A Rapid and Scalable System for Studying Gene Function in Mice Using Conditional RNA Interference
Open accessAbstract
RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19(ARF) as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PAPERCLIP:
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Fluorescent markers eGFP (for shRenilla) and TurboGFP (for shTRIM33) indicate shRNA-expressing cells following doxycycline treatment.
“Fluorescent markers (eGFP for shRenilla and TurboGFP for shTRIM33) were indicators of shRNA-expressing cells following doxycycline treatment.”
From The transcriptional cofactor TRIM33 prevents apoptosis in B lymphoblastic leukemia by deactivating a single enhancer (2015) · cites this paper as Premsrirut et al., 2011
The Results sections “Generation of a targeting construct” and “ColA1 targeted shRNAs enable potent and reversible knockdown in ES cells” describe doxycycline-inducible GFP as a visual reporter of shRNA expression. Our copy does not establish the specific assignments of eGFP to shRenilla or TurboGFP to shTRIM33. No page numbers are available in the indexed passages. (LLM, conf=0.85)
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- Authors
- Premsrirut, P. K., Dow, L. E., Kim, S. Y., Camiolo, M., Malone, C. D., Miething, C., Scuoppo, C., Zuber, J., Dickins, R. A., Kogan, S. C., Shroyer, K. R., Sordella, R., Hannon, G. J., & Lowe, S. W.
- Affiliations
- Cold Spring Harbor Laboratory (US)
- Walter and Eliza Hall Institute of Medical Research (AU)
- University of California, San Francisco (US)
- Stony Brook University Hospital (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Lowe, Scott W.
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2011-04
- Volume
- 145
- Issue
- 1
- Pages
- 145-158
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC3244080
- OpenAlex
- W2114417755
- Cited by
- 315
- References
- 43
- Citations by year
- 2026: 5 · 2025: 8 · 2024: 11 · 2023: 21 · 2022: 17 · 2021: 19 · 2020: 17 · 2019: 17 · 2018: 16 · 2017: 19 · 2016: 18 · 2015: 37 · 2014: 27 · 2013: 33 · 2012: 29
- FWCI
- 14.30
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Cold Spring Harbor Laboratory Institutional Repository (Cold Spring Harbor Laboratory) (acceptedVersion)
- Funders
- Howard Hughes Medical Institute
- National Cancer Institute (P01CA013106, P30CA008748)
- National Institute of General Medical Sciences (R01GM062534, T32GM008444)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- APC, p19ARF, p53, Oct4, tumor suppressor, gene function, therapeutic target, T-cell acute lymphoblastic leukemia, lung adenocarcinoma, Wnt signaling, paper clips
- MeSH terms
- Adenocarcinoma, Animals, Lung Neoplasms, Mice, Transgenic, RNA Processing, Post-Transcriptional, Signal Transduction, RNA Interference, RNA, Small Interfering, MicroRNAs, Wnt Proteins, Mice, Embryonic Stem Cells, Precursor T-Cell Lymphoblastic Leukemia-Lymphoma, Gene Knockdown Techniques
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