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

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

• Research Article • Previous Articles Next Articles

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)

Abstract

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

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.

Figures/Tables 8

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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

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

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