Abstract:

Lu Yuting, Wang Zhenying, Song Yali, Ji Cancan, Zhang Li Department of Dermatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China Corresponding author: Zhang Li, Email: zhangliwenzhe@medmail.com.cn 【Abstract】 Objective To construct HaCaT cell lines stably expressing the wild type human GJB6 gene or its mutant by using a Tet-On lentiviral vector, and to lay an experimental foundation for studies on pathogenesis of hidrotic ectodermal dysplasia. Methods The wild-type human GJB6 gene and its mutant （A88V） were amplified by PCR, and then inserted into the Tet-on lentivirus plasmid to construct recombinant lentivirus vectors. The recombinants were identified by gene sequencing and enzymatic digestion. Cultured HaCaT cells were classified into three groups to be transfected with a negative control lentiviral vector （NC group）, the lentivirus vector expressing the wild-type human GJB6 gene （WT group）, or the lentivirus vector expressing the mutant human GJB6 gene （MU group）. Puromycin was used to select HaCaT cell clones stably expressing the GJB6 gene which encodes the connexin 30 （Cx30） protein. The selected HaCaT cell clones were cultured with or without tetracycline for 48 hours, thereafter, real-time PCR （RT-PCR） was performed to detect GJB6 gene mRNA , Western-blot analysis to measure s of Cx30 and FLAG-tag proteins, and cell counting kit 8 （CCK8） assay to evaluate cellular proliferative activity. Results Enzymatic digestion and gene sequencing showed that recombinant lentivirus plasmids were successfully constructed. RT-PCR showed evidently increased mRNA of the GJB6 gene in stably transfected HaCaT cells. Moreover, the abundance of the GJB6 gene was 112.369 times higher in the WT group induced by tetracycline than in that without tetracycline treatment （P < 0.05）, and 2.249 times higher in the MU group induced by tetracycline than in that without tetracycline treatment （P < 0.05）. Western-blot analysis showed that Cx30 and FLAG-tag proteins were stably expressed in the WT group and MU group after induction with tetracycline, while neither of them was observed in the WT group or MU group without tetracycline treatment, or in the NC group. Significant differences were noted in cellular proliferative activity （expressed as the absorbance value at 450 nm） between the MU group with and without tetracycline treatment and between the WT group with and without tetracycline treatment at 4, 8, 12, 24, 36 and 48 hours（all P < 0.05）, but not between the NC group with and without tetracycline treatment at any of the above time points （all P > 0.05）. Conclusion HaCaT cell lines which stably express the wild-type GJB6 gene or its mutant （A88V） are successfully constructed.