Project description:We investigated the effects of tamoxifen, CreER recombination, and genetic manipulations to genes whose promoters are used to driving CreER expression on microglial gene expression across 4 different microglial inducible Cre lines.
Project description:Cre/loxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. Microglial biology has particularly benefited because microglia historically have been difficult to transduce with virus or electroporation methods for gene delivery. Here, we investigate five of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency, cell-type specificity, and spontaneous recombination, depending on the Cre line and inter-loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency, particularly in microglia. There is increasing evidence that microglia are key regulators of neural circuits and major drivers of a broad range of neurological diseases. Reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field and the development of microglia-based therapeutics.
Project description:Recent single-cell RNA sequencing studies have revealed distinct microglial states in development and disease. These include proliferative region-associated microglia (PAM) in developing white matter and disease-associated microglia (DAM) prevalent in various neurodegenerative conditions. PAM and DAM share a similar core gene signature and other functional properties. However, the extent of the dynamism and plasticity of these microglial states, as well as their functional significance, remains elusive, partly due to the lack of specific tools. Here, we report the generation of an inducible Cre driver line, Clec7a-CreERT2, designed to target PAM and DAM in the brain parenchyma. Utilizing this tool, we isolate and profile labeled cells during development and in several disease models, uncovering convergence and context-dependent differences in PAM/DAM gene expression. Through long-term tracking, we demonstrate surprising levels of plasticity in these microglial states. Lastly, we specifically deplete DAM in cuprizone-induced demyelination, revealing their roles in disease progression and recovery.
Project description:Cx3cr1CreER driven Cre-recombinase (Cre) is a widely used genetic tool for enabling gene manipulation in microglia and macrophages. However, an in-depth analysis for the possible detrimental effects of Cre-activity in microglia, surprisingly remains missing. Here we demonstrate an age-dependent sensitivity of microglia to Cx3cr1-Cre-toxicity, wherein Cre-induction specifically in early postnatal microglia is detrimental for microglial development, proliferation and function. Tamoxifen (TAM) induced Cre-activity leads to microglial activation, type1-interferon (IFN-1) signaling and increased phagocytosis, causing aberrant synaptic pruning during early postnatal period and anxiety behavior in later age. The detrimental effects of Cre-induction are caused due to DNA-damage induced toxicity in microglia, and is limited to the early postnatal period, showing no detrimental effects in adult microglia. Thus, our study reveals the age-dependent vulnerability of microglia to Cre-activity, thereby highlighting age-dependencies of Cre-action, which could be especially applicable in the broader context of environment-responsive cell-types.
Project description:We investigated the transcriptomic effect of GNAS(R201C) expression in murine cell lines derived from the Kras;Gnas model of pancreatic intraductal papillary mucinous neoplasms where transgenic mutant GNAS is doxycycline inducible (LGKC; p48(Cre), Kras(LSL-G12D), Rosa26(LSL-rtTA)), Tg(TetO-GNAS(R201C)) using bulk RNA-seq.
Project description:Transcription profiles of BV2 microglial cell lines: unstimulated, stimulated with LPS or transfected with constitutively active Stat1 and Stat3.
Project description:In order to identify the effects of the induction of the gene of interest on the mouse ES transcriptome, we performed Affymetrix Gene-Chip hybridization experiments for the different inducible cell lines Transcriptome analysis of the inducible transgenic mouse ES cell lines
Project description:Cx3cr1CreER-Eyfp/wt mice contain a subset of microglia lacking Cre and EYFP expression. These microglial escape Cre-mediated recombination and gain a repopulation advantage following Cre-driven DTA-mediated microglial depletion.
Project description:Temporally controlling cre recombinase through tamoxifen (TAM) induction has many advantages for biomedical research studies. While most projects use TAM induction of mice at early post-natal or adolescence (<2m.o.) ages, age-related neurodegeneration and aging studies can require experimental designs with cre induction in older mice (>12m.o.). While anecdotally reported to be problematic, there are no published comparisons of TAM mediated cre induction at young and older ages. Cx3cr1creERT2 mice for studying microglial were crossed to a floxed NuTRAP reporter to compare cre induction at young (3m.o.) and old (18m.o) ages. Specificity and efficiency microglial labeling was identical at 24m.o. in mice induced with TAM at 3m.o. or 18m.o. of age. Age-related microglial translatomic changes were nearly identical regardless of TAM induction age. While each cre and flox mouse line should be validated before use, these findings demonstrate that TAM induction of cres can be performed even into older mouse ages.
Project description:In order to identify the effects of the induction of the gene of interest on the mouse ES transcriptome, we performed Affymetrix Gene-Chip hybridization experiments for the different inducible cell lines Transcriptome analysis of the inducible transgenic mouse ES cell lines For the analysis on the different inducible cell lines, total RNA was extracted from three biological replicates grown in medium deprived of Tetracycline for 17 and 24 hours; RNA extracted from un-induced clones was used as control. Total RNA extracted from parental cell line EBRTcH3 (EB3) was used as additional control.