vom Berg, J. | Search bioRxiv

bioRxiv2021

Manipulating the murine Lgr5 locus using a rapid, efficient and flexible CRISPR/Cas9 pipeline

The maintenance of adult epithelial tissues, such as the lining of the intestine, depends on their periodical renewal. This is achieved through small populations of adult stem cells. Lgr5 serves as a marker for these cells. Here we report a novel non-variegated Lgr5 mouse model, which was generated via the CRISPR/Cas9 system. We show that this Lgr5-2A-CreERT2-2A-mOrange2 mouse line can be used for lineage tracing, as well as for directing gene expression to Lgr5+ cells. The introduction of the transgene affects neither the expression, nor the function of endogenous Lgr5. Therefore, this new tool will serve to mark and manipulate intestinal stem cells to gain new biological insights.

developmental biology↗

bioRxiv2020

Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37{degrees}C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4{degrees}C. These findings emphasize the risk of introducing technical biases and biological artefacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

neuroscience↗