Identification of genomic insertion of dominant-negative GHR mutation transgenes in Wuzhishan pig using whole genome sequencing method
Received date: 2021-06-24
Revised date: 2021-08-30
Online published: 2021-10-18
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)
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.
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 . DOI: 10.16288/j.yczz.21-221
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