The production of human cytokines, encoded by AAV9 vectors, was determined by infecting a murine macrophage cell line, MC57G (CRL2295TM - American Type Culture Collection, Manassas, VA), with the AAV9 vectors, followed by collecting the culture supernatant and performing ELISAs. Briefly, one day after plating MC57G cells in tissue culture plates, cells were infected with serially diluted AAV9 vectors encoding the respective human genes. Forty-eight hours after infection with AAV9 encoding each of the human cytokines, secretion of GM-CSF, IL-3, IL-4, IL-7, and IL-15 into the culture supernatant was determined using ELISAs [43] (link). Similarly, 48 hours after infecting MC57G cells with AAV9-A2, the level of A2 expression by the transduced MC57G cells was determined by FACS, using antibodies against HLA-A2 (clone BB7.2 - BioLegend, San Diego, CA) and hβ2m (Clone 2M2 - BioLegend)[35] (link), [44] (link).
Huang J., Li X., Coelho-dos-Reis J.G., Wilson J.M, & Tsuji M. (2014). An AAV Vector-Mediated Gene Delivery Approach Facilitates Reconstitution of Functional Human CD8+ T Cells in Mice. PLoS ONE, 9(2), e88205.
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