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

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

• Research Article • Previous Articles Next Articles

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 Online:2019-04-20 Published:2019-03-25
Contact: Zhang Xianwei E-mail:zxianw@163.com
Supported by:
the National Natural Science Foundation of China(31772555);the National Transgenic Major Projects(2016ZX08006002)

Abstract

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

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

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