title: Localization of transfected B7-1 (CD80) DNA in human melanoma cells after particle-mediated gene transfer.
abstract: 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.
authors.affiliation: Comprehensive Cancer Center, University of Wisconsin Hospital and Clinics, Madison, WI 53792, USA.
authors.forename: Donna O
authors.initials: DO
authors.lastname: McCarthy
authors.forename: Lorraine F
authors.initials: LF
authors.lastname: Meisner
authors.forename: Jeanne
authors.initials: J
authors.lastname: Bourdeau-Heller
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Roberts
authors.forename: Shi Qi
authors.initials: SQ
authors.lastname: Wu
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Warner
authors.forename: Mark R
authors.initials: MR
authors.lastname: Albertini
mesh_terms: 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
chemical_list: D018122:B7-1 Antigen; D004247:DNA