Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector.
Expression of multiple genes from the same target cell is required in several technological and therapeutic applications such as quantitative measurements of promoter activity or in vivo tracking of stem cells. In spite of such need, reaching independent and high-level dual-gene expression cannot be reliably accomplished by current gene transfer vehicles. To address this issue, we designed a lentiviral vector carrying two transcriptional units separated by polyadenylation, terminator and insulator sequences. With this design, the expression level of both genes was as high as that yielded from lentiviral vectors containing only a single transcriptional unit. Similar results were observed with several promoters and cell types including epidermal keratinocytes, bone marrow mesenchymal stem cells and hair follicle stem cells. Notably, we demonstrated quantitative dynamic monitoring of gene expression in primary cells with no need for selection protocols suggesting that this optimized lentivirus may be useful in high-throughput gene expression profiling studies.
Bioengineering Laboratory, Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Amherst, NY 14260, USA.
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D000328:Adult; D053687:Adult Stem Cells / Q000166:cytology / Q000378:metabolism; D001854:Bone Marrow Cells; D002294:Carcinoma, Squamous Cell; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D018859:Hair Follicle; D006801:Humans; D038101:Insulator Elements; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009928:Organ Specificity; D026723:Polyadenylation; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014161:Transduction, Genetic
D008164:Luminescent Proteins