Xu Yuanyuan, Liu Siguo, Yu Guohua, Chen Jianquan, Chen Juan, Xu Xujun, Wu Youbing, Zhang Aimin, Dowdy Steven F, Cheng Guoxiang
School of Life Science and Technology, Tongji University, 1239 Si-Ping Road, Shanghai, 200092, China.
Gene. 2008 Aug 1;419(1-2):70-4. doi: 10.1016/j.gene.2008.04.020. Epub 2008 May 13.
The Cre/loxP site-specific recombination system is a widely used tool for genetic engineering of mammalian genomes. Recombination of loxP-modified alleles is often induced by introduction of foreign DNA vector expressing Cre into the cells. But the introduced DNA vector has the potential to integrate into the genome of the cells and continuous expression of Cre recombinase from the foreign vector has the potential to yield cytotoxicity and genotoxicity in various cells. In this study, we investigate the possibility of overcoming this limitation by using a cell-permeable TAT-Cre recombinase. We found that TAT-Cre treatment of transgenic goat fibroblast cells did not compromise the development competency of reconstructed embryos by using these TAT-Cre-treated cells as nuclear donor in nuclear transfer. Finally, we obtained two live cloned goats where a selectable gene cassette was removed. Our work not only provided an efficient protein transduction-based system for removing selectable genes from transgenic goats, but also presented strong evidence that no severe damage was made to the host cells during the process of protein transduction.
Cre/loxP位点特异性重组系统是用于哺乳动物基因组基因工程的一种广泛使用的工具。loxP修饰等位基因的重组通常通过将表达Cre的外源DNA载体导入细胞来诱导。但是导入的DNA载体有可能整合到细胞基因组中，并且来自外源载体的Cre重组酶的持续表达有可能在各种细胞中产生细胞毒性和基因毒性。在本研究中，我们研究了使用细胞可渗透的TAT-Cre重组酶克服这一限制的可能性。我们发现，用TAT-Cre处理转基因山羊成纤维细胞，将这些经TAT-Cre处理的细胞用作核移植的核供体时，不会损害重构胚胎的发育能力。最后，我们获得了两只去除了选择基因盒的活克隆山羊。我们的工作不仅提供了一种基于蛋白质转导的高效系统，用于从转基因山羊中去除选择基因，而且还提供了强有力的证据，表明在蛋白质转导过程中宿主细胞没有受到严重损伤。
