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Updated: May 27, 2026
Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models
Published on: January 12, 2015
Inducible raptor and rictor knockout mouse embryonic fibroblasts
Nadine Cybulski1, Vittoria Zinzalla, Michael N Hall
1Biozentrum, University of Basel, Basel, Switzerland.
Abstract:
The mammalian Target of Rapamycin (mTOR) kinase functions within two structurally and functionally distinct multiprotein complexes termed mTOR complex 1 (mTORC1) and mTORC2. The immunosuppressant and anticancer drug rapamycin is commonly used in basic research as a tool to study mTOR signaling. However, rapamycin inhibits only, and only incompletely, mTORC1, and no mTORC2-specific inhibitor is available. Hence, a full understanding of mTOR signaling in vivo, including the function of both complexes, requires genetic inhibition in addition to pharmacological inhibition. Taking advantage of the Cre/LoxP system, we generated inducible knockout mouse embryonic fibroblasts (MEFs) deficient for either the mTORC1-specific component raptor (iRapKO) or the mTORC2-specific component rictor (iRicKO). Inducibility of the knockout was important because mTOR complex components are essential. Induction of either raptor or rictor knockout eliminated raptor or rictor expression, respectively, and impaired the corresponding mTOR signaling branch. The described knockout MEFs are a valuable tool to study the full function of the two mTOR complexes individually.
Insights
Researchers developed inducible knockout mouse embryonic fibroblasts (MEFs) to study the mammalian Target of Rapamycin (mTOR) signaling pathways. These tools enable the individual investigation of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) functions.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The mammalian Target of Rapamycin (mTOR) kinase is crucial for cell growth and metabolism, operating in two distinct complexes: mTORC1 and mTORC2.
- Rapamycin, a common drug, partially inhibits mTORC1 but not mTORC2, limiting research into mTOR signaling.
- A complete understanding of mTOR function requires methods beyond pharmacological inhibition, particularly for mTORC2.
Purpose of the Study:
- To generate novel research tools for dissecting the individual functions of mTORC1 and mTORC2.
- To overcome the limitations of current pharmacological inhibitors for studying mTOR signaling in vivo.
- To enable precise genetic inhibition of mTORC1 and mTORC2 components.
Main Methods:
- Utilized the Cre/LoxP system to create inducible knockout mouse embryonic fibroblasts (MEFs).
- Generated MEFs deficient for raptor (mTORC1 component) and rictor (mTORC2 component) via inducible knockout.
- Confirmed knockout efficiency by assessing raptor or rictor expression levels post-induction.
Main Results:
- Successfully generated inducible knockout MEFs for raptor (iRapKO) and rictor (iRicKO).
- Induction of knockout led to the specific elimination of raptor or rictor expression.
- Demonstrated impaired signaling in the corresponding mTOR complex upon knockout induction.
Conclusions:
- The developed iRapKO and iRicKO MEFs are valuable tools for studying mTORC1 and mTORC2 signaling independently.
- These genetic tools allow for a more comprehensive investigation of mTOR complex functions in biological processes.
- Facilitates deeper understanding of mTOR pathway dysregulation in diseases like cancer.
