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目的利用PiggyBAC转座酶系统及受精卵注射法,将MCK3E-OVA-pA随机插入小鼠基因组,获得骨骼肌特异表达卵清蛋白(OVA)的转基因小鼠(Muscle-OVA Tg)。方法通过体外转录,获得PiggyBAC mRNA;将PiggyBAC mRNA和含有鸡全长OVA片段、骨骼肌肌酸激酶启动子(MCK)、H-2K~b引导序列、H-2Db跨膜序列的质粒显微注射至C57BL/6J小鼠受精卵中,获得转基因阳性首建鼠。将Muscle-OVA Tg鼠与野生B6小鼠杂交建系。PCR鉴定小鼠基因型;Westernblot检测小鼠各组织中OVA蛋白表达以验证其组织特异性。结果PCR检测表明,Muscle-OVA Tg的肌组织表达OVA mRNA;Western blot进一步证实Muscle-OVA Tg小鼠骨骼肌组织特异的OVA蛋白表达,其他组织/器官,如心、脾、肺、肾组织OVA表达均呈阴性。结论利用PiggyBAC转座酶系统成功构建骨骼肌特异表达鸡OVA抗原的转基因小鼠,为相关的骨骼肌免疫反应研究提供了有价值的动物模型。
OBJECTIVE: The transgenic mouse (Muscle-OVA Tg) that specifically expressed ovalbumin (OVA) in skeletal muscle was obtained by inserting the MCK3E-OVA-pA into the genome of mice with the PiggyBAC transposase system and fertilized egg injection method. Methods PiggyBAC mRNA was obtained by in vitro transcription. PiggyBAC mRNA was microinjected into plasmid containing chicken OVA fragment, skeletal muscle creatine kinase promoter (MCK), H-2K ~ b leader sequence and H-2Db transmembrane sequence To C57BL / 6J mouse zygotes, get transgenic positive first mouse. Muscle-OVA Tg mice were crossed with wild B6 mice. PCR was used to identify the genotype of mouse; Western blot was used to detect the expression of OVA protein in each tissue to verify its tissue specificity. Results PCR showed that OVA mRNA was expressed in muscle of Muscle-OVA Tg. Western blot further confirmed skeletal muscle-specific OVA expression in Muscle-OVA Tg mice. Other tissues / organs such as OVA The expression was negative. Conclusion The construction of a transgenic mouse expressing OVA antigen specifically by skeletal muscle using PiggyBAC transposase system has provided a valuable animal model for the study of skeletal muscle immune response.