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

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。

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.