利用全基因组重测序技术鉴定五指山猪GHR突变体转基因插入位点

doi:10.16288/j.yczz.21-221

遗传 ›› 2021, Vol. 43 ›› Issue (12): 1149-1158.doi: 10.16288/j.yczz.21-221

利用全基因组重测序技术鉴定五指山猪GHR突变体转基因插入位点

魏强^1,²(), 奥岩³, 杨漫漫^1,², 陈涛^1,², 韩虎^1,², 张兴举^1,², 王然^1,², 夏秋菊¹, 姜芳芳^1,², 李勇^1,²()

1. 深圳市华大农业应用研究院,深圳 518120
2. 深圳华大生命科学研究院,深圳市动物基因组辅助育种工程实验室,深圳 518083
3. 华中农业大学,农业动物遗传育种与繁殖教育部重点实验室,武汉 430070

收稿日期:2021-06-24 修回日期:2021-08-30 出版日期:2021-12-20 发布日期:2021-10-18
通讯作者: 李勇 E-mail:weiqiang@genomics.cn;liyong3@genomics.cn
作者简介:魏强,硕士,工程师,研究方向：动物基因编辑, 动物遗传育种。E-mail: weiqiang@genomics.cn
基金资助:
广东省重点领域研发计划项目编号(2018B020203002);广东省基础与应用基础研究基金项目资助编号(2019B1515210028)

Identification of genomic insertion of dominant-negative GHR mutation transgenes in Wuzhishan pig using whole genome sequencing method

Qiang Wei^1,²(), Yan Ao³, Manman Yang^1,², Tao Chen^1,², Hu Han^1,², Xingju Zhang^1,², Ran Wang^1,², Qiuju Xia¹, Fangfang Jiang^1,², Yong Li^1,²()

1. BGI Institute of Applied Agriculture, BGI-Shenzhen, Shenzhen 518120, China
2. Shenzhen Engineering Laboratory for Genomics-Assisted Animal Breeding, BGI-Shenzhen, Shenzhen 518083, China
3. Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China

Received:2021-06-24 Revised:2021-08-30 Online:2021-12-20 Published:2021-10-18
Contact: Li Yong E-mail:weiqiang@genomics.cn;liyong3@genomics.cn
Supported by:
Supported by the Science and Technology Innovation Strategy Projects of Guangdong Province No(2018B020203002);Guangdong Province Basic and Applied Basic Research Fund No(2019B1515210028)

摘要/Abstract

摘要：

转基因插入位点及其侧翼序列的分子特征对转基因动物新品系培育和生物安全评价至关重要。GHR (growth hormone receptor)显性抑制突变体转基因猪(T274)是本实验室前期制备的一种表型显著的矮小症模型,目前已形成了多世代群体,但该模型中外源突变体的插入位点尚未鉴定。本研究利用BGI-seq500测序平台,对T274转基因猪进行全基因组重测序,获得超过289 Gb的数据(106×)。以转基因载体和猪基因组序列作为参考序列进行比对分析,获得该外源GHR突变体的插入位点,并利用PCR扩增和Sanger测序进行验证。所有的分析数据都支持外源显性抑制突变体插入在五指山猪1号染色体269,513,538~269,514,371之间。序列保守性及功能元件分析表明,该插入位点可能位于转录激活相关的基因间区域。不同世代个体的7种组织中该外源突变体的表达水平和表达一致性都较高,说明该插入位点可以作为一个潜在的转基因“友好位点”。本研究表明全基因组重测序技术可以高效的鉴定转基因插入位点,该插入位点的获得为后期转基因猪新品系的培育奠定了基础,同时也为行业提供了一个可靠的基因组定点整合位点。

关键词: 转基因插入位点, 全基因组重测序, 转基因猪, 遗传稳定性

Abstract:

Molecular characterization of sequences flanking the transgenic insertion site is essential for safety assessment and breeding a novel strain of transgenic animal. The growth hormone receptor (GHR) dominant-negative mutant transgenic pig (T274) is a Laron syndrome model, which was previously established in our laboratory and maintained in multi-generational populations. However, the insertion site of the exogenous mutant in the genome of this model has not yet been identified. In this experiment, the BGI-seq500 sequencing platform was used to re-sequence the whole genome of the T274 model. More than 289 Gb of data (106×) was obtained. Then, the transgenic vector and porcine genome sequences were used as references for alignment analysis, and the insertion site of the exogenous GHR mutant was obtained, and verified by PCR amplification and Sanger sequencing. The results showed that the insertion site of the exogenous mutant was located in chromosome 1 (269,513,538-269,514,371) of the Wuzhishan pig. Conservation and functional element analysis indicated that the insertion site could be located in the intergenic region associated with transcription activation. The expression levels of the exogenous mutant in seven tissues of offspring in different generations are high, indicating that the insertion site can be used as a potential safe site for transgene targeting. This study shows that whole-genome resequencing can efficiently identify transgenic insertion sites. The insertion site identified in T274 could be useful in establishing and breeding new transgenic pig lines, as well as a reliable safe site for transgenic pig research.

Key words: transgenic insertion site, whole genome sequencing, transgenic pig, genetic stability

魏强, 奥岩, 杨漫漫, 陈涛, 韩虎, 张兴举, 王然, 夏秋菊, 姜芳芳, 李勇. 利用全基因组重测序技术鉴定五指山猪GHR突变体转基因插入位点[J]. 遗传, 2021, 43(12): 1149-1158.

Qiang Wei, Yan Ao, Manman Yang, Tao Chen, Hu Han, Xingju Zhang, Ran Wang, Qiuju Xia, Fangfang Jiang, Yong Li. Identification of genomic insertion of dominant-negative GHR mutation transgenes in Wuzhishan pig using whole genome sequencing method[J]. Hereditas(Beijing), 2021, 43(12): 1149-1158.

图/表 8

图1

表1

表2

图2

图3

图4

图5

图6

参考文献 30

[1]	Maksimenko OG, Deykin AV, Khodarovich YM, Georgiev PG. Use of transgenic animals in biotechnology: prospects and problems. Acta Naturae, 2013,5(1):33-46.
[2]	Kong QR, Wu ML, Zhang L, Wang F, Yin Z, Mu YS, Liu ZH. Transgene insertion affects transcription and epigenetic modification of flanking host sequence in transgenic pigs. Cell Mol Biol (Noisy-le-grand), 2011,57 Suppl: OL1505-1512.
[3]	Clark J, Whitelaw B. A future for transgenic livestock. Nat Rev Genet, 2003,4(10):825-833.
[4]	Niemann H, Petersen B. The production of multi- transgenic pigs: update and perspectives for xenotransplantation. Transgenic Res, 2016,25(3):361-374.
[5]	Ruan JX, Xu J, Chen-Tsai RY, Li K. Genome editing in livestock: are we ready for a revolution in animal breeding industry? Transgenic Res, 2017,26(6):715-726.
[6]	Friedrich G, Soriano P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev, 1991,5(9):1513-1523.
[7]	Li XP, Yang Y, Bu L, Guo XG, Tang CC, Song J, Fan NN, Zhao BT, Ouyang ZM, Liu ZM, Zhao Y, Yi XL, Quan LQ, Liu SC, Yang ZG, Ouyang HS, Chen YE, Wang Z, Lai LX. Rosa26-targeted swine models for stable gene over- expression and Cre-mediated lineage tracing. Cell Res, 2014,24(4):501-504.
[8]	Ruan JX, Li HG, Xu K, Wu TW, Wei JL, Zhou R, Liu ZG, Mu YL, Yang SL, Ouyang HS, Chen-Tsai RY, Li K. Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Sci Rep, 2015,5:14253.
[9]	Ma LY, Wang YZ, Wang HT, Hu YQ, Chen JY, Tan T, Hu M, Liu XJ, Zhang R, Xing YM, Zhao YQ, Hu XX, Li N. Screen and verification for transgene integration sites in pigs. Sci Rep, 2018,8(1):7433.
[10]	Yang DS, Wang CE, Zhao BT, Li W, Ouyang Z, Liu ZM, Yang HQ, Fan P, O'Neill A, Gu WW, Yi H, Li SH, Lai LX, Li XJ. Expression of Huntington's disease protein results in apoptotic neurons in the brains of cloned transgenic pigs. Hum Mol Genet, 2010,19(20):3983-3994.
[11]	Lu YF, Tian C, Deng JX. Research progress in identification technology of genetically modified animals. Prog Biotech, 2000,20(3):60-61.
	卢一凡, 田靫, 邓继先. 转基因动物鉴定技术的研究进展. 生物工程进展, 2000,20(3):60-61.
[12]	Fujimoto S, Matsunaga S, Murata M. Mapping of T-DNA and Ac/Ds by TAIL-PCR to analyze chromosomal rearrangements. Methods Mol Biol, 2016,1469:207-216.
[13]	Groenen MAM, Archibald AL, Uenishi H, Tuggle CK, Takeuchi Y, Rothschild MF, Rogel-Gaillard C, Park C, Milan D, Megens HJ, Li ST, Larkin DM, Kim H, Frantz LAF, Caccamo M, Ahn H, Aken BL, Anselmo A, Anthon C, Auvil L, Badaoui B, Beattie CW, Bendixen C, Berman D, Blecha F, Blomberg J, Bolund L, Bosse M, Botti S, Bujie Z, Bystrom M, Capitanu B, Carvalho-Silva D, Chardon P, Chen C, Cheng R, Choi SH, Chow W, Clark RC, Clee C, Crooijmans RPMA, Dawson HD, Dehais P, De Sapio F, Dibbits B, Drou N, Du ZQ, Eversole K, Fadista J, Fairley S, Faraut T, Faulkner GJ, Fowler KE, Fredholm M, Fritz E, Gilbert JGR, Giuffra E, Gorodkin J, Griffin DK, Harrow JL, Hayward A, Howe K, Hu ZL, Humphray SJ, Hunt T, Hornshøj H, Jeon JT, Jern P, Jones M, Jurka J, Kanamori H, Kapetanovic R, Kim J, Kim JH, Kim KW, Kim TH, Larson G, Lee K, Lee KT, Leggett R, Lewin HA, Li YR, Liu WS, Loveland JE, Lu Y, Lunney JK, Ma J, Madsen O, Mann K, Matthews L, McLaren S, Morozumi T, Murtaugh MP, Narayan J, Nguyen DT, Ni PX, Oh SJ, Onteru S, Panitz F, Park EW, Park HS, Pascal G, Paudel Y, Perez-Enciso M, Ramirez-Gonzalez R, Reecy JM, Rodriguez-Zas S, Rohrer GA, Rund L, Sang YM, Schachtschneider K, Schraiber JG, Schwartz J, Scobie L, Scott C, Searle S, Servin B, Southey BR, Sperber G, Stadler P, Sweedler JV, Tafer H, Thomsen B, Wali R, Wang J, Wang J, White S, Xu X, Yerle M, Zhang GJ, Zhang JG, Zhang J, Zhao SH, Rogers J, Churcher C, Schook LB. Analyses of pig genomes provide insight into porcine demography and evolution. Nature, 2012,491(7424):393-398.
[14]	Meurens F, Summerfield A, Nauwynck H, Saif L, Gerdts V. The pig: a model for human infectious diseases. Trends Microbiol, 2012,20(1):50-57.
[15]	Park D, Park SH, Ban YW. Kim YS, Park KC, Kim NS, Kim JK, Choi IK. A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data. BMC Biotechnol, 2017,17(1):67.
[16]	Park D, Kim D, Jang G, Lim J, Shin YJ, Kim J, Seo MS, Park SH, Kim JK, Kwon TH, Choi IY. Efficiency to discovery transgenic loci in GM rice using next generation sequencing whole genome re-sequencing. Genomics Inform, 2015,13(3):81-85.
[17]	Niu L, He HL, Zhang YY, Yang J, Zhao QQ, Xing GJ, Zhong XF, Yang XD. Efficient identification of genomic insertions and flanking regions through whole-genome sequencing in three transgenic soybean events. Transgenic Res, 2021,30(1):1-9.
[18]	Guo BF, Guo Y, Hong HL, Qiu LJ. Identification of genomic insertion and flanking sequence of G2-EPSPS and GAT transgenes in soybean using whole genome sequencing method. Front Plant Sci, 2016,7:1009.
[19]	Ji Y, Abrams N, Zhu W, Salinas E, Yu ZY, Palmer DC, Jailwala P, Franco Z, Roychoudhuri R, Stahlberg E, Gattinoni L, Restifo NP. Identification of the genomic insertion site of Pmel-1 TCR α and β transgenes by next- generation sequencing. PLoS One, 2014,9(5):e96650.
[20]	Jacobsen JC, Erdin S, Chiang C, Hanscom C, Handley RR, Barker DD, Stortchevoi A, Blumenthal I, Reid SJ, Snell RG, MacDonald ME, Morton AJ, Ernst C, Gusella JF, Talkowski ME. Potential molecular consequences of transgene integration: the R6/2 mouse example. Sci Rep, 2017,7:41120.
[21]	Li FD, Li Y, Liu H, Zhang XJ, Liu CX, Tian K, Bolund L, Dou HW, Yang WX, Yang HM, Staunstrup NH, Du YT. Transgenic Wuzhishan minipigs designed to express a dominant-negative porcine growth hormone receptor display small stature and a perturbed insulin/IGF-1 pathway. Transgenic Res, 2015,24(6):1029-1042.
[22]	Liu W, Lu G. Progress in technology of transgenic animal models. Hereditas(Beijing), 2001,23(3):289-291.
	刘薇, 卢光. 转基因动物技术的研究进展. 遗传, 2001,23(3):289-291.
[23]	Chandler KJ, Chandler RL, Broeckelmann EM, Hou Y, Southard-Smith EM, Mortlock DP. Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression. Mamm Genome, 2007,18(10):693-708.
[24]	Le Saux A, Houdebine LM, Jolivet G. Chromosome integration of BAC (bacterial artificial chromosome): evidence of multiple rearrangements. Transgenic Res, 2010,19(5):923-931.
[25]	Hu W, Zhu ZY. Research progress of BAC and genetic modification. Prog Biotech, 2001,21(4):3-7.
	胡炜, 朱作言. BAC及其转基因研究进展. 生物工程进展, 2001,21(4):3-7.
[26]	Kong QR, Wu ML, Wang ZK, Zhang XM, Li L, Liu XY, Mu YS, Liu ZH. Effect of trichostatin A and 5-Aza-2'- deoxycytidine on transgene reactivation and epigenetic modification in transgenic pig fibroblast cells. Mol Cell Biochem, 2011,355(1-2):157-165.
[27]	Yin Z, Kong QR, Zhao ZP, Wu ML, Mu YS, Hu K, Liu ZH. Position effect variegation and epigenetic modification of a transgene in a pig model. Genet Mol Res, 2012,11(1):355-369.
[28]	Kong QR, Liu ZH. Inheritance and expression stability of transgene in transgenic animals. Hereditas(Beijing), 2011,33(5):504-511.
	孔庆然, 刘忠华. 外源基因在转基因动物中遗传和表达的稳定性. 遗传, 2011,33(5):504-511.
[29]	Laboulaye MA, Duan X, Qiao M, Whitney IE, Sanes JR. Mapping transgene insertion sites reveals complex interactions between mouse transgenes and neighboring endogenous genes. Front Mol Neurosci, 2018,11:385.
[30]	Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 1991,108(2):193-199.

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:

检测位置	引物名称	序列(5’→3’)	产物大小(bp)
左侧断点	TL274L1	GGCTTCAGTTACGGAGGATG	459
左侧断点	TL274R2	AACGCCTAACCCTAAGCAGAT	459
右侧断点	TR274L1	AGCGTTGGCTACCCGTGATA	1732
右侧断点	TR274R1	AGGTCCGTGACAAGACAGCA	1732
基因组序列	TL274L1	ATGGAAGACCTTGACATTGACC	1298
基因组序列	TR274R1	AGGTCCGTGACAAGACAGCA	1298

引物名称	序列(5’→3’)	产物大小 (bp)
GHR^dm -F	GTGAGATCCAGACAACGAAACT	201
GHR^dm -R	CTAATTTTCCTTCCTTTGCTGTTTA	201
QWGHR-F	GGAATATTTGGGCTAACGGTGA	243
QWGHR-R	TCAGGGTCATCAATATCGAGCT	243
pGAPDH-F	ACCTGCCGCCTGGAGAAACC	252
pGAPDH-R	GACCATGAGGTCCACCACCCTG	252

利用全基因组重测序技术鉴定五指山猪GHR突变体转基因插入位点

Identification of genomic insertion of dominant-negative GHR mutation transgenes in Wuzhishan pig using whole genome sequencing method

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 30

相关文章 5

编辑推荐

Metrics

[1]	马钧, 樊安平, 王武生, 张金川, 江晓军, 马瑞军, 贾社强, 刘飞, 雷初朝, 黄永震. 全基因组重测序解析秦川牛保种群遗传多样性和遗传结构[J]. 遗传, 2023, 45(7): 602-616.
[2]	袁金红, 李俊华, 袁娇娇, 贾克利, 李书粉, 邓传良, 高武军. 基于全基因组测序的MutMap方法在正向遗传学研究中的应用[J]. 遗传, 2017, 39(12): 1168-1177.
[3]	胡鹏飞, 徐佳萍, 艾成, 邵秀娟, 王洪亮, 董依萌, 崔学哲, 杨福合, 邢秀梅. 利用全基因组重测序分析鹿茸重量相关基因[J]. 遗传, 2017, 39(11): 1090-1101.
[4]	任才芳，孙红艳，王立中，张国敏，樊懿萱，颜光耀，王丹，王锋. iPSCs遗传稳定性与重编程机制的研究进展[J]. 遗传, 2014, 36(9): 879-887.
[5]	沈银柱，刘植义，何聪芬，黄占景，孟庆昌，柏峰，马闻师，赵松山，陆莉，张焕英. 诱发小麦花药愈伤组织及其再生植株抗盐性变异的研究[J]. 遗传, 1997, 19(6): 7-11.