Araki K, Araki M, Miyazaki J, Vassalli P
Department of Pathology, Centre Médical Universitaire, University of Geneva, Switzerland.
Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):160-4. doi: 10.1073/pnas.92.1.160.
An efficient method of transgene modulation in fertilized eggs has been developed that uses the Cre/loxP recombination system. Twelve transgenic mouse lines carrying a chicken beta-actin promoter-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase gene construct were produced. After selection of the line showing the highest expression of the CAT gene in a variety of tissues, eggs of this line were injected in the male or female pronucleus with a Cre expression vector placed under the control of the chicken beta-actin promoter and kept in a circular form to avoid genomic integration. This resulted in a transient expression of Cre in the eggs, leading to recombination of the transgene as detected by galactosidase expression and DNA analysis. Recombination was completed before the morula stage with both types of pronuclear injections and occurred with a very high frequency; no mosaicism, no incomplete recombination, and no integration of the Cre sequence were observed in 18 mice born with this modified transgene. The beta-galactosidase gene was expressed in various tissues at levels comparable to those found for the CAT gene in the founder line. This Cre transient expression system should be useful for breeding transgenic lines in which transgene expression leads to sterility or lethality--in particular, for selecting transgenic lines with high expression of a potentially lethal transgene whose full activity is difficult to explore in a conventional transgenic system because of the risk of selecting for transgenic lines carrying only poorly expressed transgenes.
一种利用Cre/loxP重组系统在受精卵中进行转基因调控的有效方法已经开发出来。构建了携带鸡β-肌动蛋白启动子-loxP-氯霉素乙酰转移酶（CAT）基因-loxP-β-半乳糖苷酶基因的12个转基因小鼠品系。在选择了在多种组织中CAT基因表达最高的品系后，将该品系的卵在雄原核或雌原核中注射一个置于鸡β-肌动蛋白启动子控制下的Cre表达载体，并保持其环状形式以避免基因组整合。这导致Cre在卵中瞬时表达，通过半乳糖苷酶表达和DNA分析检测到转基因发生重组。两种类型的原核注射在桑椹胚阶段之前重组均已完成，且重组频率非常高；在18只携带这种修饰转基因出生的小鼠中，未观察到嵌合体、未完全重组以及Cre序列的整合。β-半乳糖苷酶基因在各种组织中的表达水平与奠基品系中CAT基因的表达水平相当。这种Cre瞬时表达系统对于培育转基因品系应该是有用的，在这些转基因品系中，转基因表达会导致不育或致死——特别是对于选择具有潜在致死性转基因高表达的转基因品系，由于存在选择仅携带低表达转基因的转基因品系的风险，其全部活性在传统转基因系统中难以探究。
