Purchase this article with an account.
R.D. Dix, E.E. Kozlowska, C.O. Ekworomadu, S.W. Cousins; A Retrovirus Vector for Expression of the Immediate–Early 1 Gene of Murine Cytomegalovirus in Mouse Monocytes/Macrophages. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1664.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Although sole expression of the immediate–early 1 (ie1) gene of murine cytomegalovirus (MCMV) has been associated with latent/persistent infection of mouse monocytes, the consequence of ie1 expression alone on cytokine expression and differentiation into activated macrophages is unknown. This information is essential to our understanding of the role of monocytes/macrophages in the pathogenesis of AIDS–related human CMV (HCMV) retinitis and perhaps other retinal diseases associated with HCMV infection. To test the hypothesis that MCMV ie1 expression alone upregulates expression of proinflammatory cytokines (e.g., TNF–α) in monocytes/macrophages, we developed a retrovirus vector containing the MCMV ie1 gene. Methods: A retrovirus vector containing the MCMV ie1 gene was prepared by cloning the ie1 gene (Dr. M. Messerle, University of Halle) into a lentivirus expression vector (Invitrogen). Monolayers of a mouse macrophage cell line (IC–21) were inoculated with either the ie1–containing vector or vector alone. At 48–72 hr postinoculation, cells were harvested and processed for PCR assay for detection of ie1 DNA, RT–PCR assay for detection of ie1 mRNA, and for western blot analysis and immunohistochemical staining for detection of the ie1 gene product using a MAb to pp89. Results: Mouse macrophages inoculated with the ie1–containing retrovirus vector harbored ie1 DNA and expressed ie1–specific mRNA. Moreover, expression of the ie1 gene product (pp89) was detected in these cells and at a high frequency. As expected, ie1–specific DNA, mRNA, or protein product were not detected in macrophages inoculated with vector only . Conclusions: A mouse macrophage cell line was successfully transfected to produce the MCMV ie1 gene product using a retrovirus vector. We are now using this vector to determine if expression of MCMV ie1 alone will activate mouse monocytes to produce proinflammatory cytokines and induce differentiation into activated macrophages.
This PDF is available to Subscribers Only