中山大学学报（自然科学版）2016，Vol.55Issue(4)：100-107,8.DOI:10.13471/j.cnki.acta.snus.2016.04.017
供体同源臂长度对 ZFN 介导的同源重组效率的影响
The effect of the length of donor homologous arm on the efficiency of ZFN-induced homologous recombination
摘要
Abstract
Zinc finger nuclease (ZFN)is composed of an engineered site-specific Cys2-His2 zinc finger domain and the nonspecific restriction enzyme Fok I cleavage domain,which is able to cut at a specified genomic locus to generate double-strand break (DSB)of DNA.The DSBs induced by ZFN are subse-quently repaired through two different DNA repair mechanisms,either non-homologous end-joining (NHEJ)or homology-directed recombination (HDR).NHEJ is prone to introduce sequence insertions or deletions (indels),and can therefore produce frameshifts in open reading frames and gene loss of func-tion.HDR requires a donor template with the sequence similar to the genome to mend a lesion.By intro-ducing a DNA donor with desired modifications,precise genomic modifications can be achieved at a fre-quency improved 102 -104-fold as compared to the traditional gene targeting method.Currently,most studies have focused on screening ZFN with higher activity,and improving the delivery efficiency of ZFN and donor into host cells,less studies have investigated the relationship between the homologous arm length with the efficiency of ZFN induced homologous recombination.Here,we constructed a pair of ZFN plasmids targeting to EGFP and verified its cutting activity.Then we designed a series of donors with dif-ferent lengths of homologous arms.By introducing individual donor with ZFN into CHO cells harboring a frame-shift GFP gene,we measured the homologous recombination efficiencies through the flow cytometric analysis.We found that a 50 bp short homology arm was capable to support ZFN-mediated homologous re-combination.Increasing the length of the homologous arms could improve the efficiency of ZFN-mediated homologous recombination.A dramatic improvement (104-fold higher than traditional method)requires a homology arm longer than 1 000 bp.关键词
锌指核酸酶/DNA 双链断裂/同源重组/供体/同源臂Key words
zinc finger nuclease/double-strand break/homologous recombination/donor/homologous arm分类
生物科学引用本文复制引用
聂宇,乔艳乐,陈瑶生,何祖勇..供体同源臂长度对 ZFN 介导的同源重组效率的影响[J].中山大学学报（自然科学版）,2016,55(4):100-107,8.基金项目
国家转基因生物新品种培育重大专项资助项目（2016ZX08006003－006）；NSFC －广东省联合基金资助项目 （）
