人血管紧张素转化酶2(ACE2)BAC转基因小鼠模型的制备及鉴定

Preparation and identification of human angiotensin converting enzyme 2(ACE2)BAC transgenic mouse model

目的期望获得含有全长hACE2基因序列的hACE2人源化小鼠模型,为心血管疾病、新冠肺炎发病机制研究、药物与疫苗研发提供重要工具。方法将纯化含有hACE2完整编码序列的BAC质粒,利用小鼠受精卵显微注射和输卵管移植技术获得多个首建鼠,分别育种建系,通过杂交育种获得稳定遗传F2代小鼠品系,进一步对F2代小鼠hACE2基因整合、基因拷贝数、相对荧光定量PCR、Western Blot和免疫荧光进行分析。结果育种获得hACE2-6-9、hACE2-14-3、hACE2-15-1和hACE2-15-2共4个小鼠品系。基因整合结果显示,hACE2-6-9、hACE2-15-1和hACE2-15-2小鼠品系含有完整的hACE2基因座位点及较长的基因调控区,hACE2-14-3含有完整的hACE2基因座位点及3’UTR较短的基因调控区。相对荧光定量PCR和Western Blot检测结果显示,hACE2-6-9、hACE2-15-1和hACE2-15-2品系小鼠肠道中hACE2 mRNA和蛋白表达水平较低,而整合较短调控序列的hACE2-14-3小鼠品系则表达水平较高。免疫荧光染色结果显示hACE2-15-1品系小鼠肾小管和肺血管内皮细胞中均有hACE2表达。结论获得了含有全长基因序列的hACE2人源化BAC转基因小鼠,保留完整的hACE2启动子及基因调控区,从而为心血管疾病、新冠肺炎发病机制研究、hACE2基因表达调控机制研究和药物、疫苗的研发提供重要工具。

Objective To obtain a novel coronavirus pneumonia transgenic mouse model with constitutive human angiotensin converting enzyme 2(hACE2)gene expression,that can efficiently maintain human physiological levels of a bacterial artificial chromosome(BAC)hACE2 gene.This model could provide an important tool for research into cardiovascular disease,the pathogenesis of novel coronavirus pneumonia,and the development of drugs and vaccines.Methods A BAC plasmid containing the complete coding sequence of human ACE2(hACE2)was purified and transgenic mice were established by fertilized egg microinjection and Fallopian tube transplantation.Stable F2 mouse strains were obtained by cross-breeding and analyzed in relation to integration of the hACE2 gene,gene copy number,relative fluorescence quantitative polymerase chain reaction(PCR),Western Blot,and immunofluorescence.Results Four mouse strains(hACE2-6-9,hACE2-14-3,hACE2-15-1 and hACE2-15-2)were obtained.The result of gene integration showed that the hACE2-6-9,hACE2-15-1 and hACE2-15-2 strains contained the complete hACE2 gene locus and long gene regulatory region,while hACE2-14-3 contained the complete hACE2 gene locus and a short gene regulatory 3’untranslated region.Relative fluorescence quantitative PCR and western blot showed that hACE2 mRNA and protein levels in the intestine were lower in the hACE2-6-9,hACE2-15-1 and hACE2-15-2 strains,while expression levels were higher in the hACE2-14-3 strain with shorter regulatory sequences.Immunofluorescence staining showed that hACE2 was expressed in renal tubular epithelial cells and pulmonary vascular endothelial cells in hACE2-15-1 mice.Conclusions We obtained novel coronavirus pneumonia hACE2 transgenic mice with the full-length gene sequence and the complete promoter and gene regulatory regions.This model will provide an important tool for the study of cardiovascular diseases and the pathogenesis of novel coronavirus pneumonia,as well as for examining mechanisms regulating hACE2 gene expression and the development of drugs and vaccines.