Hrd1转基因小鼠的构建与验证

Construction and validation of Hrd1 transgenic mice

背景 内质网应激在糖尿病视网膜病变（DR）中发挥着重要作用.羟甲基戊二酰辅酶A还原酶降解蛋白1（Hrd1,又名Syvn1）作为一种内质网相关蛋白降解（ERAD）途径中的核心蛋白,能够减少内质网应激. 目的 构建Hrd1高表达小鼠,为今后研究Hrd1在DR中的作用提供动物模型. 方法 构建Hrd1系统表达重组质粒,酶切、测序后显微注射3 p1到685枚C57BL/6小鼠受精卵中,移植到代孕母鼠,得到Hrd1转基因小鼠.利用鼠尾PCR检测法鉴定F0代小鼠的基因型.得到Hrd1基因检测阳性鼠后,使其分别与野生型鼠杂交,得到F1代Hrd1转基因小鼠,取鼠尾组织,用PCR检测法再次鉴定F1代小鼠基因型. 结果 成功构建了Hrd1重组载体,重组质粒pRP.ExBi-EF1 a-Syvn1-IRES-eGFP的全序列测定结果显示,cDNA序列均正确.接受了Hrd1-pcDNA显微注射的685枚受精卵中成活598枚.将存活的受精卵移植到代孕母鼠后共产子鼠41只,获F0代子鼠8只,生理活动均良好.PCR电泳显示339 bp处阳性条带,该基因型可以传递到子代F1.结论 成功构建了Hrd1转基因小鼠.

Background Endoplasmic reticulum stress plays an important role in diabetic retinopathy （DR）.Hydroxymethyl glutaric acyl coenzyme A reductase degradation protein 1 （Hrd1） is a key protein in endoplasmic reticulum-associated degradation （ERAD）,and can reduce the excess endoplasmic reticulum stress.Objective This study was to generate Hrd1 transgenic mice and offer the animal model with Hrd1-high-expression for further pathogenesis research of DR.Methods The construct was generated by inserting the murine Hrd1 cDNA into a vector of systemic expression.The Hrd1 recombinant plasmids were then linearized,purified and microinjected 3 pl into 685 fertilized C57BL/6 mouse eggs,and the eggs were then transplanted into the pseudopregnant C57BL/6 mice to produce Hrd1 transgenic mice.Born transgenic founders were genotyped by PCR.The positive mice were mated with wild-type mice and reproduced generation was confirmed by PCR.Results The recombinant Hrdl vector was constructed successfully,and the pRP.ExBi-EF1a-Syvn1-IRES-eGFP plasmid sequencing results showed that the cDNA sequence was correct.There were 598 survival-fertilized eggs that accepted Hrd1-pcDNA microinjection.Then the ova were implanted into the pseudopregnant C57BL/6 mice and got 41 mice pups.After PCR detection,eight F0 generations Hrd1 mice were obtained,and the physiological states of the F0 mice were normal.PCR result showed positive bands of 339 bp,and the gene type can be passed on to offspring F1.Conclusions This study demonstrates the successful generation of Hrd1 transgenic mice.