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Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation.

ABSTRACT: Glial cells such as astrocytes and oligodendrocytes play crucial roles in the central nervous system. To investigate the molecular mechanisms underlying the development and the biological functions of glial cells, simple and rapid techniques for glial cell-specific genetic manipulation in the mouse cerebrum would be valuable. Here we uncovered that the Gfa2 promoter is suitable for selective gene expression in astrocytes when used with the piggyBac system and in utero electroporation. In contrast, the Blbp promoter, which has been used to induce astrocyte-specific gene expression in transgenic mice, did not result in astrocyte-specific gene expression. We also identified the Plp1 and Mbp promoters could be used with the piggyBac system and in utero electroporation to induce selective gene expression in oligodendrocytes. Furthermore, using our technique, neuron-astrocyte or neuron-oligodendrocyte interactions can be visualized by labeling neurons, astrocytes and oligodendrocytes differentially. Our study provides a fundamental basis for specific transgene expression in astrocytes and/or oligodendrocytes in the mouse cerebrum.

SUBMITTER: Hamabe-Horiike T

PROVIDER: S-EPMC7921133 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation.

Hamabe-Horiike Toshihide T Kawasaki Kanji K Sakashita Masataka M Ishizu Chihiro C Yoshizaki Tomokazu T Harada Shin-Ichi SI Ogawa-Ochiai Keiko K Shinmyo Yohei Y Kawasaki Hiroshi H

Scientific reports 20210301 1

Glial cells such as astrocytes and oligodendrocytes play crucial roles in the central nervous system. To investigate the molecular mechanisms underlying the development and the biological functions of glial cells, simple and rapid techniques for glial cell-specific genetic manipulation in the mouse cerebrum would be valuable. Here we uncovered that the Gfa2 promoter is suitable for selective gene expression in astrocytes when used with the piggyBac system and in utero electroporation. In contras ...[more]

PMID: 33649472

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Project description:BACKGROUND:Since its inception in 2001, in utero electroporation (IUE) has been widely used by the neuroscience community. IUE is a technique developed to introduce plasmid DNA into embryonic mouse brains without permanently removing the embryos from the uterus. Given that IUE labels cells that line the ventricles, including radial fibers and migrating neuroblasts, this technique is an excellent tool for studying factors that govern neural cell fate determination and migration in the developing mouse brain. Whether IUE has an effect on microglia, the immune cells of the central nervous system (CNS), has yet to be investigated. METHODS:We used IUE and the pCIG2, pCIC-Ascl1, or pRFP-C-RS expression vectors to label radial glia lining the ventricles of the embryonic cortex and/or hypothalamus. Specifically, we conducted IUE at E14.5 and harvested the brains at E15.5 or E17.5. Immunohistochemistry, along with cytokine and chemokine analyses, were performed on embryonic brains with or without IUE exposure. RESULTS:IUE using the pCIG2, pCIC-Ascl1, or pRFP-C-RS vectors alone altered microglia morphology, where the majority of microglia near the ventricles were amoeboid and displayed altered expression signatures, including the upregulation of Cd45 and downregulation of P2ry12. Moreover, IUE led to increases in P2ry12- cells that were Iba1+/IgG+ double-positive in the brain parenchyma and resembled macrophages infiltrating the brain proper from the periphery. Furthermore, IUE resulted in a significant increase in cell death in the developing hypothalamus, with concomitant increases in cytokines and chemokines known to be released during pro-inflammatory states (IL-1β, IL-6, MIP-2, RANTES, MCP-1). Interestingly, the cortex was protected from elevated cell death following IUE, implying that microglia that reside in the hypothalamus might be particularly sensitive during embryonic development. CONCLUSIONS:Our results suggest that IUE might have unintended consequences of activating microglia in the embryonic brain, which could have long-term effects, particularly within the hypothalamus.

Dataset Information

Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation.

Publications

Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation.

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