Generation of a functional cDNA encoding the LdH2 class-I molecule by using a single-LTR retroviral shuttle vector

The use of a new single long-terminal-repeat retroviral shuttle vector has allowed us to obtain copies of the Ld gene with the first seven exons spliced correctly, as well as many other partially spliced or aberrantly recombined copies. Nucleotide sequencing performed on double-stranded DNA with primers specific for the vector and for the coding region of the gene, allowed rapid screening of the recovered plasmids. Synthetic oligodeoxyribonucleotides were then used to link the 5 nt of the last exon, and the functionality of the cDNA copy was verified by expression in transfected L(TK-) cells. Cells that produced the Ld antigen were detected by immunofluorescence and were shown to synthesize an immunoprecipitable molecule of the expected size. In addition, Ld-producing cells were susceptible to killing by Ld-restricted cytotoxic T-lymphocytes. This material should prove useful for mutagenesis and for expression in cell types in which expression of the genes of the major histocompatibility complex appears to be highly regulated.