Construction of the full length and N-terminal fragment of serum response factor over-expressing lentiviral plasmid and its impacts on cardiac stem cell differentiation
10.13315/j.cnki.cjcep.2017.10.011
- VernacularTitle:血清反应因子全长及N端片段过表达慢病毒载体的构建及其对心脏干细胞分化的影响
- Author:
Hong LEI
1
;
Ke-Ke WANG
;
Yan-Ping HA
;
Ya-Nan JIA
;
Ru-Jia LI
;
Zhi-Hua SHEN
;
Jun-Li GUO
;
Wei JIE
Author Information
1. 广东医科大学基础医学院病理学系
- Keywords:
serum response factor;
N-terminal fragment of serum response factor;
plasmid construction;
cardiac stem cell;
cell differentiation
- From:
Chinese Journal of Clinical and Experimental Pathology
2017;33(10):1109-1115
- CountryChina
- Language:Chinese
-
Abstract:
Purpose To analyze the effects of full length and N-terminal fragment of serum response factor (SRF-Full and SRF-N) on TGF-β1-induced differentiation in c-Kit + cardiac stem cells (CSC).Methods Rat SRF-Full and SRF-N (1-254 aa) coding sequences were obtained from cDNA library and cloned into the linearized lentviral vector GV358 (Ubi-MCS3FLAG-SV40-EGFP-IRES-puromycin) to generate the recombinant vectors,and then positive clones were selected and sequenced after transducing the competent cells with recombinant vectors.The recombinant lentvirus were packaged through transfecting the HEK293T cells with SRF-Full,SRF-N overexpressing plasmids and viral packaging plasmids.Neonatal SD rat cKit + CSCs were isolated via magnetic activated cell sorting,and TGF-β1-induced differentiation in SRF-Full and SRF-N overexpression virus-infected CSCs was assessed by quantitative PCR.Results SRF-Full and SRF-N coding sequences were successfully obtained and properly cloned into the linearized GV358.The positive clones were selected and further confirmed by sequencing.With the help of packaging plasmids,the SRFFull and SRF-N overexpressing plasmids-transfected HEK293T cells successfully produced the lentiviral particles with the titer of 2 × 108 TU/mL,and the SRF-Full-Flag and SRF-N-Flag fusion protein were detected by Western blot in virus-infected HEK293T cells.Addition of TGF-β1 significantly induced upregulated mRNAs in cardiomyocyte markers (Nkx2.5,Gata4,cTnI) and smooth muscle cell marker (SM22α) but not the epithelia cell marker (vWF) in CSCs.Overexpression of SRF-Full facilitated TGF-β1-triggered cardiomyocyte differentiation.However,SRF-N exerted anti-differentiation effects in TGF-β1-treated cells.Conclusion The SRF-Full and SRF-N overexpressing recombinant lentiviral vectors are successfully constructed.SRF-Full facilitates while SRF-N suppresses TGF-β1-induced cardiomyocyte differentiation in c-Kit + CSCs.
