Investigating the utility of the P1 Artificial Chromosome shuttle vector pJCPAC-Mam2 for gene therapy studies
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Abstract
The goal of gene therapy is to deliver as many copies of a functional gene to a patient that lacks this protein. There have been some recent successes in rescuing some types of blindness and red blood cell disorders. Current lentivirus-based vectors can only accommodate around 10 kilobases of foreign DNA. Also, integration of the vector is random, so transcription of a given gene can vary several-fold depending on the chromosomal architecture where insertion occurs. We constructed a P1 Artificial Chromosome (PAC) shuttle vector that has a greater than 150 kilobase insert size limit and remains as an episome in human cells. We previously demonstrated that a PAC clone containing the p53 gene was transcribed and translated when transiently introduced into p53 homozygous null Saos-2 human osteosarcoma cells. Furthermore, the apoptotic pathway was triggered in some of the cells. A Saos-2 cell line that overexpresses the bcl-2 cDNA was generated using G418 selection so that the effects of different lipofection reagents on copy number and transcription and translation levels of p53 could be studied. EndoFectin Max resulted in a several-fold higher transfection efficiency than Lipofectamine 3000. Seven stable cell lines were generated from each transfection procedure using puromycin and G418 selection. Unfortunately, only 7 of 13 cell lines contained measurable levels of p53 cDNA when total RNA was subjected to reverse transcriptase polymerase chain reaction (RT-PCR). A PAC clone containing the green fluorescent gene from Pontellina plumata that is expressed from the strong cytomegalovirus promoter is being constructed to investigate whether Lipofectamine 3000 or EndoFectin Max maximizes transfection efficiency, plasmid copy number, and transcription and translation levels in the continuous cell lines HEK293, KG1 and Saos-2.
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