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.

Localized astrogenesis regulates gyrification of the cerebral cortex

“…To make pPB-gfa2-DTA, the EGFP of pPB-gfa2-EGFP was replaced with DTA. pPB-CAG-EGFP ( 53 ) and pCAG-sFGFR3 ( 31 ) were described previously. pPB-CAG-loxP-ER T2 CreER T2 -loxP-FGF8-IRES2-EGFP was made by inserting loxP-ER T2 CreER T2 -loxP developed by M. G. Holt and FGF8-IRES2-EGFP derived from pCAG-FGF8 ( 33 ) and pIRES2-EGFP (Clontech) into the pPB-CAG vector.…”

“…The sections were then subjected to Hoechst 33342 staining. The PLP probe, the FGFR2 probe, and the FGFR3 probe were described previously ( 31 , 53 ).…”

Potential of Multiscale Astrocyte Imaging for Revealing Mechanisms Underlying Neurodevelopmental Disorders

“…Furthermore, these lines may differ in multiple ways from native astrocytes and wild-type mice. In contrast, infection of postnatal brain with adeno-associated viruses (AAVs) and delivery of transposon-based reporters to embryonic brain by electroporation are relatively efficient methods for labeling astrocyte-lineage cells in vivo ( Figure 3 ) [ 141 , 142 , 143 ]. Fusing astroglial cell type-specific promoters to fluorescent proteins with piggyBac transposon can directly label targeted subpopulations permanently or within defined developmental phases [ 143 ], while recombinases driven by cell type-specific promoters can both efficiently label and alter the genome of astrocytes [ 144 ].…”

“…In contrast, infection of postnatal brain with adeno-associated viruses (AAVs) and delivery of transposon-based reporters to embryonic brain by electroporation are relatively efficient methods for labeling astrocyte-lineage cells in vivo ( Figure 3 ) [ 141 , 142 , 143 ]. Fusing astroglial cell type-specific promoters to fluorescent proteins with piggyBac transposon can directly label targeted subpopulations permanently or within defined developmental phases [ 143 ], while recombinases driven by cell type-specific promoters can both efficiently label and alter the genome of astrocytes [ 144 ]. Hamabe-Horiike et al revealed that the Gfa2 -promoter labeled nearly 80% of astrocytes, the Plp1 -promoter labeled nearly 96% of oligodendrocytes, and the Mbp -promoter labeled nearly 90% of oligodendrocytes at E15 electroporation, whereas the CAG -promoter labeled nearly 40% of neurons, 15% of astrocytes, and 8% of oligodendrocytes [ 143 ].…”

“…Fusing astroglial cell type-specific promoters to fluorescent proteins with piggyBac transposon can directly label targeted subpopulations permanently or within defined developmental phases [ 143 ], while recombinases driven by cell type-specific promoters can both efficiently label and alter the genome of astrocytes [ 144 ]. Hamabe-Horiike et al revealed that the Gfa2 -promoter labeled nearly 80% of astrocytes, the Plp1 -promoter labeled nearly 96% of oligodendrocytes, and the Mbp -promoter labeled nearly 90% of oligodendrocytes at E15 electroporation, whereas the CAG -promoter labeled nearly 40% of neurons, 15% of astrocytes, and 8% of oligodendrocytes [ 143 ]. Thus, these results suggest that cell type-specific promoter approach is efficient to label astrocyte by in utero electroporation.…”