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Aim:To construct a recombinant retrovirus vector carrying human promyelocyticleukemia(PML)cDNA and identify its expression and biology role in bladdercancer UM-UC-2 cells for future gene therapy.Methods:PML full-length cDNAwas inserted into the EcoR I and BamH I site of pLXSN vector containing the longterminal repeat(LTR)promoter.The vector was identified by restriction enzymedigestion and then transfected into PA317 packaging cell line by calcium phos-phate coprecipitation.PML cDNA was detected by polymerase chain reaction(PCR)and the protein was identified by laser confocal microscopy and Westernblot in bladder cancer cells,respectively.The morphology was observed byinverted phase contrast microscope,and MTT assay determined growth curve ofthe bladder cancer cells.Results:Restriction enzyme digestion proved that a 2.1kb PML cDNA was inserted into the pLXSN vector.PCR assay demonstrated that304 bp fragments were found in UM-UC-2/pLPMLSN transfects.Laser confocalmicroscopy showed speck dots fluorescence in the UM-UC2/pLPMLSN nucleus.A 90 kD specific brand was found by Western blot.MTT assay demonstrated theUM-UC-2/pLPMLSN bladder cancer growth inhibition.Conclusion:The retroviruspLPMLSN vector was successfully constructed and could generate high effec-tive expression of human PML in bladder cancer cell UM-UC-2,suggesting thatPML recombinant retrovirus have potential utility in the gene therapy for bladdercancer.
Aim: To construct a recombinant retrovirus vector carrying human promyelocyticleukemia (PML) cDNA and identify its expression and biology role in bladdercancer UM-UC-2 cells for future gene therapy. Methods: PML full-length cDNAwas inserted into the EcoR I and BamH I site of pLXSN vector containing the longterminal repeat (LTR) promoter. The vector was identified by restriction enzyme digestion and then transfected into PA317 packaging line by calcium phos-phate coprecipitation. PML cDNA was detected by polymerase chain reaction (PCR) and the protein was identified by laser confocal microscopy and Western blot in bladder cancer cells, respectively. The morphology was observed by inverted phase contrast microscope, and MTT assay determined growth curve of the bladder cancer cells. Results: Restriction enzyme digestion demonstrated that a 2.1 kb PML cDNA was inserted into the pLXSN vector.PCR assay for that 304 bp fragments were found in UM-UC-2 / pLPMLSN transfects.Laser confocalmicroscopy showed speck dot fluorescence in the UM-UC2 / pLPMLSN nucleus. A 90 kD specific brand was found by Western blot. MTT assay demonstrated theUM-UC-2 / pLPMLSN bladder cancer growth inhibition. Confluence: The retrovirus pLPMLSN vector was successfully constructed and could generate high effec -tive expression of human PML in bladder cancer cell UM-UC-2, suggesting that PML recombinant retrovirus have potential utility in the gene therapy for bladder cancer.