Ligand-dependent genetic recombination in fibroblasts - A potentially powerful technique for investigating gene function in fibrosis
Ligand-dependent genetic recombination in fibroblasts - A potentially powerful technique for investigating gene function in fibrosis
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DOI：
10.1016/s0002-9440(10)61108-x
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发表时间：
2002-05-01
影响因子：
6
通讯作者：
Denton, CP
中科院分区：
文献类型：
--
作者：
Zheng, B;Zhang, ZP;Denton, CP
在小鼠中条件诱导转基因表达的策略可能对于测试候选基因在疾病发病机制中的作用有价值。我们开发了一种系统，在来自 proalpha2(I) 胶原蛋白基因的成纤维细胞特异性调控序列的控制下，使用编码 Cre-ER(T) 融合蛋白的嵌合基因，在小鼠成纤维细胞中诱导谱系特异性、配体依赖性持续转基因表达。 Cre-ER(T) 作为一种他莫昔芬依赖性 DNA 重组酶来切除侧翼为特定 LoxP 共有序列的片段。为了测试该策略的效率和配体依赖性，表达 Cre-ER(T) 的小鼠与杂合性 ROSA26-LacZ 报告小鼠回交，其中 floxed-STOP 盒已被引入细菌 β-半乳糖苷酶 (LacZ) 报告基因上游普遍表达的位点。在胚胎、新生儿和成年化合物转基因小鼠中检查了组成型或他莫昔芬诱导的 LacZ 表达。当怀孕的 ROSA26-LacZ 雌性接受单剂量他莫昔芬时，在注射含有 Cre-ER(T) 转基因和目标 ROSA26-LacZ 等位基因的双转基因胚胎后 24 小时，皮肤中 LacZ 的高水平表达就可见一斑。出生后他莫昔芬还诱导 LacZ 高水平表达，特别是在真皮和内脏成纤维细胞中。通过允许对已靶向整合 LoxP 位点的等位基因进行有效的胚胎或出生后修饰，或打开 floxed-STOP 盒下游克隆的转基因，该系统将允许在预定的胚胎或出生后时间点诱导成纤维细胞特异性遗传扰动。这将极大地有助于系统性硬化症等纤维化疾病候选基因的体内功能研究。
Strategies for conditional induction of transgene expression in mice are likely to be valuable for testing the role of candidate genes in disease pathogenesis. We have developed a system for lineage-specific, ligand-dependent, induction of sustained transgene expression in fibroblastic cells in mice using a chimeric gene encoding the Cre-ER(T) fusion protein, under the control of a fibroblast-specific regulatory sequence from the proalpha2(I)collagen gene. Cre-ER(T) operates as a tamoxifen-dependent DNA recombinase to excise fragments flanked by specific LoxP consensus sequences. To test efficiency and ligand dependency of this strategy, Cre-ER(T)-expressing mice were backcrossed with heterozygous ROSA26-LacZ reporter mice, in which a floxed-STOP cassette has been introduced upstream of a bacterial beta-galactosidase (LacZ) reporter gene at a ubiquitously expressed locus. Constitutive or tamoxifen-induced LacZ expression was examined in embryonic, neonatal, and adult compound-transgenic mice. When pregnant ROSA26-LacZ females received a single dose of tamoxifen, high-level expression of LacZ in the skin was demonstrable from 24 hours after injection in double-transgenic embryos harboring both the Cre-ER(T) transgene and the target ROSA26-LacZ allele. High-level expression of LacZ was also induced postnatally by tamoxifen specifically in dermal and visceral fibroblasts. By allowing efficient embryonic or postnatal modification of alleles that have been targeted to incorporate LoxP sites, or to switch on transgenes cloned downstream of the floxed-STOP cassette, this system will allow fibroblast-specific genetic perturbations to be induced at predetermined embryonic or postnatal time points. This should greatly assist in in vivo functional studies of candidate genes in fibrotic diseases such as systemic sclerosis.