MEK抑制剂PD0325901显著提高猪胎儿成纤维细胞ssODN介导的HDR效率

doi:10.16288/j.yczz.18-294

遗传 ›› 2019, Vol. 41 ›› Issue (4): 327-336.doi: 10.16288/j.yczz.18-294

MEK抑制剂PD0325901显著提高猪胎儿成纤维细胞ssODN介导的HDR效率

欧浩¹,李国玲¹,王豪强¹,黄广燕¹,蔡更元^1,²,李紫聪¹,吴珍芳^1,²,张献伟^1,²()

^1. 华南农业大学动物科学学院,国家生猪种业工程技术研究中心,广州 510642
^2. 温氏食品集团股份有限公司,新兴 527400

收稿日期:2018-10-29 修回日期:2019-03-02 出版日期:2019-04-20 发布日期:2019-03-25
作者简介:欧浩,硕士,专业方向：动物遗传育种。E-mail: 719367112@qq.com。|李国玲,博士,研究方向：基因编辑与遗传育种。E-mail: 792268184@qq.com。
基金资助:
国家自然科学基金项目(31772555);国家转基因重大专项(2016ZX08006002)

MEK inhibitor PD0325901 significantly boosts ssODN-mediated HDR efficiency in porcine fetal fibroblasts

Hao Ou¹,Guoling Li¹,Haoqiang Wang¹,Guangyan Huang¹,Gengyuan Cai^1,²,Zicong Li¹,Zhenfang Wu^1,²,Xianwei Zhang^1,²()

^1. National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China
^2. Wens Foodstuff Group Co., Ltd, Xinxing 527400, China

Received:2018-10-29 Revised:2019-03-02 Published:2019-04-20 Online:2019-03-25
Supported by:
the National Natural Science Foundation of China(31772555);the National Transgenic Major Projects(2016ZX08006002)

摘要/Abstract

摘要：

基因组DNA发生双链断裂(double strand break, DSB)后主要通过同源定向修复(homologous-directed repair, HDR)和非同源末端连接(nonhomologous end joining, NHEJ)两种途径进行修复,其中单链寡聚核苷酸(single-stranded oligodeoxyribonucleotide, ssODN)介导的同源定向修复是动物基因组常用的基因组定点修饰技术,具有较大的科研和应用价值。为提高猪基因组ssODN介导HDR效率,本研究以猪胎儿成纤维细胞(porcine fetal fibroblasts, PFFs)为研究对象,利用丝裂原活化的细胞外信号调节激酶(mitogen-activated extracellular signal-regulated kinase, MEK)抑制剂PD0325901培养细胞,研究其对HDR效率的影响及作用分子机理。结果显示,PD0325901能显著提高PFFs的G₂期和S期细胞群百分比,减少G₁期细胞群比率,促进HDR修复因子的表达。在最适浓度250 nmol/L时,PD0325901使ssODN介导的GFP报告载体的修复效率提高了58.8%;同时使PFFs基因组位点DMD和ROSA26定点修饰效率分别提高了48.16%和17.64%。本研究表明,MEK抑制剂PD0325901能显著提高猪基因组ssODN介导的同源定向修复效率,为高效制备定点基因修饰动物模型提供了新思路。

关键词: MEK抑制剂, 同源定向修复(HDR), PD0325901, 基因编辑

Abstract:

There are two major pathways, homology-directed repair (HDR) and nonhomologous end joining (NHEJ), involved in double-strand break (DSB) repair. Single-stranded oligodeoxyribonucleotide (ssODN)-mediated homologous recombination repair is commonly used for animal site-directed genome editing, with great scientific and practical value. To improve ssODN-mediated HDR efficiency in the pig genome, we investigated the effect and molecular mechanism of mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor PD0325901 on the HDR efficiency in porcine fetal fibroblasts (PFFs). The results showed that PD0325901 obviously increased the percentage of G₂ and S phase cell populations and reduced the cell population ratio in the G₁ phase of PFFs, and promoted the expression of HDR repair factor. At the optimal concentration of 250 nmol/L, PD0325901 increased the repair efficiency of ssODN-mediated GFP reporter vector by 58.8% and the directed editing efficiency of PFF DMD and ROSA26 locus by 48.16% and 17.64%, respectively. The results show that MEK inhibitor PD0325901 significantly promotes the efficiency of ssODN-mediated homologous-directed repair in the porcine genome, thus offering a new idea to generate genetically modified pigs more effectively.

Key words: MEK inhibitor, homologous-directed repair (HDR), PD0325901, gene editing

欧浩,李国玲,王豪强,黄广燕,蔡更元,李紫聪,吴珍芳,张献伟. MEK抑制剂PD0325901显著提高猪胎儿成纤维细胞ssODN介导的HDR效率[J]. 遗传, 2019, 41(4): 327-336.

Hao Ou,Guoling Li,Haoqiang Wang,Guangyan Huang,Gengyuan Cai,Zicong Li,Zhenfang Wu,Xianwei Zhang. MEK inhibitor PD0325901 significantly boosts ssODN-mediated HDR efficiency in porcine fetal fibroblasts[J]. Hereditas(Beijing), 2019, 41(4): 327-336.

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MEK抑制剂PD0325901显著提高猪胎儿成纤维细胞ssODN介导的HDR效率

MEK inhibitor PD0325901 significantly boosts ssODN-mediated HDR efficiency in porcine fetal fibroblasts

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