Localization of transfected B7-1 (CD80) DNA in human melanoma cells after particle-mediated gene transfer.
The purpose of this study was to evaluate stable DNA transfection of M-21 human melanoma cells with particle-mediated gene transfer (PMGT) with B7-1 cDNA and to identify sites of gene integration. Stable B7-1 transfectants (M-21-B7) were obtained with PMGT using a plasmid vector containing cDNA for both B7-1 and neomycin phosphotransferase, with subsequent selection with G418. The transfected cells were flow sorted by B7-1 expression into two populations, bright and dim. The bright population had 85%-90% of cells expressing B7-1; the dim population had less than 50% of cells with B7-1 expression. Chromosome analysis with fluorescence in situ hybridization (FISH) and G-banding showed that 70% of bright cells had two main integration sites, with extensive amplification of the transgene. The dim population had random signal distribution, with little or no amplification, despite G418 selection. Because B7-1 has been mapped to 3q21, FISH was performed using a chromosome 3 painting probe (WCP) together with a probe for B7-1. In transfected bright M-21 cells, amplified genes that hybridized with the B7-1 construct were localized to chromosome 3 material inserted into marker chromosomes. These data suggest that B7-1 insertion may involve homologous recombination, but maintenance of integration and amplification required selection.
McCarthy
Donna O
DO
Comprehensive Cancer Center, University of Wisconsin Hospital and Clinics, Madison, WI 53792, USA.
Meisner
Lorraine F
LF
Bourdeau-Heller
Jeanne
J
Roberts
Timothy R
TR
Wu
Shi Qi
SQ
Warner
Thomas F
TF
Albertini
Mark R
MR
D018122:B7-1 Antigen / Q000235:genetics*; D004247:DNA / Q000032:analysis*; D022761:Gene Transfer, Horizontal; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008545:Melanoma / Q000235:genetics*; D010957:Plasmids; D014162:Transfection; D019076:Transgenes; D014407:Tumor Cells, Cultured
D018122:B7-1 Antigen; D004247:DNA