利用CRISPR/Cas9系统构建Tiki1基因修饰猪模型

Construction of Tiki1 Gene Modified Pig Model by CRISPR/Cas9 System

Tiki1基因是哈佛大学儿童医学院贺熹教授实验室发现的一个对蛙头部的诱导起到决定性作用的新基因,但Tiki1基因在小鼠等啮齿类动物中缺失,因此无法利用小鼠等小动物来研究其在哺乳动物中的作用.本文利用CRISPR/Cas9系统结合体细胞克隆技术构建Tiki1基因修饰猪模型,研究Tiki1基因在猪发育中的作用.我们利用贺熹教授团队提供的人Tiki1基因序列,在猪的基因组数据库中比对出与其同源性最高的一段序列设计2个靶位点(g1和g2).以设计的靶位点构建打靶质粒转染猪胎儿成纤维细胞,经细胞筛选、PCR扩增及测序共鉴定了52个单细胞克隆株.最终选择靶位点g1为纯合双敲的5个单细胞克隆株和靶位点g2为纯合双敲的3个单细胞克隆株作为构建Tiki1基因敲除猪的核供体.我们共计构建了720个重组胚胎,分别植入3头代孕母猪,其中有1头经B超检测成功怀孕并妊娠到期产下13头发育正常的克隆猪,经测序鉴定其中12头为Tiki1基因双敲除猪模型,Tiki1基因敲除克隆猪健康存活至今.结果表明Tiki1基因对于猪早期发育的作用机理不同于蛙,其在猪早期发育的过程中的具体作用机理有待后续进一步的深入研究.

Tiki1 gene,found and named by Professor He Xi’s team from Boston Children’s Hospital,Harvard Medical School,plays a key role in the formation of head in Xenopus.However,as Tiki1 gene is absent in rodents such as mice,it is impossible to use mice or rat to study its role in mammals.In this study,we generated Tiki1 gene modified pigs using CRISPR/Cas9 system combined with somatic cell cloning technology to study the role of Tiki1 gene in pig development.Aligned the human Tiki1 mRNA sequence provided by professor He Xi’s team with the pig genome database,we selected two target sites(g1 and g2)with the top 2 highest sequence identity at the predicted pig Tiki1 gene locus.The sgrRNA plasmid was constructed to transfect porcine fetal fibroblasts,and52 single cell clones were screened and sequenced.We finally selected 5 single-cell clones with biallelic knockout mutations at target site g1 and 3 single-cell clones with biallelic knockout mutations at target site g2 as nuclear donors for constructing Tiki1 knockout pigs.A total of 720 recombinant embryos were constructed and transferred into three surrogate sows and one of them was successfully pregnant monitored by B ultrasound.A total of 13 cloned piglets(ten living piglets and 3 dead piglets)were produced,and 12 of them were biallelic knockout mutations at Tiki1 locus.Both the living and dead Tiki1 gene knockout cloned piglets were developed normally and the living piglets have survived healthy till now.The results indicate that the role of Tiki1 gene on early development of pigs is different from that of frogs.The specific role of Tiki1 gene in the early development of pigs needs to be further investigated.