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Generation of conditionally controlled transgenic mice expressing hepatitis C virus NS3/4A protease
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摘要:
目的建立调控因素存在下表达丙型肝炎病毒（HCV）NS3/4A丝氨酸蛋白酶的转基因小鼠。方法采用分子克隆技术构建可受Tet-On调控系统和Cre-LoxP基因敲除系统双重调控表达HCV NS3/4A丝氨酸蛋白酶的真核表达质粒PBI-Ⅲ/LoxP-Luc-PolyA-LoxP-NS3/4A。该重组质粒线性化后,经显微注射制备转基因小鼠。首建鼠及经PCR检测阳性的子代鼠与C57BL/6小鼠交配传代。选择部分F2鼠与已经稳定建系的转基因小鼠Lap杂交,利用在体生物发光成像系统（BLI）检测肝脏稳定表达荧光素酶（Luc）报告基因的经盐酸强力霉素（Dox）诱导的双转基因子代鼠,并选择性针对稳定表达系传代扩群。结果酶切鉴定和测序分析显示重组载体构建成功。经PCR检测得到6只转基因首建鼠,其中3只可繁殖传代并持续检测到阳性子代鼠。BLI结果显示,由其中1只首建鼠传代而来的不同F2鼠与Lap鼠杂交得到的部分双转基因鼠肝脏部位发光信号强烈,表明这些小鼠肝细胞内报告基因Luc特异高效表达。结论建立了转基因小鼠NS3/4A,并筛选到调控因素存在时转入外源基因特异稳定表达的转基因小鼠,为进一步建立严格调控型表达...
Abstract:Objective To establish conditionally regulated transgenic mice expressing hepatitis C virus(HCV) NS3 4A serine protease.Methods A recombinant plasmid PBI-Ⅲ/LoxP-Luc-PolyA-LoxP-NS3/4A was constructed by molecular cloning and transgenic mice were generated by microinjection of fertilized ovum with the larger linearized fragment obained by enzyme digesting the recombinant vector.Founder mouse and positive offsprings detected by polymerase chain reaction(PCR) were interbred with C57BL/6 mouse to establish a defined genetic background.Part of the second generations were interbred with another transgenic mouse Lap which have been stable and systematically constructed.The interbred dual transgenic offsprings previously induced with doxycycline(Dox) were detected by in vivo bioluminescent imaging(BLI).The imaging positive dual transgenics were reserved and their NS3/4A transgenic parents were interbred with C57BL/6 instead.Results Restriction analysis and sequence analysis showed that the recombinant plasmid PBI-Ⅲ/LoxP-Luc-PolyA-LoxP-NS3/4A was successfully constructed.6 founder transgenic mouse were obtained and 3 of them have been spread to fourth generation.Transgenic mouse stably expressing the exogenous reporter gene luciferase(Luc) were selected by BLI.Conclusion Transgenic mouse conditionally expressing HCV NS3/4A serine protease were successfully generated.
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Key words:
- hepacivirus /
- models /
- animal
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