Introduction of new genetic markers on human chromosomes.
The purpose of this study was to use DNA transfection and microcell chromosome transfer techniques to engineer a human chromosome containing multiple biochemical markers for which selectable growth conditions exist. The starting chromosome was a t(X;3)(3pter----3p12::Xq26----Xpter) chromosome from a reciprocal translocation in the normal human fibroblast cell line GM0439. This chromosome was transferred to a HPRT (hypoxanthine phosphoribosyltransferase)-deficient mouse A9 cell line by microcell fusion and selected under growth conditions (HAT medium) for the HPRT gene on the human t(X;3) chromosome. A resultant HAT-resistant cell line (A9(GM0439)-1) contained a single human t(X;3) chromosome. In order to introduce a second selectable genetic marker to the t(X;3) chromosome, A9(GM0439)-1 cells were transfected with pcDneo plasmid DNA. Colonies resistant to both G418 and HAT medium (G418r/HATr) were selected. To obtain A9 cells that contained a t(X;3) chromosome with an integrated neo gene, the microcell transfer step was repeated and doubly resistant cells were selected. G418r/HATr colonies arose at a frequently of 0.09 to 0.23 x 10(-6) per recipient cell. Of seven primary microcell hybrid clones, four yielded G418r/HATr clones at a detectable frequency (0.09 to 3.4 x 10(-6)) after a second round of microcell transfer. Doubly resistant cells were not observed after microcell chromosome transfers from three clones, presumably because the markers were on different chromosomes. The secondary G418r/HATr microcell hybrids contained at least one copy of the human t(X;3) chromosome and in situ hybridization with one of these clones confirmed the presence of a neo-tagged t(X;3) human chromosome. These results demonstrate that microcell chromosome transfer can be used to select chromosomes containing multiple markers.
Satoh
H
H
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Barrett
J C
JC
Oshimura
M
M
D002460:Cell Line; D002874:Chromosome Mapping / Q000379:methods*; D002877:Chromosomes, Human / Q000648:ultrastructure*; D004351:Drug Resistance / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics*; D005821:Genetic Techniques; D006801:Humans; D006822:Hybrid Cells; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D009355:Neomycin / Q000494:pharmacology
D005819:Genetic Markers; D007041:Hypoxanthine Phosphoribosyltransferase; D009355:Neomycin