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Functional assessment of expression of Cre recombinase targeting the liver of transgenic mice regulated by Tet-on and Cre/loxP system
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摘要:
目的制备Tet-on和Cre/loxP系统双重调控下肝靶向性表达Cre重组酶的转基因小鼠rtTALAP-1/LC-1并评价其功能,为最终建立可突破胚胎期免疫耐受的双调控型HCV转基因小鼠模型奠定基础。方法选取适龄rtTALAP-1转基因小鼠与LC-1转基因小鼠交配,PCR法检测子代rtTALAP-1/LC-1转基因小鼠基因组中是否插入了rtTA元件和Cre基因片段。双阳性rtTALAP-1/LC-1小鼠dox诱导1周后,以小动物活体成像系统检测小鼠肝脏的Luc萤光素酶信号,免疫组化检测Cre重组酶在小鼠肝脏等组织中的表达状况。结果 rtTALAP-1/LC-1转基因小鼠在dox诱导后,仅在其肝脏检测到清晰的Luc萤光素酶信号,而其他脏器均未检测到萤光信号;免疫组化法也仅在小鼠肝细胞核中检测到Cre重组酶的表达,心脏、肾脏和骨骼肌等其他组织均未检测到Cre重组酶的表达。结论成功制备了rtTALAP-1/LC-1转基因小鼠,其靶基因表达的诱导反应性和肝靶向性均良好,为最终制备双调控型丙型肝炎病毒（HCV）转基因小鼠模型奠定了良好的基础。
Abstract:Objective To construct and evaluate transgenic mice rtTALAP-1/ LC-1 target expressing Cre recombinase in liver under regulation of Tet-on and Cre/loxP systems,to lay the foundation for construction of HCV transgenic mice model which can break the embryo stage immunotolerance under dual regulation systems.Methods Mating transgenic mice rtTALAP-1 and LC-1 to acquire offspring mice rtTALAP-1/LC-1,rtTA element and Cre gene fragment of them were detected by PCR analysis.Double positive rtTALAP-1/LC-1 mice were induced by dox for one week,then luciferase signal of mice liver were detected by small animal living body image-forming system and Cre recombinase expression in liver tissue etc.were detected by immunol histochemistry subsequently.Results Experimental results showed that clear luciferase signal can be detected only in liver of rtTALAP-1/ LC-1 mice after dox induction by small animal living body image-forming system.Immuno-histochemistry experiment results also showed that Cre recombinase expression only can be detected in liver of rtTALAP-1/ LC-1 mice,on the contrary Cre expression can not be detected in heart,kidney,skeletal muscle and other tissue of rtTALAP-1/ LC-1 mice.Conclusion Transgenic mice rtTALAP-1/ LC-1 were constructed successfully,the liver targeting and induced reaction of expression of targeted gene were very good as well.The above research has laid a solid foundation for construction of HCV transgenic mice model which can break the embryo stage immunotolerance under dual regulation systems.
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Key words:
- hepacivirus /
- HCV /
- mice
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