Abstract：Histone methylation is key epigenetic regulatory feature that have important function in many biological processes which include heterochromatin formation, X-chromosome inactivation and transcriptional regulation. Among them, histone H3 lysine specific demethylase 6A (KDM6A) has functions of regulating embryonic development, cell reprogramming and cell differentiation. At present, the research on KDM6A mainly focused on experiments in a non-cellular environment, and there were few studies in cells, and no animal model of KDM6A overexpression is observed. In the present study, total RNA of 293T CELL was extracted and reversely transcribed into cDNA. Specific primers were designed according to human (Homo sapiens) mRNA sequence of KDM6A gene. The coding sequences of KDM6A gene was cloned in vitro and was constructed the overexpression vector pcs2-KDM6A by genetic engineering. Then, the vector pcs2-KDM6A was transfected into mouse (Mus musculus) embryonic fibroblasts via lipofectin transfection method. The gene expression patterns were detected by qRT-PCR. The qRT-PCR assay showed that the mRNA expression level of KDM6A in transfected group was significantly higher than control group (P<0.05). In contrast, and there was no significant difference between expression of KDM6B in transfected group and control group. Furthermore, H3K27me3 expression was detected by laser confocal immunofluorescence (IF) and western blot (WB) technology respectively. These results demonstrated that the expression of H3K27me3 level after transfection of pCS2-KDM6A vector both were significantly lower than control group. Taken together, the expression vector pCS2-KDM6A was successfully constructed, and was proved to had correct enzyme activity at the cell level. The pCS2-KDM6A vector can be used to study the function of KDM6A in the development and related diseases both in vivo and in vitro. Moreover, an animal model of overexpression of KDM6A can be prepared by this vector, and a basic material for analyzing the research mechanism of KDM6A and H3K27me3 in biological events such as X chromosome inactivation, embryo development and cell reprogramming is provided.
白力格, 赵彩权, 宋丽爽, 刘雪霏, 杨磊, 李光鹏. KDM6A表达载体的构建及其对组蛋白H3K27me3修饰的影响[J]. 农业生物技术学报, 2019, 27(1): 108-117.
BAI Li-Ge, ZHAO Cai-Quan, SONG Li-Shuang, LIU Xue-Fei, YANG Lei, LI Guang-Peng. Construction of KDM6A Expression Vector and Its Effect on Histone H3K27me3 Modification. 农业生物技术学报, 2019, 27(1): 108-117.
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