Betz U A, Vosshenrich C A, Rajewsky K, Müller W
Institute for Genetics, University of Cologne, Germany.
Curr Biol. 1996 Oct 1;6(10):1307-16. doi: 10.1016/s0960-9822(02)70717-3.
The analysis of gene function based on the generation of mutant mice by homologous recombination in embryonic stem cells is limited if gene disruption results in embryonic lethality. Mosaic mice, which contain a certain proportion of mutant cells in all organs, allow lethality to be circumvented and the potential of mutant cells to contribute to different cell lineages to be analyzed. To generate mosaic animals, we used the bacteriophage P1-derived Cre-loxP recombination system, which allows gene alteration by Cre-mediated deletion of loxP-flanked gene segments.
We generated nestin-cre transgenic mouse lines, which expressed the Cre recombinase under the control of the rat nestin promoter and its second intron enhancer. In crosses to animals carrying a loxP-flanked target gene, partial deletion of the loxP-flanked allele occurred before day 10.5 post coitum and was detectable in all adult organs examined, including germ-line cells. Using this approach, we generated mosaic mice containing cells deficient in the gamma-chain of the interleukin-2 receptor (IL-2R gamma); in these animals, the IL-2R gamma-deficient cells were underrepresented in the thymus and spleen. Because mice deficient in DNA polymerase beta die perinatally, we studied the effects of DNA polymerase beta deficiency in mosaic animals. We found that some of the mosaic polymerase beta-deficient animals were viable, but were often reduced in size and weight. The fraction of DNA polymerase beta-deficient cells in mosaic embryos decreased during embryonic development, presumably because wild-type cells had a competitive advantage.
The nestin-cre transgenic mice can be used to generate mosaic animals in which target genes are mutated by Cre-mediated recombination of loxP-flanked target genes. By using mosaic animals, embryonic lethality can be bypassed and cell lineages for whose development a given target gene is critical can be identified. In the case of DNA polymerase beta, deficient cells are already selected against during embryonic development, demonstrating the general importance of this protein in multiple cell types.
如果基因破坏导致胚胎致死，那么基于胚胎干细胞中同源重组产生突变小鼠来分析基因功能的方法就会受到限制。嵌合小鼠在所有器官中都含有一定比例的突变细胞，这使得可以规避致死性，并分析突变细胞对不同细胞谱系的贡献潜力。为了产生嵌合动物，我们使用了噬菌体P1衍生的Cre-loxP重组系统，该系统允许通过Cre介导的loxP侧翼基因片段缺失来改变基因。
我们构建了巢蛋白-cre转基因小鼠品系，其在大鼠巢蛋白启动子及其第二个内含子增强子的控制下表达Cre重组酶。与携带loxP侧翼靶基因的动物杂交后，loxP侧翼等位基因在妊娠后第10.5天之前发生部分缺失，并且在所有检查的成年器官中都可检测到，包括生殖系细胞。使用这种方法，我们产生了含有白细胞介素-2受体（IL-2Rγ）γ链缺陷细胞的嵌合小鼠；在这些动物中，IL-2Rγ缺陷细胞在胸腺和脾脏中的比例较低。由于DNA聚合酶β缺陷的小鼠在围产期死亡，我们研究了嵌合动物中DNA聚合酶β缺陷的影响。我们发现一些嵌合的聚合酶β缺陷动物是存活的，但它们的大小和体重通常会减小。嵌合胚胎中DNA聚合酶β缺陷细胞的比例在胚胎发育过程中下降，推测是因为野生型细胞具有竞争优势。
巢蛋白-cre转基因小鼠可用于产生嵌合动物，其中靶基因通过Cre介导的loxP侧翼靶基因重组而发生突变。通过使用嵌合动物，可以绕过胚胎致死性，并确定给定靶基因对其发育至关重要的细胞谱系。就DNA聚合酶β而言，缺陷细胞在胚胎发育过程中就已被淘汰，这证明了该蛋白在多种细胞类型中的普遍重要性。
