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The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing arecombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 genefrom the human placent by RT-PCR.TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vectorby means of gene cloning to construct pcDNA3.1 recombinant vector.Human breast cancer cell lineMDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent.Thenthe expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined.The correctconstruction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis,PCR amplication andnucleotide sequencing.Western blotting showed that the transfected cells were able to express TIMP-3,indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructedsuccessfully.Our experiments further indicated that the potential of metastasis was significantly reduced forthe transfected cell line MDA-MB-453.Cellular & Molecular Immunology.2004;1(4):308-310.
The present study was aimed at studying the gene for TIMP-3, a mammalian tissue inhibitor, by constructing are recombinant eukaryotic cell vector for gene therapy in human breast cancer. We obtained the TIMP-3 gene from the human placent by RT-PCR. TMP- 3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector. Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent. The expression of the TIMP-3 and the effect on the metastasis of MDA-MB-453 were. The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis, PCR and nucleotide sequencing. Western blotting showed that the transfected cells were able to express TIMP -3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructedsuccessfully.Our experiments further indicated that the potential of metastasis was significantly reduced f orthe transfected cell line MDA-MB-453. Cellular & Molecular Immunology. 2004; 1 (4): 308-310.