Type
Text
Type
Dissertation
Advisor
Senthil K. Muthuswamy | Lowe, Scott W. | Greg J. Hannon | Howard C. Crawford | Raffaella Sordella | Cijiang He
Date
2010-05-01
Keywords
cancer models, genetically engineered mice, lung adenocarcinoma, recombinase-mediated cassette exchange, RNA interference, transgenic mice | Biology, Genetics -- Health Sciences, Oncology -- Biology, Molecular
Department
Department of Genetics
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/72638
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
RNA interference is a powerful tool for controlling gene expression in mammalian systems; as such, it has become an effective alternative to conventional knockout approaches. RNAi has proven to be an efficient method to inhibit tumor suppressor gene function and yield insight into the important players in cancer biology. Still, 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 fluorescence-coupled mir30-based shRNA technology with high efficiency ES cell targeting, I have developed a flexible and scalable pipeline for the rapid and reliable production of doxycycline-regulated shRNA transgenic mice. These RNAi mice contain single copy DOX-regulatable shRNAs downstream of the endogenous Collagen Type 1 locus, allowing for spatial, temporal and reversible gene expression in mice. Using this platform, I generated novel DOX-regulated shRNA transgenic lines targeting the bioluminescence reporter Luciferase and endogenous tumor suppressor genes, including Trp53, INK4a and ARF, each showing strong doxycycline-dependent knockdown of its target protein, without disrupting processing of endogenous miRNAs. To study the role of these TSGs in the maintenance of KrasG12D driven lung adenocarcinomas, I crossed these to produce mice bearing an shRNA, CCSP-rtTA (clara cell specific promoter. reverse tet-transactivator), LSL-KrasG12D and LSL-Luciferase alleles. However, owing to the slow rate and high expense of producing quadruple transgenic mice, I later devised a strategy for"speedy" mouse model production. This approach entailed re-derivation of embryonic stem cells harboring the relevant alleles and subsequent generation of"mosaic" models produced by blastocyst injection. Using these RNAi mouse models, I show that INK4a/ARF or Trp53 downregulation by RNAi cooperates with KrasG12D to accelerate lung tumorigenesis and recapitulate the phenotypes of knockout models. Additionally, I investigate whether INK4a/ARF or Trp53 knockdown is required for tumor maintenance. Together, this work built a platform that greatly accelerates the rate at which one can study genetic interactions and tumor maintenance genes and also identify and validate new drug targets in vivo. This approach can be applied to build many other complex cancer models and thus may have significant implications for guiding future therapies.
Recommended Citation
Premsrirut, Prem K., "Spatial, temporal and reversible regulation of endogenous gene in vivo using RNA interference" (2010). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 1841.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/1841