ISSN 1674-3865  CN 21-1569/R
主管：国家卫生和计划生育委员会
主办：中国医师协会
　　　辽宁省基础医学研究所
　　　辽宁中医药大学附属医院
Chinese Pediatrics of Integrated Traditional and Western Medicine

Chinese Pediatrics of Integrated Traditional and Western Medicine ›› 2021, Vol. 13 ›› Issue (4): 286-.

Previous Articles     Next Articles

Construction of Atg7-gene-knockout human brain microvascular endothelial cell line based on CRISPR/cas9 technology

Objective:To construct Atg7-gene-knockout human brain microvascular endothelial cells(HBMEC) by CRISPR/cas9 technology, so as to provide an in vitro cell model for the role of Atg7 in the development of cerebral vessels, the mechanism of Atg7 participating in the formation of blood-brain barrier and thestudy of cerebrovascular diseases.#br# Methods:Two pairs of guiding RNA sequences(sgRNA) were designed according to exon 1 of human Atg7. HBMEC was transfected with lentivirus; puromycin and Pyricularia oryzae were used to screen for cells successfully transfected. RT-qPCR and Western blotting were used to detect the mRNA and protein of the target gene, and the morphological changes were observed under the microscope.#br# Results:The CRISPR/cas9 plasmid targeting Atg7 was constructed. The results of RT-qPCR, Western blot and immunofluorescence showed that stable Atg7-knockout human brain microvascular endothelial cell line was  obtained by monoclonal screening. No obvious morphological changes were observed under light microscope.#br# Conclusion:The successful construction of human brain microvascular endothelial cells with Atg7 gene knockout by CRISPR/cas9 technology is beneficial to the development of cerebral vessels, the mechanism of blood-brain barrier morphogenesis and the study of cerebrovascular diseases.   

  1. China Medical University, Shenyang 110032,China
  • Online:2021-08-25 Published:2021-12-07

PDF (PC)

44

Abstract: Objective:To construct Atg7-gene-knockout human brain microvascular endothelial cells(HBMEC) by CRISPR/cas9 technology, so as to provide an in vitro cell model for the role of Atg7 in the development of cerebral vessels, the mechanism of Atg7 participating in the formation of blood-brain barrier and thestudy of cerebrovascular diseases.
Methods:Two pairs of guiding RNA sequences(sgRNA) were designed according to exon 1 of human Atg7. HBMEC was transfected with lentivirus; puromycin and Pyricularia oryzae were used to screen for cells successfully transfected. RT-qPCR and Western blotting were used to detect the mRNA and protein of the target gene, and the morphological changes were observed under the microscope.
Results:The CRISPR/cas9 plasmid targeting Atg7 was constructed. The results of RT-qPCR, Western blot and immunofluorescence showed that stable Atg7-knockout human brain microvascular endothelial cell line was  obtained by monoclonal screening. No obvious morphological changes were observed under light microscope.
Conclusion:The successful construction of human brain microvascular endothelial cells with Atg7 gene knockout by CRISPR/cas9 technology is beneficial to the development of cerebral vessels, the mechanism of blood-brain barrier morphogenesis and the study of cerebrovascular diseases.

Key words: HBMEC, Atg7 gene, CRISPR/cas9 technology, Gene knockout