条件基因打靶研究存在的主要问题及对策

Issues and solutions of conditional gene targeting

基于Cre-loxP等位点特异性重组系统的条件基因打靶技术在解析基因功能和制备疾病小鼠模型方面发挥着极其重要的作用。但包括Cre重组酶转基因的表达模式不理想、重组效率存在差异、Cre重组酶的毒性等在内的Cre-loxP重组系统相关的缺陷以及条件基因打靶技术本身存在的问题,如遗传背景、育种策略、实验对照、基因代偿等方面的影响往往被忽略,严重影响基因功能和小鼠表型解析的准确性。针对这些问题,精细调控实现Cre重组酶的理想时空特异性表达、详尽地分析Cre重组酶的重组效率、降低Cre重组酶的毒性、遗传背景单一化、优化育种策略、设立严格实验对照、多基因联合条件基因打靶等诸多解决方案应予以采纳。

Conditional gene targeting,based on the site-specific recombination system such as Cre-loxP,plays a vital role in the study of gene functions and the generation of disease mouse models.It was always under consideration that there were problems in the Cre-loxP recombination system,such as illegal expression pattern of Cre transgene,variation of Cre recombination efficiency and toxicity of Cre recombinase,as well as the potential influences of genetic background,breed-ing strategy,experimental control and gene compensation.Oversights of these issues may have a profound influence on the accuracy of gene functional dissection and conditional gene targeting mice phenotypic interpretation.Accordingly,solutions should be adopted including delicate regulative control of temporal-spatial specific Cre expression,detailed detection of Cre recombination efficiency,reduction of Cre toxicity,simplification of mouse genetic background,optimization of breeding,setting up of proper control and combined conditional gene targeting.