Inducible shRNA expression for application in a prostate cancer mouse model.
RNA interference (RNAi) is a powerful tool to induce loss-of-function phenotypes by inhibiting gene expression post-transcriptionally. Synthetic short interfering RNAs (siRNAs) as well as vector-based siRNA expression systems have been used successfully to silence gene expression in a variety of biological systems. We describe the development of an inducible siRNA expression system that is based on the tetracycline repressor and eukaryotic RNA polymerase III promoters (U6 and 7SK). For proof of concept we selectively inhibited expression of two catalytic subunits of the phosphatidylinositol 3-kinase (PI 3-kinase), p110alpha and p110beta, by using vector-derived short hairpin RNAs (shRNAs). Stable pools of human prostate cancer cells (PC-3) exhibiting reduced levels of both PI 3-kinase catalytic subunits due to the expression of corresponding shRNAs in an inducible fashion were established and analyzed for their invasive potential in vitro as well as in an orthotopic metastatic mouse model. This inducible system for RNAi allows an unbiased and comparable analysis of loss-of-function phenotypes by comparing selected isogenic cell populations on the induced and non-induced level. In addition, conditional RNAi allows the study of essential and multifunctional genes involved in complex biological processes by preventing inhibitory and compensatory effects caused by constitutive knockdown.
Czauderna
Frank
F
Atugen AG, Otto Warburg Haus (No. 80), Robert-Roessle-Strasse 10, 13125 Berlin, Germany.
Santel
Ansgar
A
Hinz
Michael
M
Fechtner
Melanie
M
Durieux
Birgit
B
Fisch
Gerald
G
Leenders
Frauke
F
Arnold
Wolfgang
W
Giese
Klaus
K
Klippel
Anke
A
Kaufmann
Jörg
J
D000818:Animals; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019869:Phosphatidylinositol 3-Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000235:genetics*; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D034622:RNA Interference; D012320:RNA Polymerase III / Q000235:genetics; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology
D021122:Protein Subunits; D034741:RNA, Small Interfering; D019869:Phosphatidylinositol 3-Kinases; D012320:RNA Polymerase III; D013752:Tetracycline