Construction of eukaryotic expression vector pcDNA3.1/Hygro(+)-Lefty A and establishment of cell line stably expressing Lefty A / 中国组织工程研究

ABSTRACT

BACKGROUND: Lefty gene could attenuate renal fibrosis by inhibiting transforming growth factor-?1 (TGF-?1) signal transduction. OBJECTIVE: To construct eukaryotic expression vector pcDNA3.1/Hygro (+)-Lefty A and to establish a cell line that can stably express Lefty A in order to verify the effects on inhibiting renal fibrosis. DESIGN, TIME AND SETTING: Gene cloning and single sample experiment was performed at Key Laboratory of Renmin Hospital of Wuhan University from April to July 2008. MATERIALS: Human renal tubule epithelial cells (HK-2 cell) were purchased from Peking Union Medical College, pcDNA3.1/Hygro(+) was given by professor Siamak Tabibzadeh in Stony University as a gift. Clone vector of Lefty A (pCMV-SPORT6, GenBank Accession is BC035718) was purchased from Wuhan Genesil Biotechnology Co., Ltd. METHODS: The coding sequence of Lefty A was gained from pCMV-SPORT6 through PCR and then was used to construct eukaryotic expression vector in pcDNA3.1/Hygro(+). The recombinant pcDNA3.1/Hygro (+)-Lefty A was transfected into human kidney tubular epithelial-2 cells (HK-2 cells) through LipofectamineTM 2000. After screening culture by Hygromycin, stable transfected cell line was established and the expression of Lefty A was identified by Hygromycin. MAIN OUTCOME MEASURES: The results identified by electrophoresis of PCR products, the results identified by recombinant enzyme incision, the results of recombinant gene sequence, the filtration of Lefty A stable expression cell line and the expression of mRNA Lefty A in cells. RESULTS: The coding sequence of Lefty A was gained from pCMV-SPORT6 by PCR using specific primers, and was confirmed by 10 g/L gel electrophoresis. There was a little bigger than 1.0 kb specific PCR band at the 1.0 kb of the bands. The results were consistent with 1.1 kb location. The recombinant plasmid was confirmed by Hind III, BamH I single enzyme digestion respectively, and each segment was 6.7kb. 5.6 kb and 1.1 kb segments were gained respectively after double digestion, and consistent with the segment of pcDNA3.1/Hygro(+) and Lefty A. The positive recombinant plasmids were sequenced for bioinformatics analysis. The results were consistent with expectation. The high expression of Lefty A mRNA in transfected tubular epithelial cell line was found. CONCLUSION: The eukaryotic expression vectors of pcDNA3.1/Hygro(+)-Lefty A were constructed successfully and the gene of Lefty A was stably expressed in stable transfected HK-2 cell line.