Simultaneous transfer of four functional genes from the HLA class II region into mammalian cells by fusion with yeast spheroplasts carrying an artificial chromosome
Demmer, L.A.; Chaplin, D.D.
Journal of Immunology 150(12): 5371-5378
1993
ISSN/ISBN: 0022-1767 PMID: 8515065 Document Number: 418322
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-DQAI was retrofitted by homologous recombination with a neomycin-r 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-r 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.