间质细胞双尾C基因条件性敲除小鼠模型建立及其表型分析

Establishment and phenotypic analysis of mesenchymal cell bicaudal-C gene conditional knockout mice models

目的构建间质细胞双尾C(Bicc1)基因条件性敲除小鼠模型,并对其表型进行分析。方法将CRISPR Cas9方法构建的Bicc1f/+小鼠子代与Pdgfra启动子驱动的Cre小鼠进行杂交,获得子代小鼠,饲养1~2周后提取其脚趾及鼠尾组织基因组DNA,经PCR扩增及琼脂糖凝胶电泳后,在DNA水平检测小鼠基因型;选取经鉴定后生长至3周龄的Bicc1基因条件性敲除小鼠(实验组)、野生型小鼠(对照组)各3只进行后续实验:通过腹腔注射他莫昔芬进行诱导敲除Bicc1基因,于1周后取小鼠肾脏、骨骼肌、皮肤及脂肪组织,一部分组织通过实时定量PCR(RT-qPCR)鉴定其中的Bicc1 mRNA表达水平;另一部分组织以10%多聚甲醛固定,随后进行HE染色和Masson染色,在光镜下观察分析。应用SPSS 28.0软件对数据进行分析,组间比较采用t检验,P<0.05表示差异有统计学意义。结果通过繁育获得间质细胞Bicc1基因条件性敲除小鼠,基因型为Bicc1^(f/f)Cre+/-,野生型小鼠基因型为Bicc1^(f/f)Cre^(-/-);RT-qPCR检测结果显示,实验组小鼠肾脏、骨骼肌、皮肤、脂肪组织中Bicc1 mRNA表达水平均显著低于对照组(P均<0.01);HE染色和Masson染色显示,与对照组相比,实验组小鼠肾小球萎缩,肾小囊形状不规则,部分肾小囊消失;骨骼肌纤维排列疏松散乱,肌纤维堆积不致密;皮肤及脂肪组织未见明显差别。结论成功建立了间质细胞Bicc1基因条件性敲除小鼠模型,可以为研究Bicc1基因在组织器官间质细胞中的作用提供动物模型;3周龄小鼠间质细胞中敲除Bicc1后1周,其肾脏和骨骼肌发生病理性改变。

Objective To establish mesenchymal cell bicaudal-C(Bicc1)gene conditional knockout mice models and analyze their phenotypes.Methods Bicc1f/+mice were crossed with Pdgfra promoter-driven Cre mice to obtain the offspring mice.Genomic DNA was extracted from the toe and tail tissues from 1-2 weeks old mice,amplified by PCR and detected at the DNA level by agarose gel electrophoresis.Three Bicc1 gene conditional knockout mice(experimental group)and three wild-type mice(control group)were selected after identification and grew to 3 weeks of age for follow-up experiments.The Bicc1 gene was knocked out by the induction of tamoxifen intraperitoneal injection.After 1 week,the kidney,skeletal muscle,skin and adipose tissue samples were collected.Real-time quantitative PCR(RT-qPCR)was performed to determine the expression levels of Bicc1 mRNA in the collected tissue samples.HE and Masson staining were performed with tissue samples fixed in 10%paraformaldehyde,and observed with a light microscope.The SPSS 28.0 software was used to analyze the data,t-test was used for comparison between groups,and P<0.05 was considered statistically significant.Results Mesenchymal cell Bicc1 gene conditional knockout mice models were obtained by breeding,and the genotype was Bicc1^(f/f)Cre+/-.The genotype of the wild-type mice was Bicc1^(f/f)Cre^(-/-).RT-qPCR showed that the expression levels of Bicc1 mRNA in kidney,skeletal muscle,skin and adipose tissue of the experimental mice were significantly lower than those of the control group(all P<0.01).HE staining and Masson staining showed that compared with the control group,glomerular atrophy could be observed in the experimental group,renal capsules were irregular in shape,and some renal capsules disappeared.The arrangement of skeletal muscle fibers were loose and scattered,and the accumulation of muscle fibers was not dense.There were no significant differences between the skin and adipose tissue.Conclusion Mesenchymal cell Bicc1 gene conditional knockout mice models were successfully established,which could provide models for studying the mechanisms of action of Bicc1 gene in different tissues and organs.Mesenchymal cell conditional Bicc1 gene knockout affected the phenotypes of kidney and skeletal muscle in mice.