Date of Award
Master of Science
High levels in both class I MHC and susceptibility to gene delivery were seen in adherent cell cultures isolated from peripheral blood (PBAC). The morphology, patterns of expression, and response to functional assays of the PBAC cultures were characteristic of high endothelial cells. To characterize PBAC for expression of retroviral vector-delivered genes, PBAC cells were infected with recombinant retroviral vector carrying the gene for either green fluorescent protein (GFP) or bovine immunodeficiency virus (BIV) CA. A low level of GFP expression was detected in one to three percent of cells after two weeks, in ten percent of cells after three weeks, and in one hundred percent of cells after six weeks. Low levels of BIV CA expression were also detected in approximately fifty percent of infected PBAC after four weeks.These results indicated that retroviral vectors can deliver and express viral and non-viral genes in CTL target cells, though at a low level. Because CTL can be stimulated by very small quantities of protein, the use of retroviral vectors for delivery of viral genes to bovine high endothelial cells is a promising method for assessment of cellmediated immunity in cattle.In domestic animal species, assessment of cell-mediated immune responses to virus infection is hampered by the requirement for class I MHC compatibility between target and effector cells. An inability to infect target cells in vitro, or alternatively virus-induced lysis of infected target cells can also be complicating factors. One way to circumvent these problems is to use retrovirus-mediated gene transfer to deliver individual viral genes to autologous primary target cells. Several primary bovine cell cultures were assessed as potential target cells for cytotoxic T lymphocyte (CTL) assays. Class I MHC expression was analyzed by FACS using an anti-class I heavy chain antibody. Susceptibility to gene delivery was assayed by titration of retroviral vector on the primary cultures. All cells tested were susceptible to gene delivery and expressed class I MHC, though levels differed greatly among different primary cultures.
Joel Visser Earnest-DeYoung
Earnest-DeYoung, Joel Visser, "The use of retroviral gene delivery for development of a bovine cytotoxic T cell assay system" (1997). Retrospective Theses and Dissertations. 18421.