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目的建立TNNT3(R69H)突变转基因小鼠模型。方法构建pEGFP-TNNT3(R69H)转基因构件,通过原核显微注射方法将线性化、纯化后的外源质粒pEGFP-TNNT3(R69H)注射入BDF1小鼠受精卵中,胚胎移植至同期发情的假孕受体母鼠输卵管内,获得子代小鼠。用PCR和Southern blot方法检测子代鼠尾基因组DNA,通过RT-PCR及Western blot的方法检测TNNT3基因表达。结果 8只假孕小鼠共移植注射后的胚胎82枚,出生40只子代鼠,经PCR和Southern方法检测得到5只转基因阳性小鼠。对其子代小鼠进行RT-PCR、Western blot检测结果显示,TNNT3在转基因小鼠心脏和骨骼肌中表达量明显增多。结论通过显微注射法使外源基因pEGFP-TNNT3(R69H)在小鼠基因组中整合,成功建立了TNNT3(R69H)突变转基因小鼠模型。
Objective To establish a transgenic mouse model of TNNT3 (R69H) mutation. Methods The recombinant plasmid pEGFP-TNNT3 (R69H) was constructed and injected into the fertilized egg of BDF1 mice by pronuclear and purified plasmid pEGFP-TNNT3 (R69H) Recipient mother rat fallopian tube, obtained offspring mice. The tail genomic DNA was detected by PCR and Southern blot. The expression of TNNT3 gene was detected by RT-PCR and Western blot. Results Eight dummy embryos were transplanted in 82 embryos and 40 offspring were born. Five transgenic mice were obtained by PCR and Southern blot. RT-PCR was performed on the offspring mice. Western blot results showed that the expression of TNNT3 was significantly increased in the heart and skeletal muscle of transgenic mice. Conclusion Transgenic mouse model of TNNT3 (R69H) was successfully constructed by integrating the exogenous gene pEGFP-TNNT3 (R69H) into mouse genome by microinjection.