Gene targeting at the human CD4 locus by epitope addition.
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-1+ cell lines expressed protein only from the targeted allele; thus, these cells were functionally CD4-.
Department of Biochemistry, Beckman Center, Stanford University School of Medicine, California 94305-5307.
M
M
Jasin
S J
SJ
Elledge
R W
RW
Davis
P
P
Berg
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics; D015139:Blotting, Southern; D015704:CD4 Antigens / Q000235:genetics*; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics; D006801:Humans; D051379:Mice; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013601:T-Lymphocytes / Q000276:immunology; D018800:Thy-1 Antigens
D000954:Antigens, Surface; D015704:CD4 Antigens; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D018800:Thy-1 Antigens