2019年第46卷第9期目录
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封面故事:对人类疾病相关的研究,受到取材和伦理道德的限制,无法直接在人体中进行,必须依靠动物模型进行实验. 利用小鼠制备的人类疾病模型往往不能真实反映人类疾病的发生和发展,猪在生理代谢及免疫和神经系统等方面与人类非常接近,涉及的伦理问题少,被普遍认为是理想的人类疾病模型. 长期以来基因打靶技术大多只能在小鼠上完成,这是因为小鼠的胚胎干细胞能在体外培养并无限增殖. 但迄今为止猪的胚胎干细胞尚未成功建系,而常规同源重组结合体细胞克隆进行猪基因打靶的效率极低,导致众多人类疾病猪模型无法建立和应用. 近年来CRISPR/Cas9技术的出现为猪等大动物基因敲除技术带来了革命性的的突破,极大地促进了基因靶向修饰猪等大动物的研究,为这些克隆效率低下又缺少全能性胚胎干细胞物种的基因编辑开辟了新渠道. 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. 吴彩霞等利用CRISPR/Cas9 技术结合体细胞克隆技术成功构建了Tiki1基因敲除猪模型,为进一步深入研究 Tiki1基因在哺乳动物乃至人脑部发育中的作用提供了可靠的动物模型.
(吴彩霞,刘朝明,颜泉梅,张全军,赵宇,欧阳振,樊娜娜,赖良学. 利用CRISPR/Cas9系统构建Tiki1基因修饰猪模型, 本期第900~908页)
Cover Story: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, and 52 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.
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