Stable transfection of rat preporinsulin II gene into rat hematopoietic stem cells via recombinant adeno-associated virus.
We investigated the ability of recombinant adeno-associated virus (rAAV), to mediate the transfer of rat preproinsulin II (rI2) gene into rat hematopoietic stem cells in vitro and expression of rI2 following intra-venous (i.v.) injection of infected stem cells into syngeneic rats. The pLP-1 recombinant plasmid containing rI2 was engineered as follows: rI2 with RSV-promoter was released from pBC 12BI (ATCC), purified, and inserted into BamH1 site of rAAV vector plasmid pWP-19. Plasmid pLP-1, together with pAAV¿AD (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 (neoR) 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 neoR and rI2 was analyzed by RT-PCR. At week 1, neoR and rI2 were expressed in liver, spleen, thymus, peripheral blood lymphocytes and bone marrow. At week 2, neoR was expressed in spleen and brain, while at week 6, thymus, lymph nodes, bone-marrow, liver, spleen, and brain expressed neoR. rI2 was not detected after week 1. In summary, we showed that rAAV was efficient for transferring neoR and rI2 into rat hematopoietic stem cells.
Shah
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Department of Surgery, Indiana University School of Medicine, Indianapolis, USA.
Jindal
R M
RM
D000818:Animals; D015139:Blotting, Southern; D017931:DNA Primers / Q000032:analysis; D004279:DNA, Viral / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000821:virology; D006801:Humans; D007328:Insulin / Q000096:biosynthesis; D011384:Proinsulin / Q000096:biosynthesis / Q000235:genetics*; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D011920:Rats, Inbred WF; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004279:DNA, Viral; D007328:Insulin; D011498:Protein Precursors; C013737:preproinsulin; D011384:Proinsulin