title: A retro-lentiviral system for doxycycline-inducible gene expression and gene knockdown in cells with limited proliferative capacity.
abstract: Currently, there is no reliable system for regulated gene expression and regulated gene knockdown in cells with finite lifespan. In this manuscript, we describe a vector system, consisting of a retrovirus for the delivery of rtTA, and a lentivirus for the delivery of either a transgene or a miR-shRNA for the modification of primary cells. Primary rat pulmonary microvascular endothelial cells (PMVEC) modified by these vectors for the inducible expression of Gaussia luciferase or DsRed Express demonstrated greater than 100-fold induction of the transgene expression with doxycycline. The system works reliably in both sequential and simultaneous infection modes, with about 95% of the sells selected with two antibiotics being inducible in each mode. The lentiviral vector for gene knockdown allows for the direct cloning of shRNA oligos using alpha-complementation, and for the monitoring of induction of RNA interference with fluorescent reporter, mCherry. The gene knockdown vector was validated by knocking down beta-actin expression in PMVECs, with two of the four constructs showing 59 and 75% knockdown, respectively, compared to uninduced controls. The vectors described here were successfully used for the modification of various primary and established cell lines for regulated gene expression and regulated knockdown.
authors: Department of Cell Biology and Neuroscience, University of South Alabama, 307 University Blvd., Mobile, AL 36688, USA. malexeye@jaguar1.usouthal.edu
authors: Mikhail F
authors: MF
authors: Alexeyev
authors: Rafik
authors: R
authors: Fayzulin
authors: Inna N
authors: IN
authors: Shokolenko
authors: Viktoriya
authors: V
authors: Pastukh
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology*; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008168:Lung / Q000098:blood supply; D051379:Mice; D055806:Microvessels / Q000166:cytology; D051381:Rats; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D000199:Actins; D008164:Luminescent Proteins; C413663:fluorescent protein 583; D008156:Luciferases; D004318:Doxycycline