Construction of pDsVEGF165Red1-N1 and pIRES2-BMP2-EGFP and their co-expression using RFP and EGFP as reporter gene in HEK 293-T cells

Haibo ZOU; Hong AN; Dianming JIANG

ABSTRACT

BACKGROUND:

It has been proved that vascular endothelial growth factor 165(VEGF165) and bone morphogenetic protein 2(BMP2) can accelerate the vascularization synergistically.

OBJECTIVE:

To construct the vectors, pDsVEGF165Red1-N1 and pIRES2-BMP2-enhanced green fluorescent protein(EGFP) followed by co-transfected into HEK 293-T cells,and study their expression and location of VEGF165and BMP2 in the cells.

DESIGN:

A randomized and controlled experiment.

SETTING:

National Key Laboratory,Institute of Surgery Research,Daping Hospital,Third Military Medical University.MATERIALS The experiment was conducted at National Key Laboratory,Institute of Surgery Research,Daping Hospital,Third Military Medical University from September 2002 to March 2004. pcDNA3.1\BMP2 ( gift of Dr.Bostrom, UCLA School of Mediine, Los Angeles,USA).pDsRed1-N1(gift of Pro. Roger Y.Tisen,University of California,San Diego,USA). pUC18/VEGF165,293-T cells(preserved by our Laboratory).

METHODS:

According to the nucleotide sequence of hVEGF165, the primers were designed.The hVEGF165 gene without stop codon was amplified by polymerase chain reaction (PCR).The fragment digested was cloned into the expression vector pDsRed1-N1.Meantime,the pIRES2-BMP2-EGFP expression vector was constructed.The two plasmids were co-transfected into HEK 293-T cells.The co-expression and distribution of the VEGF165and BMP2 were observed with confocal laser microscopy (CLSM) and detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blotting.MAIN OUTCOME

MEASURES:

Identification of the recombinant plasmids and the expressing mRNA and protein in 293-T cells.

RESULTS:

The recombinant plasmids were verified correct construction by restriction enzyme analysis, PCR and sequencing. The two genes which were co-transfected could express in HEK 293-T cells.The co-expression of the report genes,RFP and EGFP, were found over the cytoplasm and in the nuclei by CLSM.

CONCLUSION:

Two report gene expression vectors contain VEGF165 and BMP2 have been constructed successfully, which can be co-expressed in HEK 293-T cells. Thus, they can provide important and convenient tool to study intracellular interaction of VEGF165 and BMP2.