Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9

Abstract

Functional studies of resident microglia require molecular tools for their genetic manipulation. Here we show that microRNA-9-regulated lentiviral vectors can be used for the targeted genetic modification of resident microglia in the rodent brain. Using transgenic reporter mice, we demonstrate that murine microglia lack microRNA-9 activity, whereas most other cells in the brain express microRNA-9. Injection of microRNA-9-regulated vectors into the adult rat brain induces transgene expression specifically in cells with morphological features typical of ramified microglia. The majority of transgene-expressing cells colabels with the microglia marker Iba1. We use this approach to visualize and isolate activated resident microglia without affecting circulating and infiltrating monocytes or macrophages in an excitotoxic lesion model in rat striatum. The microRNA-9-regulated vectors described here are a straightforward and powerful tool that facilitates functional studies of resident microglia.

MiR-9 promotes microglial activation by targeting MCPIP1

“…In addition to its involvement in neurogenesis, miR-9 expression also plays a role in gastric adenocarcinoma39. While most cells of the CNS express miR-9, a recent study indicates the lack of miR-9 activity in murine microglia under basal conditions40. In agreement with the findings of Akerblom et al ., our study also demonstrated no expression of miR-9 RNA in microglial cells in brain sections from control mice, however, following LPS injection, there was induction of mature miR-9 in Iba-1 positive microglia (Supplementary Fig.…”

A new approach for ratiometric in vivo calcium imaging of microglia

“…This leads to degradation of the transgene messenger RNA specifically in cells expressing microRNA-9 (miR-9). As we have shown previously, rodent microglia lacks miR-9 expression, and therefore miR-9-regulated vectors can be used for selective labelling of microglia 26 . To begin with, we transduced cortical cells in adult mice with the original miR-9-regulated vector in which the expression of green fluorescent protein (GFP) was driven by the phosphoglycerate kinase (PGK) promoter (LV.miR-9.T construct 26 ; Fig.…”

“…Although currently used in mice, we anticipate that the same or similar vectors will also work in other rodent species (see ref. 26). …”

“…On the other hand, in vitro data suggest that well known PAMPs, such as LPS, cause chronic elevations of [Ca 2+ ] i and that these chronic elevations are required for effector responses of microglia such as release of NO or certain cytokines and chemokines 13, 14 . To enable reliable in vivo measurements of steady state intracellular Ca 2+ level as well as Ca 2+ transients we chose to express in microglia our novel ratiometric GECI Twitch-2B 25 by means of a microRNA-9-regulated lentiviral vector 26 .…”

Direct Neural Conversion from Human Fibroblasts Using Self-Regulating and Nonintegrating Viral Vectors

“…We, and others, have previously used miRNA-regulated vectors to control expression and efficiently visualize and isolate different cell populations (Å kerblom et al, 2013;Brown et al, 2007;Gentner and Naldini, 2012;Sachdeva et al, 2010). In this study, we used miR-124 to regulate neural conversion genes.…”