AAV2-CMV-mCherry

Microglia are a specialized population of myeloid cells that mediate innate immune responses in the central nervous system. Cultured microglia are refractory to most chemical and electrical transfection methods, resulting in little gene delivery and toxic and/or inflammatory activation. Recombinant adeno-associated viral (rAAV) vectors are non-enveloped, single-stranded DNA vectors that are commonly used to transduce many primary cell types and tissues. Here, researchers evaluated the feasibility and efficiency of using rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 produces high transduction rates and causes minimal toxicity or inflammatory responses in both newborn and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, researchers successfully excised the LoxP-flanked miR155 gene in cultured microglia using rAAV2 expressing Cre recombinase. rAAV serotypes 5, 6, 8, and 9 were further evaluated and found to be able to transduce cultured microglia efficiently to varying degrees with little to no changes in inflammatory gene expression. These results provide strong encouragement for applying rAAV-mediated gene expression to microglia for mechanistic and therapeutic purposes.

The researchers tested the viability of rAAV on microglia isolated from adult mouse brain. Adult microglia were used for viral infection starting at day 7 after isolation. The rAAV2-CMV-mCherry vector was diluted and added to the culture medium. Approximately 7 days after vector addition, cultured microglia were fixed and stained with an anti-RFP antibody, followed by microscopic examination to detect RFP expression (Figure 1a). Greater than 98% of newborn microglia expressed RFP, with no obvious morphological changes compared to uninfected cells. RFP expression was plotted onto a standard curve generated by titrating serial amounts of rAAV2-CMV-mCherry DNA into total purified cellular RNA (Figure 1b). The average Ct value for RFP detected from uninfected cells was 36.3, while the average Ct value for RFP detected from rAAV2-CMV-mCherry infected cells was 25.84 (Figure 3b). Similarly, more than 99% of adult microglia successfully expressed RFP 7 days after infection, with no obvious morphological changes or reduction in cell number (Figure 1c). The average Ct value for RFP mRNA in uninfected cells was 36.04, and the average Ct value for RFP mRNA in rAAV2-CMV-mCherry-infected cells was 27.59 (Figure 1d). These results show that both newborn and adult microglia can be transduced to express foreign genes using rAAV2 without morphological evidence of inflammatory activation or toxicity.

Figure 1. Recombinant adeno-associated viral (rAAV)2 is an effective vector for gene delivery in cultured neonatal and adult microglia. (Su W, et al., 2016)