Sakai K, Miyazaki J i
Department of Nutrition and Physiological Chemistry, Osaka University Medical School, 2-2 Yamadaoka, Osaka, Suita, 565, Japan.
Biochem Biophys Res Commun. 1997 Aug 18;237(2):318-24. doi: 10.1006/bbrc.1997.7111.
The Cre/loxP site-specific recombination system derived from bacteriophage P1 provides a convenient tool for directed modifications of genomes in various organisms. To exploit Cre-mediated manipulation of mouse genomic sequences at the zygote stage, we have developed a transgenic mouse line carrying the CAG-cre transgene in which the cre gene is under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid (CAG) promoter. The activity of the Cre recombinase at early stages of development was examined by crossing the CAG-cre transgenic mice to another transgenic mouse line carrying a reporter gene construct, CAG-CAT-Z, which directs expression of the E. coli lacZ gene upon Cre-mediated excision of the loxP-flanked chloramphenicol acetyltransferase (CAT) gene located between the CAG promoter and the lacZ gene. PCR-based analysis of F1 progeny from CAG-cre males x CAG-CAT-Z females showed that transmission of the CAG-cre transgene was accompanied by the complete deletion of the CAT gene of the CAG-CAT-Z transgene in all tissues, and that this deletion was never observed in the progeny without transmission of the CAG-cre gene. On the other hand, analysis of F1 mice from CAG-CAT-Z males x CAG-cre females showed that the CAG-CAT-Z transgene had undergone complete deletion of the CAT gene in all tissues irrespective of the cotransmission of the CAG-cre gene. This Cre-mediated recombination in F1 mice occurred before the two-cell stage of embryonic development, as shown by X-gal staining. The results suggest that the CAG-cre transgene is expressed in developing oocytes of CAG-cre transgenic mice, and Cre mRNA and/or protein are retained in mature oocytes irrespective of the transmission of the CAG-cre transgene, resulting in efficient Cre-mediated recombination of paternally derived target genes upon fertilization. The CAG-cre transgenic mouse should serve as a useful tool to introduce prescribed genetic modifications into the mouse embryo at the zygote stage.
源自噬菌体P1的Cre/loxP位点特异性重组系统为各种生物体基因组的定向修饰提供了便利工具。为了在合子期利用Cre介导的对小鼠基因组序列的操控，我们构建了一个携带CAG-cre转基因的转基因小鼠品系，其中cre基因受巨细胞病毒立即早期增强子-鸡β-肌动蛋白杂交（CAG）启动子的控制。通过将CAG-cre转基因小鼠与另一个携带报告基因构建体CAG-CAT-Z的转基因小鼠品系杂交，检测了发育早期Cre重组酶的活性。CAG-CAT-Z报告基因构建体在CAG启动子和lacZ基因之间含有loxP侧翼的氯霉素乙酰转移酶（CAT）基因，在Cre介导切除该CAT基因后可指导大肠杆菌lacZ基因的表达。对CAG-cre雄性小鼠与CAG-CAT-Z雌性小鼠杂交产生的F1后代进行基于PCR的分析表明，CAG-cre转基因的传递伴随着CAG-CAT-Z转基因的CAT基因在所有组织中的完全缺失，而在未传递CAG-cre基因的后代中从未观察到这种缺失。另一方面，对CAG-CAT-Z雄性小鼠与CAG-cre雌性小鼠杂交产生的F1小鼠的分析表明，无论CAG-cre基因是否共传递，CAG-CAT-Z转基因的CAT基因在所有组织中都发生了完全缺失。如X-gal染色所示，F1小鼠中的这种Cre介导的重组发生在胚胎发育的二细胞期之前。结果表明，CAG-cre转基因在CAG-cre转基因小鼠的发育卵母细胞中表达，并且Cre mRNA和/或蛋白保留在成熟卵母细胞中，而与CAG-cre转基因的传递无关，从而在受精时导致父源靶基因的有效Cre介导的重组。CAG-cre转基因小鼠应作为一种有用的工具，用于在合子期将特定的基因修饰引入小鼠胚胎。
