A system for Cre-regulated RNA interference in vivo.

TitleA system for Cre-regulated RNA interference in vivo.
Publication TypeJournal Article
Year of Publication2008
AuthorsStern, P, Astrof, S, Erkeland, SJ, Schustak, J, Sharp, PA, Hynes, RO
JournalProc Natl Acad Sci U S A
Date Published2008 Sep 16
KeywordsAnimals, B-Lymphocytes, Cells, Cultured, Genetic Engineering, Integrases, Lentivirus, Mice, Mice, Inbred C57BL, MicroRNAs, Oncogene Proteins, RNA Interference, Spleen

We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct, allowing its regulated expression (and, at the same time, deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes, miRNA-based RNAi (including two shRNA constructs at once), and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture, efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny, and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo.

Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID18779577
PubMed Central IDPMC2532697
Grant ListP01-HL066105 / HL / NHLBI NIH HHS / United States
R01-GM34277 / GM / NIGMS NIH HHS / United States
U54-CA112967 / CA / NCI NIH HHS / United States
U54-CA126515 / CA / NCI NIH HHS / United States
/ / Howard Hughes Medical Institute / United States