Studies On Transferring Novel Gene Into Human Hematopoietic Cells By Adeno-associated Virus

Transfer of the human multi-drug resistance 1 (MDR1) gene to hematopoietic stem cells offers an approach to overcome the myelosuppression caused by a number of anti-neoplastic drugs. Adeno- associated virus is a human parvovirus with many interesting features in its life cycle that can be exploited in human gene therapy~ So it was chosen as a vector in hopes of developing a safe and efficient viral vector for human gene therapy. This study was designed to construct the recombinant AAV-MDR 1 vector and to package the AAV-MDR1 vector into viruses, then transfer mdrl into human hematopoietic cells of peripheral blood. By using adeno-associated virus vectors, we have transferred human mdr-1 gene into human hematopoietic cells and investigated the characteristics of human hematopoietic cells modified with mdr-l gene in drug resistance.The adeno-associated vector , containing human mdrl eDNA ,was transfected by lipofection into NIH3T3 .The transfected NIH3T3 cells were then selected in the culture system containing colchicine at a concertration of 6Onglml. By co-transfecting the helper plasmid, pAAV/Ad(AdS), and the vector ,rAAVMDRI containing the mdrl eDNA into the packaging cell line 293 cell,in the presence of adenovirus (Ad5) concentrated crude virus stocks were prepared from infected 293 cells by three cycles of freeze-thawing Then Nll-13T3 cells were infected by the crude exact of the infected 293 cells. PCR analysis confirmed that mdrl eDNA had been successfully transferred into NIH3T3 cells.An assay of MTT proved that NIH3T3 cells modified by mdrl gene had resistance to colchicine. But in our study , the resistance of hematopoietic cells was not increased parently when the cells were infected by the crude virus stocks. S Green fluorescent proteins (GFPs) aie presently attracting tremendous interest as the first general method to created strong visible fluorescence by purely molecular biological means. So far, they have been used as reporters of gene expression. In this study , the GFP gene was transferred into the human hematopoietic cells by AAV vectors. It was about 30% of the hematopoietic cells that expressed the green fluorescent proteins. It is conducted that the AAV vector can successfully transfer the foreign gene into the human hematopoietic cells. The cells modified with mdrl gene has increased the resistance to drugs, but there are a few problems in transferring mdrl gene into human hematopoietic stem cells..