ESTABLISHMENT OF A LYMPHOBLASTOID CELL LINE USING A MUTANT MDV CONTAINING A GREEN FLUORESCENT PROTEIN EXPRESSION CASSETTE

We have previously described the construction and characterization of mutant Marek’s disease viruses (MDVs) having mutations within the unique-short (US) region of the genome that have retained oncogenicity (Anderson et al., 1998; Parcells et al., 1995). We have also reported the characterization of lymphoblastoid cell lines (LBCLs) derived using these mutant viruses (Parcells et al., 1998). These mutant MDVs were constructed using a lacZ expression cassette. Expression of lacZ was found to be constitutive during lytic infection but was found to be tightly repressed in tumors and the derived LBCLs. The construction of these viruses and the analysis of lacZ induction required the use of toxic substrates or antibody staining to detect lacZ expression. We now report the establishment of an MDV lymphoblastoid cell line, MDCC-UA04, that was derived from a tumor induced by an MDV having an insertion of a green fluorescent protein expression cassette into the US2 gene. Like previous mutant-derived LBCLs, expression of the marker cassette is constitutive in lytic infection, but repressed in tumors and in the UA04 cells. UA04 cells express CD3low, CD4, TCR-2 low, MHC class II, and CD28 antigens on their surface. The percentage of UA04 cells expressing GFP is generally low (5–7%), but increases markedly within 48 hrs of 5ʹiododeoxyuridine (IUdR) treatment (20–30%) in a manner similar to many MDV lytic antigens. Thus, induction of GFP expression in UA04 cells can serve as a non-invasive marker for MDV reactivation from latency.