摘要
利用斑马鱼作为体内模型,研究旨在提高斑马鱼原始生殖细胞(Primordial germ cells,PGCs)中同源重组(Homologous recombination,HR)的效率。首先,将UAS:m RFP-nos1载体显微注射到Tg(kop:Kal TA4)转基因胚胎中标记转基因PGCs,结果表明筛选PGCs特异表达m RFP的胚胎能够相对提高转基因的生殖系传递效率。随后建立了PGCs中HR效率的评估体系,并且证明抑制DNA ligase IV(Lig4)和Xrcc6(曾用名Ku70)的活性不但在全胚胎水平,而且在PGCs水平都能够显著提高HR的效率。研究表明Tg(kop:Kal TA4)转基因品系是开展HR介导的基因打靶的一个有效平台。
Primordial germ cells （PGCs） give rise to gametes which transmit the genetic information to next generation, therefore PGCs provide us an ideal cell type for genetic manipulation. Homologous recombina- tion （HR） is the most efficient technique to create designed genetic modifications, however, its efficiency is rather low in vertebrates. In this study, by using zebrafish as an in vivo model, we aimed to enhance the effi- ciency of HR in zebrafish PGCs. First, we injected UAS：mRFP-nosl construct into Tg （kop：KalTA4） embryos to label the transgenic PGCs, and we showed that screening of PGCs-specific mRFP expression led to rela- tively high-efficient germline transmission of transgene. Then we established an in vivo assay to evaluate the HR frequency in PGCs. We further revealed that suppression of the activities of DNA ligase IV （Lig4） and Xrcc6 （previously known as Ku70） could significantly increase the HR efficiency, not only at whole embryo level but also in PGCs. We proposed that the Tg（kop：KalTA4） line could be used as an effective platform for HR-mediated gene targeting.
出处
《水生生物学报》
CAS
CSCD
北大核心
2015年第2期339-348,共10页
Acta Hydrobiologica Sinica
基金
Foundation item:The National Basic Research Program of China(grant numbers 2010CB126306&2012CB944504)
the National Science Fund for Excellent Young Scholars of NSFC(grant number 31222052)
the FEBL grant 2011FBZ23
