Astrocyte Specificity and Coverage of hGFAP-CreERT2 [Tg(GFAP-Cre/ERT2)13Kdmc] Mouse Line in Various Brain Regions

Abstract

Astrocyte is the most abundant cell type in the central nervous system and its importance has been increasingly recognized in the brain pathophysiology. To study in vivo function of astrocyte, astrocyte-specific gene-targeting is regarded as a powerful approach. Especially, hGFAP-CreERT2, which expresses tamoxifen-inducible Cre recombinase under the human GFAP promoter, has been developed and characterized from several research groups. However, one of these mouse lines, [Tg(GFAP-Cre/ERT2)13Kdmc] from Ken McCarthy group has not been quantitatively analyzed, despite its frequent use. Here, we performed comprehensive characterization of this mouse line with quantitative analysis. By crossing this mouse line with Ai14 (RCL-tdTomato), a very sensitive Cre reporter mouse line, we visualized the Cre-expressing cells in various brain regions. For quantitative analysis, we immunostained S100β as an astrocytic marker and NeuN, tyrosine hydroxylase or calbindin as a neuronal marker in different brain regions. We calculated ‘astrocyte specificity’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of tdTomato positive cells and the ‘astrocyte coverage’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of S100β positive cells. Interestingly, we found varying degree of astrocyte specificity and coverage in each brain region. In cortex, hypothalamus, substantia nigra pars compacta and cerebellar Purkinje layer, we observed high astrocyte specificity (over 89%) and relatively high astrocyte coverage (over 70%). In striatum, hippocampal CA1 layer, dentate gyrus and cerebellar granule layer, we observed high astrocyte specificity (over 80%), but relative low astrocyte coverage (50–60%). However, thalamus and amygdala showed low astrocyte specificity (about 65%) and significant neuron specificity (over 30%). This hGFAP-CreERT2 mouse line can be useful for genetic modulations of target gene either in gain-of-function or loss-of-function studies in the brain regions with high astrocyte specificity and coverage. However, the use of this mouse line should be restricted to gain-of-function studies in the brain regions with high astrocyte specificity but low coverage. In conclusion, hGFAP-CreERT2 mouse line could be a powerful tool for gene-targeting of astrocytes in cortex, striatum, hippocampus, hypothalamus, substantia nigra pars compacta and cerebellum, but not in thalamus and amygdala.

Tamoxifen induction of Cre recombinase does not cause long-lasting or sexually divergent responses in the CNS epigenome or transcriptome: implications for the design of aging studies

“…To address this question, we used C57BL/6 J male and female mice, subjected to a routinely used dose and regime of Tam i.p. injections to induce cre recombinases in a variety of tissues, including CNS [ (Srinivasan et al 2016;Jahn et al 2018;Park et al 2018)]. In the absence of cre expression, we were able to interrogate side effects of TAM on cytosine methylation and hydroxymethylation levels, in both C G a n d n o n -C G c o n t e x t s a n d R N A -s e q transcriptomic profiles of three commonly studied CNS tissues: hippocampus, cortex, and retina.…”

Conversion of Reactive Astrocytes to Induced Neurons Enhances Neuronal Repair and Functional Recovery After Ischemic Stroke

“…The specificity of Cre recombinase under a human GFAP promoter in mice has been thoroughly characterized. Specificity defined as the proportion of co-labeled S100β and reporter positive cells over the total number of reporter positive cells in the mouse has been reported as high as 96.02% (Park et al, 2018 ).…”

Neural Cadherin Plays Distinct Roles for Neuronal Survival and Axon Growth under Different Regenerative Conditions

“…We did not quantify the percentage of astrocytes that express YFP. Nonetheless, previous reports have shown that approximately 80-90% of astrocytes successfully express the transgene (Casper et al, 2007;Park et al, 2018). To confirm that Cre recombination does not occur in RGCs, we performed immunohistochemistry on the sectioned retinas of GFAPCreERT; Rosa26-YFP mice.…”