Stable transfection of rat preproinsulin ii gene into rat hematopoietic stem cells via recombinant adeno-associated virus

摘要

We investigated the ability of recombinant adeno-associated virus (rAAV), to mediate the transfer of rat preproinsulin II (rI_2) gene into rat hematopoietic stem cells in vitro and expression of rI_2 following intra-venous (i. v.) injection of infected stem cells into syngeneic rats. The pLP-1 recombinant plasmid containing rI_2 was engineered as follows: rI_2 with RSV-promoter was released from pBC12BI(ATCC), purified, and inserted into BamH1 site of rAAV vector plasmid pWP-19. Plasmid pLP-1, together with pAAVAD (Somatix Corp.), was used to co-transfect cell line 293 (ATCC). The rAAV genome was rescued using helper adenovirus and packaged into mature rAAV virions (vLP-1). Bone-marrow from female Wistar-Furth rats was enriched for stem cells by using plastic adherence and negative selection with monoclonal anti-rat CD3 and CD45RA to deplete T and B cells. The remaining cells were exposed to vLP-1 (moi=50: 1) for 2 hours. Transfection was confirmed by PCR of neomycin resistance gene (neo~R) after 8 days in culture. For in vivo studies, ten million exposed stem cells were injected i. v. into syngeneic rats (n=3). The results represent 3 identical experiments. Expression of neo~R and rI_2 was analyzed by RT-PCR. At week 1, neo~R and rI_2 were expressed in liver, spleen, thymus, peripheral blood lymphocytes and bone marrow. At week 2, neo~R was expressed in spleen and brain, while at week 6, thymus, lymph nodes, bone-marrow, liver, spleen, and brain expressed neo~R. rI_2 was not detected after week 1. In summary, we showed that rAAV was efficient for transferring neo~R and rI_2 into rat hematopoietic stem cells.