三种小分子化合物对猪胎儿成纤维细胞DNA精确修复效率的影响

Effect of Three Small Molecule Compounds on Precise DNA Repair Efficiency in Porcine Fetal Fibroblasts

旨在研究3种小分子化合物(RS-1、Chir99021和PD173074)对猪胎儿成纤维细胞DNA精确修复效率的影响。本研究以猪胎儿成纤维细胞(porcine fetal fibroblasts,PFFs)为试验对象,利用HDR通路关键因子Rad51激活剂RS-1、Wnt信号通路关键因子糖原合成酶激酶-3(glycogen synthase kinase 3,GSK-3)抑制剂Chir99021和成纤维细胞生长因子受体(fibroblast growth factor receptor,FGFR1)抑制剂PD173074培养细胞,通过流式细胞术检测绿色荧光细胞数百分比来验证3种小分子化合物对DNA精确修复效率的影响。结果显示:1)经不同剂量的Chir99021和PD173074处理后,PFFs DNA损伤修复因子LIG4、NHEJ1、XRCC5、XRCC6和Rad51表达水平都显著下调(P <0. 05),而经不同浓度PD173074(0. 1～0. 5μmol·L^(-1))处理后,PNKP因子表达上调(P <0. 05)。2) Chir99021低剂量时可显著提高同源重组修复(homologous recombination,HR)介导的同源重组效率,Chir99021高剂量时可显著提高单链退火修复(single strand annealing,SSA)介导的同源重组效率; RS-1和PD173074低剂量时可显著提高HR效率(P <0. 05),但RS-1在高浓度时显著下调SSA修复效率(P <0. 05),而PD173074对SSA效率无显著影响(P>0. 05);此外单链寡核苷酸介导的DNA修复(single-stranded oligonucleotide,ss ODN)效率并不受小分子化合物处理的影响(P>0. 05)。本研究结果表明,适当浓度小分子化合物的应用有利于不同策略的基因敲入细胞系的产生,为获得精确的遗传修饰的动物模型打基础。

The aim of this study was to investigate the effects of3small molecule compounds(RS-1,Chir99021and PD173074)on the DNA precise repair efficiency in porcine fetal fibroblasts.In this study,the activator RS-1for Rad51,the inhibitor Chir99021for glycogen synthase kinase3(GSK-3)and the inhibitor PD173074for fibroblast growth factor receptor(FGFR1)were used to culture porcine fetal fibroblasts,and then the percentage of green fluorescent cells was detected by flow cytometry to verify the effect of3small molecule compounds on DNA repair efficiency.The results showed that:1)After treatment with different doses of Chir99021and PD173074,the expression levels of PFFs DNA damage repair factors LIG4,NHEJ1,XRCC5,XRCC6and Rad51were significantly down-regulated(P<0.05),and after treatment with different doses of of PD173074,the expression level of PNKP factor was significantly up-regulated(P<0.05).2)The low dose of Chir99021significantly increased the efficiency of homologous recombination(P<0.05)mediated by homologous recombination(HR)repair(P<0.05).High dose of Chir99021could significantly improve the efficiency of homologous recombination mediated by single strand annealing(SSA)repair(P<0.05).The low doses of RS-1and PD173074significantly increased HR efficiency(P<0.05),but RS-1significantly down-regulated SSA repair efficiency at high concentration(P<0.05),while PD173074had no significant effect on SSA efficiency(P>0.05).Furthermore,the single-stranded oligonucleotide-mediated DNA repair(ssODN)was not affected(P>0.05)by small molecule compounds.This study indicates that the application of small molecule compounds with appropriate concentrations will facilitate the generation of gene knock-in cell lines,which laying the foundation for obtaining genetic modification animal models.