To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly ...To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.展开更多
The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the go...The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.展开更多
In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected...In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein （GFP） positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.展开更多
Retinoids mediate their actions via RARs(retinoic acid receptors)and RXRs(retinoid X receptors).Each classes of these nuclear retinoid receptor is further subdiviede into three species namelyα,βand γ.Recent studies...Retinoids mediate their actions via RARs(retinoic acid receptors)and RXRs(retinoid X receptors).Each classes of these nuclear retinoid receptor is further subdiviede into three species namelyα,βand γ.Recent studies demonstrate that ER-positive HBC cell lines are sensitive and ER-negative cell lines are resistant to growth inhibitory effects of retinoic acid(RA). In this study we look at the expresion of RARs and RXRs in 6 HBC cell lines, we found only RARαmRNA level was strong correlated with ER-status. To further inestigate the major role of RARαin retinoidmediated inhibition of growth, we transfected RARαcDNA in two RA-resistant ER-negative HBC cell lines.Analyses of different clonal populations of RARα transfectants from each cell line revealed growth inhibition by retinoids. Our results demonstrates that RARα Plays a major role in mediating retinoids inhibition of growth in HBC cells and adequate levels are required for such actions.展开更多
Breast cancer containing estrogen receptors (ER ) are responslve to antiestrogen treatment and have a better prognosis compared with ER-negative tumors. The loss of estrogen receptors appears to be associated with a p...Breast cancer containing estrogen receptors (ER ) are responslve to antiestrogen treatment and have a better prognosis compared with ER-negative tumors. The loss of estrogen receptors appears to be associated with a progression to less clifferentiated cells. We transfected the human ER into the ER-negative breast cancer cell line MDA MD 231 cells. We found that expresslon of adequate ER is strong associated with the ability of human breast cancer cell growth inhibition and progression. Compared with nontransfected or mock-trans fected cells, ER-transfected cells exhibited growth slower, forming smaller colonles in soft agar and growth inhibited by estrogen and tamoxifen. Therefore reactivation or transfection of the estrogen receptor gene can be considered as therapeutic approaches to hormone-independent breast cancer.展开更多
文摘To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.
基金Supported by Key Teachers Foundation of Education Office of Heilongjiang Province, 2005 (1055G005)
文摘The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.
文摘In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein （GFP） positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.
文摘Retinoids mediate their actions via RARs(retinoic acid receptors)and RXRs(retinoid X receptors).Each classes of these nuclear retinoid receptor is further subdiviede into three species namelyα,βand γ.Recent studies demonstrate that ER-positive HBC cell lines are sensitive and ER-negative cell lines are resistant to growth inhibitory effects of retinoic acid(RA). In this study we look at the expresion of RARs and RXRs in 6 HBC cell lines, we found only RARαmRNA level was strong correlated with ER-status. To further inestigate the major role of RARαin retinoidmediated inhibition of growth, we transfected RARαcDNA in two RA-resistant ER-negative HBC cell lines.Analyses of different clonal populations of RARα transfectants from each cell line revealed growth inhibition by retinoids. Our results demonstrates that RARα Plays a major role in mediating retinoids inhibition of growth in HBC cells and adequate levels are required for such actions.
文摘Breast cancer containing estrogen receptors (ER ) are responslve to antiestrogen treatment and have a better prognosis compared with ER-negative tumors. The loss of estrogen receptors appears to be associated with a progression to less clifferentiated cells. We transfected the human ER into the ER-negative breast cancer cell line MDA MD 231 cells. We found that expresslon of adequate ER is strong associated with the ability of human breast cancer cell growth inhibition and progression. Compared with nontransfected or mock-trans fected cells, ER-transfected cells exhibited growth slower, forming smaller colonles in soft agar and growth inhibited by estrogen and tamoxifen. Therefore reactivation or transfection of the estrogen receptor gene can be considered as therapeutic approaches to hormone-independent breast cancer.
