Vallier L, Mancip J, Markossian S, Lukaszewicz A, Dehay C, Metzger D, Chambon P, Samarut J, Savatier P
Laboratoire de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Unité Mixte de Recherche, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France.
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2467-72. doi: 10.1073/pnas.041617198. Epub 2001 Feb 20.
We have developed a universally applicable system for conditional gene expression in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expression vectors that allow transgene expression from endogenous cellular promoters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the beta-galactosidase/neo(R) fusion protein and the Cre-ER(T2) (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bicistronic gene-trap vector encoding the hygro(R) protein and the human alkaline phosphatase (hAP), the expression of which is prevented by tandemly repeated stop-of-transcription sequences flanked by loxP sites. In selected clones, hAP expression was shown to be regulated accurately by 4'hydroxy-tamoxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitro in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtually all the tissues of the 10-day-old chimeric fetus (after injection of 4'hydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can be applied to drive conditional expression of virtually any transgene in a large variety of cell types, both in vitro and in vivo.
我们开发了一种在胚胎干细胞（ES细胞）中进行条件性基因表达的通用系统，该系统依赖于他莫昔芬依赖性的Cre重组酶-loxP位点介导的重组以及双顺反子基因捕获表达载体，后者允许从内源性细胞启动子进行转基因表达。将两种载体先后导入受体ES细胞基因组：（i）一种双顺反子基因捕获载体，编码β-半乳糖苷酶/新霉素抗性（neo(R)）融合蛋白和Cre-ER(T2)（与人类雌激素受体的突变配体结合域融合的Cre重组酶）；（ii）一种双顺反子基因捕获载体，编码潮霉素抗性（hygro(R)）蛋白和人碱性磷酸酶（hAP），其表达受位于loxP位点两侧的串联重复转录终止序列抑制。在选定的克隆中，hAP表达显示受4-羟基他莫昔芬精确调控。hAP实现了严格的激素依赖性表达：（i）在体外未分化的ES细胞和胚状体中；（ii）在体内几乎10日龄嵌合胎儿的所有组织中（向代孕母体注射4-羟基他莫昔芬后）；（iii）在从嵌合胎儿分离的原代胚胎成纤维细胞中进行离体实验。因此，这种方法可用于在多种细胞类型中驱动几乎任何转基因的条件性表达，无论是在体外还是体内。
