Gene targeting at the human CD4 locus by epitope addition

Abstract

Homologous recombination at the CD4 locus in a human T-cell line has been achieved by an approach called epitope addition. The endogenous CD4 gene provided transcription, translation, and leader sequences to a crippled introduced Thy-1 gene, resulting in the expression of murine Thy-1 epitopes on the surface of the human cells. Thy-1+ cells were selected using the Fluorescence Activated Cell Sorter (FACS). An estimated 700-fold enrichment for homologous versus nonhomologous integration events was obtained, such that 70% of cells scoring positive for Thy-1 were derived from gene targeting. Three of the Thy-l+ cell lines expressed protein only from the targeted allele; thus, these cells were functionally CD4-.