MEK inhibitor PD0325901 significantly boosts ssODN-mediated HDR efficiency in porcine fetal fibroblasts
Received date: 2018-10-29
Revised date: 2019-03-02
Online published: 2019-03-25
Supported by
the National Natural Science Foundation of China(31772555);the National Transgenic Major Projects(2016ZX08006002)
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 . DOI: 10.16288/j.yczz.18-294
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