Construction and identification of LSD1 overexpression and demethylation disfunction plasmids
10.3760/cma.j.issn.1673-4181.2018.01.004
- VernacularTitle:LSD1过表达及去甲基化作用缺失质粒的构建与鉴定
- Author:
Lili WU
1
;
Yijie LIU
;
Bingyan CAO
;
Qiaoman FEI
;
Xiujuan ZHAO
;
Qian LI
;
Yating HAN
;
Lei CAO
;
Bing YANG
;
Ling ZHANG
Author Information
1. 300070,天津医科大学基础医学院
- Keywords:
Lysine-specific histone demethylase 1;
Demethylation;
Recombinant plasmid
- From:
International Journal of Biomedical Engineering
2018;41(1):26-31,37
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct rat lysine-specific histone demethylase 1 (LSD1) overexpression plasmids and LSD 1 demethylation fragment disfunction plasmids,and to evaluate their expression levels in HEK293T cells.Methods LSD1 fragments were amplified by PCR,and LSD1 demethylation disfunction fragments were amplified by overlap PCR.Rat-specific LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids were constructed and verified by agarose gel electrophoresis and sequencing.HEK293T cells were infected using the validated recombinant plasmids and blank vectors,and the stably expressed cells were selected by puromycin.The expression of LSD1 in the stably expressed HEK293T was detected by Western Blot and real-time quantitative PCR.Results The results of agarose gel electrophoresis and sequencing showed that LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids were successfully constructed.The Western Blot and real-time quantitative PCR results showed that compared with the blank group,the relative expression of LSD1 and mRNA in the LSD1 overexpression group and the LSD1 demethylation disfunction group were up-regulated,and the differences were statistically significant(all P＜0.01).Conclusions The constructed LSD1 overexpression plasmids and LSD1 demethylationi disfunction plasmids can achieve overexpression of LSD1 gene in HEK293T cells.This paper lays a foundation for further study of the relationship between LSD 1 gene and related diseases and demethylation of LSD 1.
