Construction of islet beta cell-specific PDHA1 knockout mice by Cre/loxP system

doi:10.3969/j.issn.2095-4344.1985

Abstract

Abstract:

BACKGROUND: In rodent models of type 2 diabetes mellitus and human, the expression level of pyruvate dehydrogenase complex is decreased, and PDHA1 deficiency is the most common cause of changed pyruvate dehydrogenase complex activity.
OBJECTIVE: To construct and identify the inducible islet-specific PDHA1 knockout mice, so as to provide basis for the study on the role of PDHA1 in pathogenesis of diabetes mellitus.
METHODS: The study was approved by the Ethics Committee of Southern Medical University. By using Cre-loxP recombination system, PDHA1flox/floxCre+/- mice were generated by crossing PDHA1flox/flox mice with Cre+/- mice. Genotypic identification was performed by PCR at the age of 3-4 weeks and the PDHA1flox/floxCre+/- mice were the required mouse model. PDHA1flox/floxCre+/- mice (βKO group, n=5) and Cre+/- mice (control group, n=5) at the age of 4 weeks were selected and received the same water and feed. Tamoxifen (50 mg/kg) was intraperitoneally injected at the age of 8 week to induce gene knockout. Three weeks later, pancreatic tissue, adipose tissue, and liver tissue were removed under anesthesia. qPCR, western blot and immunohistochemical staining were applied to identify the PDHA1 knockout effect and to observe the mouse phenotype.
RESULTS AND CONCLUSION: (1) PCR analysis selected PDHA1flox/floxCre+/- mice. (2) qPCR method was used to test the pancreas, fat and liver tissues of mice, and it was confirmed that the knockout effect had tissue specificity. The expression level of PDHA1 mRNA in islet in the βKO group was significantly lower than that in the control group. (3) The results of western blot and immunohistochemical scanning showed a significant decrease in PDHA1protein in the βKO group compared with the control group, suggesting the PDHA1 knockout effect was obvious. (4) In summary, the β cell-specific deletion of the PDHA1 gene is successfully constructed by Cre-loxP system, which provides a novel target for studying the pathogenesis of diabetes at animal level.

Key words: diabetes, PDHA1, Cre-loxP system, beta cells, gene

CLC Number:

Wang Xiao1, Lai Shuchang1, Ye Yanshi1, Xu Xuejuan1, Bai Xiaochun2, Shen Jie1 . Construction of islet beta cell-specific PDHA1 knockout mice by Cre/loxP system[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(31): 5023-5029.

Figures/Tables 2

2.1 PDHA1+/+、Cre+/-小鼠基因鉴定情况将引进的4月龄的PDHA1flox/flox纯合子小鼠自交，5个月后繁殖出29只小鼠，基因型均为PDHA1flox/flox纯合子。将PDHA1flox/flox小鼠与Cre+/-小鼠杂交，繁殖F1代小鼠。根据孟德尔遗传定律，子代小鼠的基因表型为PDHA1flox/-Cre+/-或PDHA1flox/flox (Cre纯合子小鼠具有胚胎致死性)。在子一代小鼠4周龄时PCR实验筛选基因型为PDHA1flox/-Cre+/-小鼠。部分小鼠鉴别结果见图1，2，扩增后的DNA分子在电场中向正极移动，根据分子量的差别，电泳后可呈现迁移位置的差异，条带类型对应基因型。进一步与PDHA1flox/flox纯合子小鼠杂交，F2代小鼠再次PCR基因筛选，PDHA1flox/floxCre+/-小鼠即为所需要的模型小鼠。部分子代小鼠的基因型鉴定结果见图3。选取周龄相同、体质量相近的5只PDHA1flox/floxCre+/-小鼠为实验组(βKO组)，5只Cre+/-小鼠为对照组。被选取小鼠在8周龄时，连续7 d注射药物他莫昔芬(50 mg/kg)进行诱导敲除。 2.2 qPCR法验证敲除效果的组织特异性用qPCR技术分别检测了胰岛、肝脏、脂肪组织中PDHA1 mRNA的相对表达，结果见图4。两组小鼠在肝脏、脂肪组织中PDHA1的mRNA相对表达水平并无明显差异；而在胰岛中βKO组小鼠PDHA1 mRNA的相对含量明显下降(P < 0.01)。证实PDHA1的敲除效应具有胰岛特异性。 2.3 Western Blot、免疫组织化学实验验证PDHA1的敲除效应 2.3.1 Werstern Blot实验结果见图5A。敲除小鼠PDHA1蛋白表达较对照组小鼠明显降低，说明在实验中，利用Cre-Loxp系统原理成功将小鼠胰腺组织中的PDHA1基因敲除，使其蛋白转录和翻译水平下降，表达量降低。因为仅敲除胰腺β细胞中的PDHA1基因，所以在胰腺总蛋白中仍存在部分PDHA1蛋白表达。 2.3.2 免疫组织化学结果见图5B。观察胰岛细胞团可以发现，PDHA1蛋白主要分布于小鼠胰腺的腺管细胞中，在胰岛中也有分布。βKO组小鼠胰岛中PDHA1的表达降低，提示βKO组小鼠胰岛细胞中PDHA1基因敲除成功。此外，实验发现βKO组小鼠体积部分增大，胰岛内细胞排列松散，内有空泡存在，提示PDHA1的敲除作用引起了胰岛β细胞形态的改变，为后续的进一步研究奠定基础。 "

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