Construction of dedicator of cytokinesis 4 over-expressed lentivirus vector and establishment of stable transfected Neuro-2a cells
10.13481/j.1671-587X.20240516
- VernacularTitle:胞质分裂作用因子4过表达慢病毒载体的构建和稳定转染Neuro-2a细胞的建立
- Author:
Shengnan LI
1
,
2
;
Jiawen HE
;
Keqi LIAO
;
You LI
Author Information
1. 广东医科大学广东省衰老相关心脑疾病重点实验室,广东湛江 524002
2. 广东医科大学附属医院神经病学研究所,广东湛江 524002
- Keywords:
Dedicator of cytokinesis 4;
Over-expression lentivirus vector;
Neuro-2a cell;
Stable transfection;
Over-expression lentivirus
- From:
Journal of Jilin University(Medicine Edition)
2024;50(5):1322-1329
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct an over-expression lentiviral vector of the dedicator of cytokinesis 4(DOCK4),and to establish DOCK4 stably over-expressing Neuro-2a cells.Methods:The DOCK4 sequence was searched in the National Center for Biotechnology Information(NCBI)and primers were designed and synthesized;polymerase chain reaction(PCR)method was used to amplify the DOCK4 gene sequences.After digestion with BamH Ⅰ and Age Ⅰ restriction endonucleases,the DOCK4 gene sequences were ligated with the digested lentiviral vector GV492 to construct the GV492-DOCK4 over-expression recombinant plasmid.The positive clones with a similar length to the target gene fragment were screened and identified by PCR method.The GV492-control plasmid and GV492-DOCK4 over-expression recombinant plasmid were transfected into the HEK293T cells,and the lentivirus was collected and titered 48 h after transfection.The Neuro-2a cells were divided into GV492-control group and GV492-DOCK4 group,and the cells were infected with GV492-control lentivirus and GV492-DOCK4 over-expression lentivirus,respectively,and the multiplicity of infection(MOI)was 100.After 72 h of infection,the successfully infected Neuro-2a cells were screened by using puromycin(10 mg·L-1).The growth status of Neuro-2a cells and the expression of green fluorescent protein in various groups were observed under fluorescence microscope.Real-time quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of DOCK4 mRNA and DOCK4 protein in the Neuro-2a cells in various groups.Results:The PCR results showed that the gene fragment length of the GV492-DOCK4 over-expression recombinant plasmid was approximately 691 bp.The sequencing results showed that the gene sequence of the GV492-DOCK4 over-expression recombinant plasmid was consistent with the designed over-expression sequence of DOCK4.The titers of the lentiviruses in GV492-control group and GV492-DOCK4 over-expression group were 2.5×108 TU·mL-1 and 2.5×108 TU·mL-1,respectively.The fluorescence microscope observation results showed that Neuro-2a cells in various groups grew well and expressed green fluorescent protein.The RT-qPCR results showed that compared with GV492-control group,the expression level of DOCK4 mRNA in the Neuro-2a cells in GV492-DOCK4 group was significantly increased(P＜0.01).The Western blotting results showed the specific bands near the relative molecular mass of 225 000 in various groups.Compared with GV492-control group,the expression level of DOCK4 protein in the Neuro-2a cells in GV492-DOCK4 group was significantly increased(P＜0.01).Conclusion:This study successfully constructs the DOCK4 over-expression lentiviral vector and establishes the Neuro-2a cells stably over-expressing DOCK4.
