Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures

Abstract

We have developed methods to achieve efficient CRISPR-Cas9 mediated gene knockout in ex vivo mouse embryonic salivary epithelial organoids. Salivary epithelial organoids 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 buds can be cultured in low-concentration, non-solidified Matrigel suspensions in 96-well plates, which greatly increases sample throughput compared to conventional cultures embedded in solidified Matrigel. We then describe an efficient workflow to produce experiment-ready, high-titer lentiviruses within 1 week after molecular cloning. To track transduced cells, we designed the lentiviral vector to co-express a nuclear fluorescent reporter with the guide RNA. We routinely achieve 50-80% transduction efficiency as indicated by the fluorescent reporter. Importantly, we detected robust loss of targeted protein products when testing 8 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 organoids, which will greatly facilitate studies of specific gene functions using this system.