A Double Fluorescence Screening Strategy to Enhance the Efficiency of Gene Targeting
CHEN Jian-wu;REN Hong-yan;HUA Wen-jun;LIU Xi-mei;QI Shi-jin;ZHOU Li;OU Yang-yan;BI Yan-zhen;YANG Ye;ZHENG Xin-min;College of Animal Sciences,Yangtze University;Hubei Key Lab of Animal Embryo Engineering and Molecular Breeding,Institute of Animal Science and Veterinary,Hubei Academy of Agro Sciences;College of Agriculture,Hubei Three Groges Vocational and Technical College;
Gene targeting technology mediated by homologous recombination is a powerful tool for gene function analyze,because of its high accuracy and the mutation we introduced can be predicted. While in eukaryotes,the ratio of homologous recombination is too low to do further screening and identification. For this reason,establishing a high efficiency and widely used screening strategy becomes important. A new screening strategy which can dramatically improve the homologous recombination efficiency based on visual "double fluorescence"screening was established. The green fluorescent protein and neomycin gene were used as positive selection markers,and red fluorescent protein gene for negative selection marker. After screening by G418,the cell clones which just expressing the green fluorescent protein gene were selected and detected by transboundary PCR. The new strategy was used to detecting the gene targeting of pig MSTN gene in pig cells combined with CRISPR / Cas9 technology. In the two target sites T1 and T2,the targeting efficiency screening by "double establishing"reached up to 80. 5% and 86. 7%,which the was dramatically improved. This visual "double fluorescent"screening strategy established will become a useful tool for animal gene editing in the future.