Nanjidsuren Tsevelmaa, Park Chae-Won, Sim Bo-Woong, Kim Sun-Uk, Chang Kyu-Tae, Kang Myung-Hwa, Min Kwan-Sik
a Animal Biotechnology, Graduate School of Future Convergence Technology , Institute of Genetic Engineering, Hankyong National University , Anseong , Republic of Korea.
b National Research Center , Korea Research Institute of Bioscience and Biotechnology , Ochang , Republic of Korea.
Anim Biotechnol. 2016 Oct;27(4):223-30. doi: 10.1080/10495398.2016.1176032.
Transcription activator-like effector nucleases (TALENs) are a new type of engineered nuclease that is very effective for directed gene disruption in any genome sequence. We investigated the generation of mice with genetic knockout (KO) of the G protein-coupled receptor kinase (GRK) 5 gene by microinjection of TALEN mRNA. TALEN vectors were designed to target exons 1, 3, and 5 of the mouse GRK5 gene. Flow cytometry showed that the activity of the TALEN mRNAs targeted to exons 1, 3, and 5 was 8.7%, 9.7%, and 12.7%, respectively. The TALEN mRNA for exon 5 was injected into the cytoplasm of 180 one-cell embryos. Of the 53 newborns, three (5.6%) were mutant founders (F0) with mutations. Two clones from F028 showed a 45-bp deletion and F039 showed the same biallelic non-frame-shifting 3-bp deletions. Three clones from F041 were shown to possess a combination of frame-shifting 2-bp deletions. All of the mutations were transmitted through the germline but not to all progenies (37.5%, 37.5%, and 57.1% for the F028, F039, and F041 lines, respectively). The homozygote GRK5-KO mice for 28 and 41 lines created on F3 progenies and the homozygous genotype was confirmed by PCR, T7E1 assay and sequencing.
转录激活样效应核酸酶（TALENs）是一种新型的工程核酸酶，对任何基因组序列中的定向基因破坏都非常有效。我们通过显微注射TALEN mRNA研究了G蛋白偶联受体激酶（GRK）5基因敲除（KO）小鼠的产生。TALEN载体被设计用于靶向小鼠GRK5基因的外显子1、3和5。流式细胞术显示，靶向外显子1、3和5的TALEN mRNA的活性分别为8.7%、9.7%和12.7%。将外显子5的TALEN mRNA注射到180个单细胞胚胎的细胞质中。在53只新生小鼠中，有3只（5.6%）是带有突变的突变奠基者（F0）。来自F028的两个克隆显示有45 bp的缺失，F039显示有相同的双等位基因非移码3 bp缺失。来自F041的三个克隆显示具有移码2 bp缺失的组合。所有突变都通过种系传递，但并非传递给所有后代（F028、F039和F041系分别为37.5%、37.5%和57.1%）。在F3后代中产生了28和41系的纯合GRK5-KO小鼠，并通过PCR、T7E1分析和测序确认了纯合基因型。
