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Retroviral vector mediated coexpression of MIPlα and B71 in rat breast cancer cells
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Abstract:
To establish a rat breast cancer cell line stably coexpressing mouse MIP1α and B71，and to assay its in vitro biological activity．Methods: mMIP1αcDNA was cloned into retrovirus vector pBabe puro to construct pBahe puro/mMIP1α, then pBabe puro/mMIP1α was used to transfect packaging cells and the antipuromycin PA317 packaging cells were proliferated. Meanwhile, pLXSN/mB71 was constructed and the antiG418 cells were proliferated. Finally, the two supernatants were used to infect SHZ88 together and the cotransfected cells were selected with 1 mg/ml G418 and 2 μg/ml puromycin together. Expression of mM1Plα mRNA and protein in SHZ88 and SHZ88/mB71+mM1P1α cells were analyzed by RTPCR and immunocytochemistry, respectively. Expression of mB71mRNA and protein was analyzed by RTPCR and flow cytometry, respectively. Lymphocyte proliferation activity of SHZ88/B71+mM1P1α was detected by MTT assay; chemotactic activity of MIP1α was measured by chemotaxis assay. Results：A titer of 4.6×107 CFU/L was obtained after transfection with recombinant retroviral vector. The growth curve of cells showed that the recombinant retroviral had no effect on the growth of rat breast cancer cells. There was expression of B71 and MIP1αmRNA/protein in SHZ88/mMIP1α+mB71 cells. The proliferation indices(PI) in mMIP1α+mB71 group (1.95±0.31) was significantly higher than that in SHZ88/PLXSN group (0.76±0.25)(P<0.01). Chemotaxis assay showed that chemotactic activity of lymphocytes in mMIP1α+mB71 group (3.88±0.33) was significantly higher than that in SHZ88/pBabe puro group (0.99±0.19)(P<0.001).Conclusion: A new rat breast cancer cel1 line SHZ88/mMIP1α+mB71 has been established, which can stably coexpress MIP1α and B71 gene and possesses biologic activity in vitro.
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