A system for Cre-regulated RNA interference in vivo.
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.
Stern
Patrick
P
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Astrof
Sophie
S
Erkeland
Stefan J
SJ
Schustak
Joshua
J
Sharp
Phillip A
PA
Hynes
Richard O
RO
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D002478:Cells, Cultured; D005818:Genetic Engineering; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D013154:Spleen / Q000201:enzymology
D035683:MicroRNAs; D015513:Oncogene Proteins; C045073:Cre recombinase; D019426:Integrases
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