Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Abstract

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Tropism, intracerebral distribution, and transduction efficiency of HIV- and SIV-based lentiviral vectors after injection into the mouse brain: a qualitative and quantitative in vivo study

“…However, according to our results, highly infective viruses (HIV-1SEW, HIV-1SEW(Vpr), PBjSEW(Vpr)) are not only able to enter ependymal cells of the ipsilateral lateral ventricle, but they seem to cross these cells, and reach the contralateral site of the third and via foramen interventriculare even the contralateral lateral ventricle by liquor (HIV-1SEW). Here, they also infected ependymal cells but did not enter the nervous tissue, a finding that was supported by our own observations after injection of the vector particles into the lateral ventricles (data not shown; see also Baekelandt et al 2002 ; Porlan et al 2016 ). Since especially for both HIV-1SEW derived vectors, a marked accumulation of microglial cells around the ventricles could be observed, and a certain contribution of these cells to vector transport into the liquor cannot be excluded.…”

Intron detention tightly regulates the stemness/differentiation switch in the adult neurogenic niche

“…ACS-4500) and concentrated by ultracentrifugation in an Optima XL-100K centrifuge (Beckman Coulter), as previously described in ref. 60 . For overexpression studies, mouse Scratch1 cDNA was cloned in the pRRL-SIN-PPT.PGK.EGFP.WPRE expression vector, followed by P2A and tdTomato fluorescent protein.…”