Efficiency of recombination by Cre transient expression in embryonic stem cells: Comparison of various promoters
Efficiency of recombination by Cre transient expression in embryonic stem cells: Comparison of various promoters
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DOI：
10.1093/oxfordjournals.jbchem.a021860
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发表时间：
1997-11-01
影响因子：
2.7
通讯作者：
Yamamura, K
中科院分区：
文献类型：
--
作者：
Araki, K;Imaizumi, T;Yamamura, K
噬菌体P1的Cre - loxP重组系统在胚胎干细胞（ES）的基因操作中经常被使用。Cre的表达水平对于诱导ES细胞中loxP位点特异性重组至关重要。为了比较重组效率，我们构建了由不同启动子驱动的四种Cre表达载体：巨细胞病毒/鸡β - 肌动蛋白（CAG）启动子、人多肽链延伸因子1α（hEF - 1α）启动子、小鼠磷酸甘油酸激酶 - 1（mPGK）启动子以及多瘤增强子/单纯疱疹病毒胸苷激酶（MC1）启动子。我们通过电穿孔将这些Cre表达载体导入携带单拷贝CAG - loxP - 氯霉素乙酰转移酶（CAT）基因 - loxP - β - 半乳糖苷酶（β - gal）基因构建体的三种ES细胞系。由于Cre介导的重组会导致CAT基因的切除，重组效率可以通过β - gal的表达来监测。实验中未使用选择系统。当使用CAG启动子时获得了最高的重组频率，其次依次是hEF - 1α启动子、mPGK启动子和MC1启动子。这些结果表明，瞬时表达系统中的重组效率与Cre表达载体的启动子活性相关。因此，为了通过Cre进行有效的重组，选择启动子是很重要的。
The Cre-loxP recombination system of bacteriophage P1 is frequently utilized in genetic manipulation in embryonic stem (ES) cells, The level of Cre expression is critical to induce loxP site-specific recombination in ES cells, To compare the efficiency of recombination, we constructed four Cre expression vectors driven by different promoters: cytomegarovirus/chicken beta-actin (GAG) promoter, human polypeptide chain elongation factor 1 alpha (hEF-1 alpha) promoter, mouse phosphoglycerate kinase-1 (mPGK) promoter, and polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter, We introduced these Cre expression vectors by electroporation into three ES cell lines carrying a single copy of CAG-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase (beta-gal) gene construct, Since the Cre-mediated recombination leads to excision of the CAT gene, the efficiency of recombination can be monitored as beta-gal expression. No selection system was used in the experiments, The maximum recombination frequency was obtained when the CAG promoter was used, followed by the hEF-1 alpha promoter, the mPGK promoter and the MC1 promoter in order, These results indicate that the efficiency of recombination in transient expression system correlates with the promoter activity of Cre expression vector. Thus, it is important to choose the promoter for effective recombination by Cre.