title: Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts.
abstract: In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to effectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene provides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts.
authors.affiliation: 1. State Key Laboratory for Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China; 2. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China;
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, China Agricultural University, Beijing 100193, China.
authors.forename: Yu-shuang
authors.initials: YS
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China.
authors.forename: Lan-lan
authors.initials: LL
authors.lastname: Huang
authors.affiliation: College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China.
authors.forename: Zi-jian
authors.initials: ZJ
authors.lastname: Jian
authors.affiliation: State Key Laboratory for Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China.
authors.forename: Xin-hua
authors.initials: XH
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China.
authors.forename: Shou-ren
authors.initials: SR
authors.lastname: Liu
authors.affiliation: State Key Laboratory for Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China.
authors.forename: Wen-hui
authors.initials: WH
authors.lastname: Pi
mesh_terms: D000818:Animals; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage / Q000235:genetics; D011088:DNA Ligases / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods; D059765:Homologous Recombination / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D059767:Recombinational DNA Repair / Q000235:genetics*; D012756:Sheep / Q000235:genetics* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D011088:DNA Ligases