Recombinant adeno-associated virus-mediated gene transfer into human leukemia cell lines.
Adeno-associated virus (AAV)-based vector is a promising gene transfer vehicle by virtue of the characteristics of wild-type AAV:tropism to a wide range of human tissues and locus-specific integration at chromosome 19q13.3. To elucidate the nature of the recombinant AAV (rAAV), transduction of neomycin phosphotransferase enzyme gene (NeoR gene) into seven human leukemia cell lines was performed. Transduction efficiencies were assessed by colony formation assay and limiting dilution assay. The results suggested that both assays are comparable. Transduction efficiencies of the NeoR gene into K-562, MEG-O1, Raji, MOLT-3, HL-60, U937 and NKM-1 at a multiplicity of infection (MOI) of 0.1 were 0.27, 0.25, 0.015, 0.009, < 0.0025 and < 0.0025%, respectively. After purification and concentration of rAAV, 27% efficiency was observed in K562 at an MOI of 7 and a linear relationship between MOI and efficiency was confirmed, suggesting that this system may be useful for gene transduction into leukemia cells. Integration of the NeoR gene into the host genome was detected by Southern blotting analysis, which showed various sizes of digested fragments. A fluorescent in situ hybridization (FISH) study was carried out on 11 clones, in all of which the NeoR gene was integrated out of chromosome 19q13.3. In five of the clones, whole chromosome painting probes revealed that the integration sites were chromosomes 1q, 2q, 2q, 11p, 12p and 13q.
Itou
T
T
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Miyamura
K
K
Abe
A
A
Emi
N
N
Tanimoto
M
M
Terasaki
H
H
Shimadzu
M
M
Saito
H
H
D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D006801:Humans; D007938:Leukemia / Q000235:genetics*; D011995:Recombination, Genetic; D014407:Tumor Cells, Cultured