Generation of a self-cleaved inducible Cre recombinase for efficient temporal genetic manipulation
Generation of a self-cleaved inducible Cre recombinase for efficient temporal genetic manipulation
复制标题
生成自切割诱导型 Cre 重组酶，用于有效的时间遗传操作
DOI：
10.15252/embj.2019102675
复制
发表时间：
2020-01-14
期刊：
影响因子：
11.4
通讯作者：
Zhou, Bin
中科院分区：
文献类型：
--
作者：
Tian, Xueying;He, Lingjuan;Zhou, Bin
位点特异性重组酶介导的遗传技术，如诱导型Cre-loxP重组（CreER），被广泛用于具有时间控制的体内遗传操作。Cre-loxP技术提高了我们对特定基因在器官发育、组织再生和疾病进展中的体内功能的理解。然而，诱导型CreER在基因缺失中通常仍然是低效的。为了提高基因操作的效率，我们产生了自切割的诱导型CreER（sCreER），其将诱导型CreER自身转换成组成型活性的Cre。我们构建了内皮细胞驱动因子Npr 3-sCreER和成纤维细胞驱动因子Col 1a 2-sCreER，并分别与传统的Npr 3-CreER和Col 1a 2-CreER进行了比较。对于易于重组的等位基因，如R26-tdTomato，sCreER和常规CreER之间的重组效率没有显著差异。然而，对于重组相对惰性的等位基因，例如R26-Confetti、R26-LZLT、R26-GFP或VEGFR 2（flox/flox）等位基因，与心内膜细胞或成纤维细胞中的传统CreER相比，sCreER显示出显着更高的重组效率。与传统的CreER相比，sCreER显著提高了重组诱导基因表达或基因缺失的效率，允许暂时但有效的体内基因组修饰，用于研究特定细胞谱系中的基因功能。
Site-specific recombinase-mediated genetic technology, such as inducible Cre-loxP recombination (CreER), is widely used for in vivo genetic manipulation with temporal control. The Cre-loxP technology improves our understanding on the in vivo function of specific genes in organ development, tissue regeneration, and disease progression. However, inducible CreER often remains inefficient in gene deletion. In order to improve the efficiency of gene manipulation, we generated a self-cleaved inducible CreER (sCreER) that switches inducible CreER into a constitutively active Cre by itself. We generated endocardial driver Npr3-sCreER and fibroblast driver Col1a2-sCreER, and compared them with conventional Npr3-CreER and Col1a2-CreER, respectively. For easy-to-recombine alleles such as R26-tdTomato, there was no significant difference in recombination efficiency between sCreER and the conventional CreER. However, for alleles that were relatively inert for recombination such as R26-Confetti, R26-LZLT, R26-GFP, or VEGFR2(flox/flox) alleles, sCreER showed a significantly higher efficiency in recombination compared with conventional CreER in endocardial cells or fibroblasts. Compared with conventional CreER, sCreER significantly enhances the efficiency of recombination to induce gene expression or gene deletion, allowing temporal yet effective in vivo genomic modification for studying gene function in specific cell lineages.