Construction of a Tc1-like transposon Sleeping Beauty-based gene transfer plasmid vector for generation of stable transgenic mammalian cell clones.
We have constructed a single plasmid-, Tc1-like transposon-based gene transfer vector, termed the Prince Charming vector (pPC). The pPC vector was constructed by ligating the CMV-driven "Sleeping Beauty" transposase gene downstream to the Tc1-like transposon inverted repeat (IR) elements and by inserting the RSV promoter (to drive expression of the gene-of-interest) along with a multiple cloning site (MCS), a polyadenylation signal, and the SV40 promoter-driven neomycin gene, at a site flanked by the transposon IR elements. To assess the utility of the pPC vector, we cloned a red fluorescent protein (RFP) gene into the pPC vector at the MCS and transfected human TE85 osteosarcoma cells with the pPC-RFP expression vector using Effectene. Stable transgenic cell clones expressing RFP were selected with G418 sulfate and individual clones were isolated. After 4 weeks of clonal isolation and expansion, 99% of cells in each randomly selected clone expressed RFP strongly. Aliquots of each clone were then maintained in either the presence or the absence of G418 sulfate and were passaged weekly. Even after 6 months in culture in the absence of G418 sulfate, approximately 90% of the cells in each clone still maintained a strong expression level of RFP, indicating that these transgenic cell clones were stable and that the clonal stability of these clones did not require a constant selection pressure. In conclusion, we have developed a single plasmid-, Tc1-like transposon-based gene transfer vector that can be used to generate stable transgenic mammalian cell clones.
Harris
Joseph W
JW
Musculoskeletal Disease Center, Jerry L. Pettis Memorial VA Medical Center, Loma Linda, CA 92357, USA.
Strong
Donna D
DD
Amoui
Mehran
M
Baylink
David J
DJ
Lau
K-H William
KH
D007446:Chromosome Inversion; D002999:Clone Cells / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional / Q000235:genetics; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection / Q000379:methods; D014170:Transformation, Genetic / Q000235:genetics*; D019895:Transposases / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000097573:Red Fluorescent Protein
D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D019895:Transposases; D000097573:Red Fluorescent Protein