Main Menu (Mobile)- Block
- Overview
-
Support Teams
- Overview
- Anatomy and Histology
- Cryo-Electron Microscopy
- Electron Microscopy
- Flow Cytometry
- Gene Targeting and Transgenics
- Immortalized Cell Line Culture
- Integrative Imaging
- Invertebrate Shared Resource
- Janelia Experimental Technology
- Mass Spectrometry
- Media Prep
- Molecular Genomics
- Primary & iPS Cell Culture
- Project Pipeline Support
- Project Technical Resources
- Quantitative Genomics
- Scientific Computing Software
- Scientific Computing Systems
- Viral Tools
- Vivarium
- Open Science
- You + Janelia
- About Us
Main Menu - Block
- Overview
- Anatomy and Histology
- Cryo-Electron Microscopy
- Electron Microscopy
- Flow Cytometry
- Gene Targeting and Transgenics
- Immortalized Cell Line Culture
- Integrative Imaging
- Invertebrate Shared Resource
- Janelia Experimental Technology
- Mass Spectrometry
- Media Prep
- Molecular Genomics
- Primary & iPS Cell Culture
- Project Pipeline Support
- Project Technical Resources
- Quantitative Genomics
- Scientific Computing Software
- Scientific Computing Systems
- Viral Tools
- Vivarium

Note: Research in this publication was not performed at Janelia.
Abstract
We have developed methods to achieve efficient CRISPR-Cas9–mediated gene knockout in ex vivo mouse embryonic salivary epithelial explants. Salivary epithelial explants provide a valuable model for characterizing cell signaling, differentiation, and epithelial morphogenesis, but research has been limited by a paucity of efficient gene perturbation methods. Here, we demonstrate highly efficient gene perturbation by transient transduction of guide RNA–expressing lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic mice. We first show that salivary epithelial explants can be cultured in low-concentration, nonsolidified Matrigel suspensions in 96-well plates, which greatly increases sample throughput compared to conventional cultures embedded in solidified Matrigel. We further show that salivary epithelial explants can grow and branch with FGF7 alone, while supplementing with insulin, transferrin, and selenium (ITS) enhances growth and branching. We then describe an efficient workflow to produce experiment-ready, high-titer lentiviruses within 1 wk after molecular cloning. To track transduced cells, we designed the lentiviral vector to coexpress a nuclear fluorescent reporter with the guide RNA. We routinely achieved 80% transduction efficiency when antibiotic selection was used. Importantly, we detected robust loss of targeted protein products when testing 9 guide RNAs for 3 different genes. Moreover, targeting the β1 integrin gene (Itgb1) inhibited branching morphogenesis, which supports the importance of cell–matrix adhesion in driving branching morphogenesis. In summary, we have established a lentivirus-based method that can efficiently perturb genes of interest in salivary epithelial explants, which will greatly facilitate studies of specific gene functions using this system.