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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase.
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Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase.

机译：Cre重组酶的瞬时表达可在受精卵中进行转基因的位点特异性重组。

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摘要

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 bornwith 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.
机译：已经开发了使用Cre / loxP重组系统在受精卵中进行转基因调控的有效方法。产生了十二个携带鸡β-肌动蛋白启动子-loxP-氯霉素乙酰转移酶（CAT）基因-loxP-β-半乳糖苷酶基因构建体的转基因小鼠品系。选择表现出在各种组织中CAT基因最高表达的品系后，将该品系的卵注射到在鸡β-肌动蛋白启动子的控制下的Cre表达载体的雄性或雌性前核中，并保存在避免基因组整合的圆形形式。这导致蛋中Cre的瞬时表达，导致通过半乳糖苷酶表达和DNA分析检测到的转基因重组。两种类型的前核注射均在桑stage之前完成重组，发生频率很高。在18只经此修饰的转基因出生的小鼠中，未观察到镶嵌现象，未完成重组以及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可与奠基人行中CAT基因的表达水平相媲美。这种Cre瞬时表达系统应可用于育种转基因表达会导致不育或致死性的转基因品系-特别是对于选择具有高致死力的转基因品系的转基因品系而言，其在常规转基因系统中难以探索其全部活性因为选择只携带表达不佳的转基因的转基因品系的风险。

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