2022, Vol. 26 ›› Issue (20): 3207-3211

Construction, phenotype and functional identification of vascular endothelial cell-specific PDHA1 knockout mice

Hao Wei1, Sun Yue1, Yang Anning2, Liu Taiyang1, Bao Rui1, Liu Yaoyang1, Wang Qiushi1, Wang Meng1, Chang Sirong1, Li Yuanyuan1, Liu Zhihong1     

Abstract: BACKGROUND: PDHA1 gene is an indispensable gene in the aerobic respiratory chain. The energy metabolism of vascular endothelial cells is related to many diseases. Construction of vascular endothelial cell-specific PDHA1 knockout mice helps to study the role of energy metabolism in diseases related to vascular endothelial cells.
OBJECTIVE: To breed vascular endothelial cell-specific PDHA1 knockout mice and identify their phenotypes.
METHODS: PDHA1(iΔEC/ iΔEC) mice were co-bred with PDHA1(iΔEC/-) mice, and more PDHA1(iΔEC/ iΔEC) mice were obtained for subsequent experiments. PCR and agarose gel electrophoresis were used for gene identification. RT-PCR and western blot were used to detect the mRNA and protein expression of genes related to energy metabolism after PDHA1 knockout, respectively. The protein expression of PDHA1 after PDHA1 conditional knockout was determined by double immunofluorescence staining of VE-cadherin and PDHA1.
RESULTS AND CONCLUSION: Genotypes of offspring mice were successfully detected by agarose gel electrophoresis, including 15 PDHA1(iΔEC/ iΔEC) and 2 PDHA1(iΔEC/-) mice. Results of reverse transcription PCR and western blot assay showed that the transcription and translation levels of PDHA1 were decreased, the protein and mRNA expressions of HK2 in the glycolysis pathway were increased, and the protein and mRNA expression of IDH in the aerobic phosphorylation pathway were decreased. Double immunofluorescence staining of VE-cadherin and PDHA1 showed a decrease in the expression of PDHA1.
Key words: PDHA1, double immunofluorescence, CreERT2 gene, phenotypic validation, gene identification, energy metabolism, vascular endothelial cell, pulmonary fibrosis