Early phase karyotype analysis of chromosome segregation after formation of mouse-mouse hybridomas with chromosome painting probes

FISH analysis with chromosome painting probes allows, better than karyotypi ng after Giemsa banding, the study of chromosome segregation after hybridom a formation. FISH is particularly useful for intraspecies hybrids and allow s visualization of small chromosome fragments. Cell hybrids were constructe d between P3 x 63Ag8.653 mouse myeloma cells and lymphocytes from BALB/c mi ce by PEG fusion and by selection in hypoxanthine-azaserine medium. Three h ybridomas (A4, D8, F10) were selected and, after cloning, the cells were cu ltivated in vitro over a period of 28 days. During this time in culture, ai r-dried metaphase spreads were prepared by standard methods. For FISH chrom osome painting, digoxigenin- and biotin-labeled mouse chromosome painting p robes and rhodamine-antidigoxigenin antibodies and fluorescein-avidin were used for dual color detection. Total chromosome numbers and the numbers of mouse chromosomes 1, X, 6 and 12 were estimated as function of days in cult ure. Mean chromosome numbers of 78 (D8), 82 (F10) and 150 (A4) were observe d. The major rearrangements of chromosome numbers occured in the first 28 d ays in culture and did not change significantly between day 28 and day 56. Mouse chromosome #12, which had the largest chromosome fragments in the par ent myeloma, remained stable while the number of X chromosomes, which were significantly fragmented already in the parent myeloma, decreased by approx imately 50%.