A pipeline for the generation of shRNA transgenic mice
Open accessAbstract
RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches-inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNAs to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNAs to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.
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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 Dow et al., 2012
The Introduction (The basics of Col1a1-targeted shRNA transgenic mice; page unavailable) describes a fluorescent reporter linked to inducible shRNA expression, and the Procedure (Testing ES cell clones; page unavailable) uses GFP fluorescence to check induction after doxycycline treatment. Our copy does not establish the specific eGFP–shRenilla or TurboGFP–shTRIM33 pairings. (LLM, conf=0.87)
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“Renilla and TRIM33 shRNA targeted ES cells were derived by Mirimus (Cold Spring Harbor, NY) as described (Premsrirut et al., 2011; Dow et al., 2012), using site-specific integration of the shRNA cassette into the Col1a1 locus of KH2 ES cells (Beard et al.”
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“This is consistent with the reported inefficient expression of shRNA in the brain using this system [12].”
BackgroundMethodResultInfluential
- Authors
- Dow, L. E., Premsrirut, P. K., Zuber, J., Fellmann, C., McJunkin, K., Miething, C., Park, Y., Dickins, R. A., Hannon, G. J., & Lowe, S. W.
- Affiliations
- Cold Spring Harbor Laboratory (US)
- Memorial Sloan Kettering Cancer Center (US)
- University of Zurich (CH)
- Walter and Eliza Hall Institute of Medical Research (AU)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Lowe, Scott W
- ORCID
- Venue
- Nature Protocols
- Published
- 2012-02
- Volume
- 7
- Issue
- 2
- Pages
- 374-393
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC3724521
- OpenAlex
- W2047793174
- Cited by
- 159
- References
- 32
- Citations by year
- 2026: 2 · 2025: 7 · 2024: 7 · 2023: 11 · 2022: 10 · 2021: 11 · 2020: 9 · 2019: 14 · 2018: 13 · 2017: 11 · 2016: 11 · 2015: 17 · 2014: 22 · 2013: 7 · 2012: 7
- FWCI
- 6.81
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Cold Spring Harbor Laboratory Institutional Repository (Cold Spring Harbor Laboratory) (acceptedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institutes of Health (5p30ca045508-35)
- National Cancer Institute (U01CA105388, P01CA087497, P30-CA045508, P01CA013106, P30CA008748)
- National Institute of General Medical Sciences (T32GM008444, R37GM062534)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Keywords
- RNA interference, COL1A1, type I collagen, shRNA design, inducible gene silencing
- MeSH terms
- Collagen Type I, alpha 1 Chain, Animals, Cloning, Molecular, Genetic Engineering, Mice, Transgenic, Collagen Type I, RNA Interference, RNA, Small Interfering, MicroRNAs, Mice, Genotyping Techniques
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