Inducible gene knockouts in the small intestinal and colonic epithelium
Inducible gene knockouts in the small intestinal and colonic epithelium
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DOI：
10.1074/jbc.274.53.38071
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发表时间：
1999-12-31
影响因子：
4.8
通讯作者：
Gordon, JI
中科院分区：
文献类型：
--
作者：
Saam, JR;Gordon, JI
我们已经开发了两种用于在快速自我更新的小鼠小肠和结肠上皮中进行Cre介导的靶基因重组的系统。当Cre重组酶的表达直接置于来自脂肪酸结合蛋白基因（Fabp）的转录调控元件的控制下时，早在胚胎第13.5天就开始缺失loxP侧翼（floxed）DNA序列，在肠道形态发生完成之前。到胚胎第16.5天，Fabp-Cre还指导排列在肾盏和肾盂、米和膀胱的移行上皮的所有细胞层中的重组，Fabp-Cre表达和重组在整个成年期在两种上皮中维持。第二个系统允许重组仅在肠道中和成年期的任何时期被诱导。该系统使用Fabp调节元件来指导反向四环素调节的反式激活因子（rtTA）的表达。另一个转基因在泰特操纵子序列和来自人巨细胞病毒的最小启动子（tetO-P-hCMV-Cre）的控制下编码Cre。在不存在强力霉素诱导剂的情况下，在含有Fabp-rtTA、tetO-P-hCMV-Cre加上floxed报告基因的成年三转基因小鼠的肠道中没有检测到基础重组。在口服施用多西环素4天后，报告基因的重组在小肠、盲肠和结肠上皮中是明显的。在多西环素被撤回后，重组的基因座持续至少60天，表明重组已经发生在上皮细胞祖细胞中，所述上皮细胞祖细胞在肠的增殖单位中具有长的停留时间这种诱导系统应该有许多应用，用于在选定的生理或病理生理条件下，在出生后生活的选定时间检查基因功能。
We have developed two systems for performing Cre-mediated recombination of target genes in the rapidly self-renewing mouse small intestinal and colonic epithelium, When expression of Cre recombinase is placed directly under the control of transcriptional regulatory elements from a fatty acid-binding protein gene (Fabp), deletion of loxP flanked (floxed) DNA sequences is initiated as early as embryonic day 13.5, well before completion of intestinal morphogenesis. By embryonic day 16.5, Fabp-Cre also directs recombination in all cell layers of the transitional epithelium that lines the renal calyces and pelvis, meters, and bladder, Fabp-Cre expression and recombination are maintained in both epithelia throughout adulthood. The second system allows recombination to be induced only in the gut and at any period during adulthood. This system uses Fabp regulatory elements to direct expression of a reverse tetracycline-regulated transactivator (rtTA), Another transgene encodes Cre under the control of tet operator sequences and a minimal promoter from human cytomegalovirus (tetO-P-hCMV-Cre). In the absence of a doxycycline inducer, no basal recombination is detectable in the gut of adult tri-transgenic mice containing Fabp-rtTA, tetO-P-hCMV-Cre, plus a floxed reporter gene. After 4 days of oral administration of doxycycline, recombination of the reporter is apparent in the small intestinal, cecal, and colonic epithelium, After doxycycline is withdrawn, the recombined locus persists for at least 60 days, indicating that recombination has occurred in epithelial cell progenitors that have long residency times in the proliferative units of the intestine (crypts of Lieberkuhn), This inducible system should have a number of applications for examining gene function at selected times in postnatal life, under selected physiologic or pathophysiologic conditions.