Prem K. Premsrirut
B.A. University of California-Berkeley, 2001
Ph.D. Stony Brook University, 2010
10th Year MSTP; 4th Year Medical Student
Advisor: Scott Lowe, PhD
Cold Spring Harbor Laboratory
Graduate Program: Genetics
Title: Conditional Regulation of Tumor Suppressor Genes in vivo using RNA interference
Abstract:
Prem K. Premsrirut1 , Cornelius Miething1, Johannes Zuber1, Sang Yong Kim1, Gregory J. Hannon1, and Scott W. Lowe1,2
1 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 2Howard Hughes Medical Institute, Cold Spring Harbor, NY
RNA interference (RNAi) is a powerful tool for controlling gene expression in mammalian systems. To date though, the promise of regulatable RNAi transgenic mice has yet to be realized because the reproducible generation of these animals remains a significant technical limitation. By combining optimized miR30-based shRNA technology with high efficiency ES cell targeting, we developed a flexible and scalable pipeline for the rapid and reliable production of tet-regulated shRNA transgenic mice. These second-generation conditional RNAi mice contain single copy tet-regulatable shRNAs downstream of the endogenous Collagen Type 1 (ColA1) locus, allowing for spatial, temporal and reversible gene expression in mice. Using this platform, we first generated 5 novel doxycycline-regulated shRNA transgenic lines targeting the bioluminescence reporter Luciferase, pluripotency regulator Pou5f1 and the tumor suppressor genes Trp53, INK4a and ARF, each showing strong doxycycline-dependent knockdown of its target protein, without disrupting processing of endogenous miRNAs. Next, to study the role of KrasG12D
Trp53, INK4a and ARF in the maintenance of driven lung adenocarcinomas, we crossed these mice to produce quadruple transgenic mice bearing an shRNA, CCSP-rtTA (clara cell specific promoter -reverse tet-transactivator), LSL-Kras and LSL-luciferase alleles. However, owing to the slow rate and high expense of producing quadruple transgenic mice, we devised a strategy for “speedy” mouse model production. This approach entailed re-derivation of embryonic stem (ES) cells harboring the relevant alleles and subsequent generation of “mosaic” models produced by blastocyst injection. Using these RNAi mouse models, we showed that INK4a/ARF, ARF or Trp53 downregulation by RNAi cooperates with KrasG12D to accelerate lung tumorigenesis and recapitulated the phenotypes of knockout models. Additionally, we determined whether INK4a/ARF, ARF or Trp53 reactivation lead to tumor regression. Together, our work built a new platform that greatly accelerates the rate at which we can study genetic interactions and tumor maintenance genes and also identify and validate new drug targets in vivo. Our approach can be applied to build many other complex cancer models and thus may have significant implications for guiding future cancer therapies.
Publications:
(pre-MSTP publications indicated with an *)
Premsrirut, P.K. et al. A rapid and scalable system for studying gene function in mice using conditional RNA interference. (2010) in submission.
*Dickins RA, McJunkin K, Hernando E, Premsrirut PK, Krizhanovsky V, Burgess DJ, Kim SY, Cordon-Cardo C, Zender L, Hannon GJ, Lowe SW. (2007). Tissue-specific and reversible RNA interference in transgenic mice. Nat Genet. 39(7):914-21. PMID: 17572676.
