science , 2002, 295(5557): 1089-1092.
[3] Dai YF, 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 αl, 3-galactosyltransferase gene in cloned pigs. Nat Biotechnol , 2002, 20(3): 251-255.
[4] Phelps CJ, Koike C, Vaught 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 AJ, Rudert WA, Bottino R, Bertera S, Trucco M, Starzl TE, Dai YF, Ayares DL. Production of α1, 3-galactosyltransferase-deficient pigs. Science , 2003, 299(5605): 411-414.
[5] Kolber-Simonds D, Lai LX, Watt SR, Denaro M, Arn S, Augenstein ML, Betthauser J, Carter DB, Greenstein JL, Hao YH, Im G-S, Liu ZH, Mell GD, Murphy CN, Park K-W, Rieke A, Ryan DJ, Sachs DH, Forsberg EJ, Prather RS, Hawley RJ. Production of α-1, 3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proc Nati Acad Sci USA , 2004, 101(19): 7335-7340.
[6] 郑道山, 冯冲, 朱彦宾, 龙川, 冯书堂, 潘登科, 马文丽. 利用启动子缺陷型打靶载体敲除五指山小型猪 GGTA1 基因. 生物技术通讯, 2011, 22(4): 458-462.
[7] Tseng Y-L, Dor FJMF, Kuwaki K, Ryan D, Wood J, Denaro M, Giovino M, Yamada K, Hawley R, Patience C, Schuurman H-J, Awwad M, Sachs DH, Cooper DKC. Bone marrow transplantation from α 1, -3galactosyltransferase gene-knockout pigs in baboons. Xenotransplantation , 2004, 11(4): 361-370.
[8] Shigeta T, Hsu H-C, Enosawa S, Matsuno N, Kasahara M, Matsunari H, Umeyama K, Watanabe M, Nagashima H. Transgenic pig expressing the red fluorescent protein kusabira-orange as a novel tool for preclinical studies on hepatocyte transplantation. Transplant Proc , 2013, 45(5): 1808-1810.
[9] Yamada K, Yazawa K, Shimizu A, Iwanaga T, Hisashi Y, Nuhn M, O'Malley P, Nobori S, Vagefi PA, Patience C, Fishman J, Cooper DKC, Hawley RJ, Greenstein J, Schuurman H-J, Awwad M, Sykes M, Sachs DH. Marked prolongation of porcine renal xenograft survival in baboons through the use of α1, 3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med , 2005, 11(1): 32-34.
[10] Kuwaki K, Tseng YL, Dor FJMF, Shimizu A, Houser SL, Sanderson TM, Lancos CJ, Prabharasuth DD, Cheng J, Moran K, Hisashi Y, Mueller N, Yamada K, Greenstein JL, Hawley RJ, Patience C, Awwad M, Fishman JA, Robson SC, Schuurman H-J, Sachs DH, Cooper DKC. Heart transplantation in baboons using α1, 3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med , 2005, 11(1): 29-31.
[11] Cooper DKC. A milestone in xenotransplantation research. Xenotransplantation , 2014, 21(1): 13-15.
[12] 潘登科, 张莉, 周艳荣, 冯冲, 龙川, 刘晓, 董恩球, 王树臣, 万荣, 张健, 陈红星. 体细胞核移植生产转ω-3脂肪酸去饱和酶基因s Fat-1 克隆猪. 中国科学(C辑: 生命科学), 2009, 39(3): 295-302.
[13] Park K-W, Lai LX, Cheong H-T, Cabot R, Sun QY, Wu GM, Rucker EB, Durtschi D, Bonk A, Samuel M, Rieke A, Day BN, Murphy CN, Carter DB, Prather RS. Mosaic gene expression in nuclear transfer-derived embryos and the production of cloned transgenic pigs from ear-derived fibroblasts. Biol Reprod , 2002, 66(4): 1001-1005.
[14] Naruse K, Ishikawa H, Kawano H-O, Ueda H, Kurome M, Miyazaki K, Endo M, Sawasaki T, Nagashima H, Makuuchi M. Production of a transgenic pig expressing human albumin and enhanced green fluorescent protein. J Reprod Dev , 2005, 51(4): 539-546.
[15] Cabot RA, Kühholzer B, Chan AWS, Lai LX, Park K-W, Chong K-Y, Schatten G, Murphy, CN, Abeydeera LR, Day BN, Prather RS. Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector. Anim Biotechnol , 2001, 12(2): 205-214.
[16] Lai LX, Park K-W, Cheong H-T, Kühholzer B, Samuel M, Bonk A, Im G-S, Rieke A, Day BN, Murphy CN, Carter DB, Prather RS. Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells. Mol Reprod Dev , 2002, 62(3): 300-306.
[17] Hofmann A, Kessler B, Ewerling S, Weppert M, Vogg B, Ludwig H, Stojkovic M, Boelhauve M, Brem G, Wolf E, Pfeifer A. Efficient transgenesis in farm animals by lentiviral vectors. EMBO Rep , 2003, 4(11): 1054-1058.
[18] Hyun S, Lee G, Kim D, Kim H, Lee S, Nam D, Jeong Y, Kim S, Yeom S, Kang S, Han J, Lee B, Hwang W. Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein. Bio Reprod , 2003, 69(3): 1060-1068.
[19] Whitelaw CB, Radcliffe PA, Ritchie WA, Carlisle A, Ellard FM, Pena RN, Rowe J, Clark AJ, King TJ, Mitrophanous KA. Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector. FEBS Lett , 2004, 571(1-3): 233-236.
[20] Watanabe S, Iwamoto M, Suzuki S-I, Fuchimoto D, Honma D, Nagai T, Hashimoto M, Yazaki S, Sato M, Onishi A. A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin. Bio Reprod , 2005, 72(2): 309-315.
[21] Webster NL, Forni M, Bacci ML, Giovannoni R, Razzini R, Fantinati P, Zannoni A, Fusetti L, Dalpra L, Bianco MR, Papa M, Seren E, Sandrin MS, Mc Kenzie IFC, Lavitrano M. Multi-transgenic pigs expressing three fluorescent proteins produced with high efficiency by sperm mediated gene transfer. Mol Reprod Dev , 2005, 72(1): 68-76.
[22] Yong HY, Hao YH, Lai LX, Li RF, Murphy CN, Rieke A, Wax D, Samuel M, Prather RS. Production of a transgenic piglet by a sperm injection technique in which no chemical or physical treatments were used for oocytes or sperm. Mol Reprod Dev , 2006, 73(5): 595-599.
[23] 刘忠华, 宋军, 王振坤, 田江天, 孔庆然, 郑重, 尹智, 高力, 马海鹍, 孙爽, 李玉田, 王洪斌. 体细胞核移植生产绿色荧光蛋白转基因猪. 科学通报, 2008, 53(5): 556-560.
[24] 张鹏, 杨珍珍, 窦红伟, 李伟杭, 律波, Lars B, 杜玉涛, 谭萍萍, 马润林. 利用改进的手工克隆技术生产转GFP基因猪克隆胚胎. 遗传, 2011, 33(5): 527-532.
[25] Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. Green mice as a source of ubiquitous green cells. FEBS Lett , 1997, 407(3): 313-319.
[26] Hakamata Y, Tahara K, Uchida H, Sakuma Y, Nakamura M, Kume A, Murakami T, Takahashi M, Takahashi R, Hirabayashi M, Ueda M, Miyoshi I, Kasai N, Kobayashi E. Green fluorescent protein-transgenic rat: a tool for organ transplantation research. Biochem Biophys Res Commun , 2001, 286(4): 779-785.
[27] Takahashi R-I, Kuramochi T, Aoyagi K, Hashimoto S, Miyoshi I, Kasai N, Hakamata Y, Kobayashi E, Ueda M. Establishment and characterization of CAG/EGFP transgenic rabbit line. Transgenic Res , 2007, 16(1): 115-120.

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

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

Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	莫健新,王豪强,黄广燕,蔡更元,吴珍芳,张献伟. 微生物源果胶酶在猪PK15细胞中异源表达及其酶学性质分析[J]. 遗传, 2019, 41(8): 736-745.
[2]	周李生, 赵为民, 涂枫, 吴云鹤, 任守文, 方晓敏. 猪乳头性状生理学和遗传学研究进展[J]. 遗传, 2019, 41(5): 384-390.
[3]	赵剑超, 柴壮, 郭诗萌, 刘忠华. 猪早期胚胎发育中SOX2基因启动子活性分析[J]. 遗传, 2019, 41(10): 950-961.
[4]	黄耀强,李国玲,杨化强,吴珍芳. 基因编辑猪在生物医学研究中的应用[J]. 遗传, 2018, 40(8): 632-646.
[5]	刘海龙, 谌阳, 高杨, 周玲, 韩晓松, 赵长志, 杨高娟, 陈毅龙, 杨慧, 谢胜松. 靶向miRNA前体不同类型sgRNA的丰度及特异性评估[J]. 遗传, 2018, 40(7): 561-571.
[6]	冉茂良, 董莲花, 翁波, 曹蓉, 彭馥芝, 高虎, 罗荟, 陈斌. miR-362靶向ZNF644基因调控猪未成熟支持细胞的增殖和凋亡[J]. 遗传, 2018, 40(7): 572-584.
[7]	黄万龙,张秀秀,李嫒,苗向阳. 利用RNA-seq技术筛选大白猪皮下和肌内脂肪组织差异表达基因[J]. 遗传, 2017, 39(6): 501-511.
[8]	黄娇娇, 曹春伟, 郑国民, 赵建国. 基因组编辑技术在猪遗传改良中的应用[J]. 遗传, 2017, 39(11): 1078-1089.
[9]	钟翠丽, 李国玲, 莫健新, 全绒, 王豪强, 李紫聪, 吴珍芳, 张献伟. 不同电转仪的电转参数、质粒用量和拓扑结构对猪胎儿成纤维细胞转染效率的影响[J]. 遗传, 2017, 39(10): 930-938.
[10]	张轲, 冯光德, 张宝云, 向伟, 陈龙, 杨芳, 储明星, 王凭青. 表观遗传标记在猪分子育种中的研究与应用前景[J]. 遗传, 2016, 38(7): 634-643.
[11]	李国玲, 钟翠丽, 倪生, 刘德武, 蔡更元, 李紫聪, 杨化强, 吴珍芳. 利用CRISPR/Cas9系统建立Xist基因敲除猪模型[J]. 遗传, 2016, 38(12): 1081-1089.
[12]	李利族, 韩丽鑫, 王金奎, 汪亮, 刘娣, 杨秀芹. 猪PILRA基因克隆及变异剪接体鉴定[J]. 遗传, 2015, 37(9): 926-931.
[13]	刘静, 王亚楠, 孙亚奇, 王洪洋, 汪超, 彭中镇, 刘榜. 猪13号染色体上拷贝数变异区内基因信息发掘及遗传规律分析[J]. 遗传, 2014, 36(4): 354-359.
[14]	付言峰, 周艳红, 王爱国, 李兰, 刘红林, 李碧侠, 任守文. 梅山猪胚胎附植期EphB2的组织表达及RNA-seq分析[J]. 遗传, 2014, 36(12): 1243-1248.
[15]	宿英,龙毅,阮国荣,吴丽花,张志燕,肖石军,邓玮芸,吕显山,胡豆,吴国灶,沈虎群,廖信军,丁能水,黄路生. 利用传递不平衡分析在多群体中鉴别猪脐疝易感基因[J]. 遗传, 2014, 36(10): 995-1005.