Lantinga-van Leeuwen Irma S, Leonhard Wouter N, van de Wal Annemieke, Breuning Martijn H, Verbeek Sjef, de Heer Emile, Peters Dorien J M
Leiden University Medical Center, Center for Human and Clinical Genetics, Leiden, The Netherlands.
Genesis. 2006 May;44(5):225-32. doi: 10.1002/dvg.20207.
Gene inactivation often leads to an embryonic-lethal phenotype. In focal diseases like renal cell carcinomas and polycystic kidney disease, somatic gene inactivation in subsets of cells is likely to occur at later stages. We generated a transgenic mouse line with an inducible form of Cre recombinase for conditional gene modifications in kidney epithelial cells. To this end a 1.4-kb promoter fragment of the kidney-specific cadherin gene (KspCad) was cloned upstream of a tamoxifen-inducible Cre recombinase (CreER(T2)) encoding sequence. Expression and activity of Cre was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) analysis and by crossbreeding to Z/EG reporter mice. One KspCad-CreER(T2) line showed kidney-specific Cre expression and mediated recombination upon tamoxifen treatment in Z/EG reporter mice. No reporter gene expression was detected in untreated animals or in extrarenal tissues upon treatment. Within the kidneys, enhanced green fluorescent protein (EGFP) fluorescence was observed in epithelial cells in several nephronic segments. In addition, the system successfully recombined a floxed Pkd1 gene.
基因失活通常会导致胚胎致死表型。在诸如肾细胞癌和多囊肾病等局部疾病中，细胞亚群中的体细胞基因失活可能在后期发生。我们构建了一种转基因小鼠品系，其携带可诱导形式的Cre重组酶，用于在肾上皮细胞中进行条件性基因修饰。为此，将肾特异性钙黏蛋白基因（KspCad）的1.4 kb启动子片段克隆到编码他莫昔芬诱导型Cre重组酶（CreER(T2)）的序列上游。使用逆转录聚合酶链反应（RT-PCR）分析并通过与Z/EG报告基因小鼠杂交来评估Cre的表达和活性。一个KspCad-CreER(T2)品系显示出肾特异性Cre表达，并在他莫昔芬处理后介导了Z/EG报告基因小鼠中的重组。在未处理的动物或处理后的肾外组织中未检测到报告基因表达。在肾脏内，在几个肾单位节段的上皮细胞中观察到增强型绿色荧光蛋白（EGFP）荧光。此外，该系统成功地重组了一个floxed Pkd1基因。
