Biology subjects

Mehlferber, M. M.

Publications and source records attributed to Mehlferber, M. M..

2 recordsLinked to original sources

bioRxiv2022

Characterization of protein isoform diversity in human umbilical vein endothelial cells (HUVECs) via long-read proteogenomics

Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by protein isoforms. To characterize the isoform landscape within ECs, we applied a long read proteogenomics approach to analyze human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream MS analysis to infer protein isoform expression. We detected 53,836 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted "reference isoform" 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signaling pathways. Finally, we identified novel protein isoforms arising from a diversity of splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/490813v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@86ca88org.highwire.dtl.DTLVardef@15ab038org.highwire.dtl.DTLVardef@e7553borg.highwire.dtl.DTLVardef@18cbae8_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2022

Efficient Gene Knockout in Salivary Gland Epithelial Organoid Cultures

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, non-solidified 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 ITS (insulin, transferrin, and selenium) enhances growth and branching. 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 80% transduction efficiency when antibiotic selection is used. Importantly, we detected robust loss of targeted protein products when testing 9 guide RNAs for 3 different genes. Moreover, targeting the {beta}1 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.

genetics↗