Simultaneous transfer of four functional genes from the HLA class II region into mammalian cells by fusion with yeast spheroplasts carrying an artificial chromosome.
Gene transfer using yeast artificial chromosome (YAC) clones provides an opportunity to study the expression of several linked genes within an environment more closely approximating their normal chromosomal context. A YAC clone spanning 330 kb of the HLA class II region from centromeric of TAP 1 to telomeric of HLA-DQA1 was retrofitted by homologous recombination with a neomycin plasmid targeted either to an Alu repeat sequence within the YAC genomic insert or to the Ura-3 gene within the right arm of the YAC vector. The modified YAC clones were transferred to Chinese hamster ovary or L cells by spheroplast fusion. Eight of 14 Alu-retrofitted and 10 of 15 right arm-retrofitted neomycin clones retained the six human loci known to be encoded by the YAC as well as portions of the left and right YAC vector arms. All tested L cell transformants showed IFN-gamma-inducible TAP 1 and TAP 2 mRNA expression. Two of eight analyzed clones expressed HLA-DQB mRNA and one of four expressed HLA-DQA. Cells expressing both the HLA-DQA and -DQB mRNA showed HLA-DQ cell surface expression. These studies establish the feasibility of introducing groups of functional genes into mammalian cells by spheroplast fusion with a single YAC clone.
Demmer
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LA
Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110.
Chaplin
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DD
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002459:Cell Fusion; D015825:Chromosomes, Fungal; D006224:Cricetinae; D005802:Genes, MHC Class II; D006683:HLA-DQ Antigens / Q000032:analysis / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection; D015003:Yeasts / Q000235:genetics
D006683:HLA-DQ Antigens; D012333:RNA, Messenger; D007371:Interferon-gamma