Simultaneous transfer of four functional genes from the HLA class II region into mammalian cells by fusion with yeast spheroplasts carrying an artificial chromosome

Academic Article

Abstract

  • 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(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-γ-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.
  • Published In

    Author List

  • Demmer LA; Chaplin DD
  • Start Page

  • 5371
  • End Page

  • 5378
  • Volume

  • 150
  • Issue

  • 12