Objective To transfect a recombinant pcDNA3.1-DCN into HepG2 cells and detect the expressions of decorin(DCN) and p21WAF1/CIP1 so as to investigate the mechanism of tumor suppression of DCN.Methods HepG2 cells were divided into pcDNA3.1-dec-HepG2 group(transfected group) and pcDNA3.1-HepG2 group(control group).After the recombinant pcDNA3.1-DCN and pcDNA3.1 were transfected into HepG2 hepatoma cells by liposome,the stably transfected cell lines were established using G418 screening.RT-PCR method was used to detect the expressions of DCN and p21WAF1/ CIP1 mRNA;Western blotting method was used to detect the expressions of DCN and p21WAF1/CIP1 protein;immunohistochemistry was performed to detect the expression of DCN protein.Results Compared with control group,the expressions of DCN and p21WAF1/CIP1 mRNA were significantly increased in transfected group by RT-PCR(P

Objective To construct the vector for ?-galactosidase (?-gal) gene expression regulation with tetracycline-regulated gene expression system,and to control ?-galactosidase gene expression in hepG2 cells with the vector.So that offer a experimental base for regulating gene expression for liver cancer gene therpy with this system.Methods The primers for explanding two tetracycline operator (TetO2) gene according to the gene nucleotide sequences of TetO2 gene and pcDNA3.1 vector were designed.TetO2 gene was amplified by PCR with pcDNA3.1 plasmid as template.The TetO2 PCR products were cloned into pcDNA3.1 and the vector was named as pcDNA3.1-TetO2.The pcDNA3.1-Teto2 with TetO2 PCR products sequence was analyzed by ABI3130 sequencing analysis.Then ?-gal gene was cloned into pcDNA3.1-TetO2,the vector was named as pcDNA3.1-TetO2-?-gal.The pcDNA6/TR plasmid vector with tetracycline repressor(TR) was transfected into HepG2 cells by Lipotap,and the stable transfection cells were screened by blasticidin.TR gene expression was detected by RT-PCR in HepG-2 cells with and without pcDNA6/TR plasmid transfection.The pcDNA3.1-TetO2-?-gal plasmid vector was transfected into HepG2 cells with and without pcDNA6/TR plasmid transfection,and 3 to 4 d later, these transfected gene cells were treated with doxycline(4 mg?L-1).After 48 h,?-gal gene expression was detected with ?-gal cell staining.Results The pcDNA3.1-TetO2 sequencing analysis results showed that a cassette was made for a cytomegalovirus-type 2 tetracycline operator (TetO2)-TetO2 promoter in pcDNA3.1-Teto2.The double digestion reslut of pcDNA3.1-TetO2-?-gal plasmid vector demonstrated that ?-gal gene was successfully cloned into pcDNA3.1-TetO2 vector .The RT-PCR result of TR gene showed TR gene could be expressed in HepG2 cells with pcDNA6/TR plasmid transfection and TR gene didn’t express in HepG2 cells without pcDNA6/TR plasmid transfection.?-gal gene expressed in HepG2 cells without pcDNA6/TR plasmid transfection,but didn’t express in HepG2 cells with pcDNA6/TR plasmid transfection.However,?-gal gene expression could be induced with doxycline in HepG2 cells with pcDNA6/TR plasmid transfection.Conclusion The tetracycline-regulated gene expression system vector for regulating ?-gal gene expression is successfully constructed and the vector system can control ?-gal gene expression in HepG2 cells.

Enhanced green fluorescent protein( EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin.Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo - peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his -EGFP B in frame with EGFP, myc and 6 × His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6 × his did not interfere each other and octo- peptide linker owned certain flexibility. The results suggest that pcDNA6/myc-his-EGFP B may be useful as a genetic tool for mammalian cells and a vector for gene therapy.

DNA ; isolation & purification ; Formaldehyde ; Genes, erbB-1 ; genetics ; Genetic Techniques ; Humans ; Mutation ; Paraffin ; Tissue Fixation