| OBJECTIVE: It is to investigate cadherin-5 N-terminal CED-4 gene transfer of human breast cancer cell line MDA-MB-435 and the effect of the transfected CED-4 gene on the proliferation of MDA-MB-435 cells. METHODS: ①Competent E. coli (strain XL1-Blue) was prepared using calcium chloroid. ② Transformation of the competent bacteria: We transformed the competent bacteria with the plasmids pVSV-G, pGAG-POL, pMSCV and CED-4pMSCV containing CED-4 gene. The appropriate volume of the transformed bacteria was transfered and spreaded onto agar LB medium to select the ampicillin-resistant colonies. ③ Extraction and restriction endonuclease analysis of the plasmids: The plasmids pVSV-G, pGAG-POL, pMSCV, CED-4pMSCV were extracted from the transformed bacteria with Qiagen plasmid purification kit, and they were cut with restriction endonucleases BspH ?,Bgl II,Afl II ,EcoR? and BglП, respectively. The plasmids cut by these restriction endonucleases were examined using agar electrophoresis. ④ Packaging of the retrovirus in 293 cells: Before package of the retrovirus, 293 cells were screened with G418. 293 cells were respectively transfered by the plasmids pVSV-G, pGAG-POL, CED-4pMSCV and the plasmids pVSV-G, pGAG-POL, pMSCV plus lipofectamine. The recombinant retrovirus containing CED-4 target gene and the backbone recombinant retrovirus were packaged by 293 cells. ⑤ Gene transfection of MDA-MB-435 cells: The experiment group and the experiment control group cells were transfected with the recombinant retrovirus respectively. EGFP(enhanced green fluorescent protein, EGFP) gene inserted in pMSCV is the positive marker of the positive transfected cells. The green fluorescent of the transfected cells was detected after transfection of 3 days and the morphology of the transfected cells was intensively observed after transfection of 2 weeks. ⑥ Experiment of the proliferation of cells: The quantities of absorbance of the 3 group cells were measured by MTT assay. ⑦ RT-PCR: The priners were designed. The total RNA of three groups was extracted. The integrin β1 expression of 3 groups was investigated with RT-PCR and agar electrophoresis method. RESULTS: ①Ampicillin resistant tranformed bacteria colonies growth: After transformation with pMSCV , CED-4pMSCV, pGAG-POL and pVSV-G, 76,20,28,30 colonies were observed on the plate containing ampicillin, and the colonies were not found in the negative control groups. ② The normal size of the fragments from the plasmids cut by endonucleases: The results of restriction endonuclease analysis and agar electrophoresis indicated that size of plasmids pMSCV, CED-4pMSCV, pGAG-POL and pVSV-G was normal. ③The recombinant retrovirus packaged by 293 cells: Green fluorescent was detected in 293 cells after transfection of 24 hours. NIH 3T3 cells were transfected with the recombinant retrovirus after the media containing the recombinant retrovirus were concentrated. Green fluorescent was detected in NIH 3T3 cells 3 days later. ④ High-efficient genetic transduction of MDA-MB-435 cells with the recombined retrovirus: Green fluorescent was detected in the transfected MDA-MB-435 cells after transfection of 3 days.The ratio of EGFP positive MDA-MB-435 cells was up to 97.54%. ⑤ The transfected CED-4 gene inhibition of the MDA-MB-435 cell proliferation: The quantities of absorbance of the experiment group was significantly lower than that of the experiment control group and the blank control groups(p<0.05), while there was no significant difference between the experiment control group and the blank control group(p>0.05). The cells of the experiment group began to shrink, and looked like pearls in line after transfection of 2 weeks. The gap among the cells became wider. Then the cells detached from the plate, suspended in media and died after transfection of 4 weeks. ⑥ CED-4 gene upregulation of the integrin β1 expression of the transfected MDA-MB-435 cells: The results of RT-PCR demonstrated that the expression of integrin β1 of the transfected MDA-MB-435 cells was upregul...
|
PDF Full Text Request