AAV4-Cre-GFP

Quiescent neural stem cells (qNSCs) with radial morphology are the only proven source of new neurons in the adult mammalian brain. Although various strategies have been developed to target and manipulate adult hippocampal qNSCs, they often suffer from long propagation times, low recombination efficiency, and nonspecific labeling. Therefore, there is an urgent need to develop an easily manufactured viral vector that allows for flexible packaging and stable expression of various transgenes in qNSCs. Here, researchers report a recombinant adeno-associated virus serotype 4 (rAAV4)-based toolkit that preferentially targets hippocampal qNSCs and allows lineage tracing, functional analysis, and activity manipulation of adult qNSCs. Importantly, targeting qNSCs in a non-Cre-dependent manner opens the possibility to study qNSCs in genetically refractory animal species and may have a translational impact in gene therapy by preferentially targeting qNSCs.

The researchers packaged the CMV-GFP/Cre plasmid into rAAV4 capsid to generate rAAV4-CMV-GFP/Cre (Figure 1A). rAAV4-CMV-GFP/Cre was injected into the DG of Ai9 reporter mice, which contain a loxP-flanked STOP cassette that prevents transcription of the downstream RFP variant (tdTomato) in the absence of Cre recombinase (Figure 1B). Three days after viral injection, the researchers examined the expression of GFP (for Cre expression) and tdTomato (for Cre-dependent recombination) in AAV4-labeled cells and observed that both GFP and tdTomato signals were strong, and all tdTomato+ cells co-localized with GFP (Figure 1C), indicating that Cre-mediated recombination using CMV-GFP/Cre is highly efficient. Importantly, the majority of rAAV4-labeled tdTomato+ cells were rNSCs (Figure 1D) and colocalized with NESTIN and GFAP markers (Figures 1E and 1F).

Figure 1. rAAV4 Allows for Genetic Manipulation of Quiescent NSCs in the Adult Hippocampus. (Crowther A J, et al., 2018)

The researchers injected rAAV4-CMV-GFP/Cre into another reporter line called mTmG, a dual fluorescent Cre reporter mouse that expresses membrane-targeted tdTomato (mT) before Cre-mediated excision and membrane-targeted GFP (mG) after excision (Figure 1G). Since GFP/Cre is localized in the nucleus, the membrane-targeted GFP signal around the NSC soma and radial processes in the mTmG reporter mouse indicated that recombination had occurred. rAAV4-GFP/Cre efficiently targeted rNSCs (Figure 1H), indicating that the rAAV4-Cre system can target rNSCs using different Cre-inducible reporter lines.