Establishment of a galactocerebrosidase-deficient twitcher mouse cell line that expresses galactocerebrosidase activity in hybrids with control human fibroblasts.
Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of a twi+/twi X twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression.
Konola
J T
JT
Department of Biology, Clark University, Worcester, Massachusetts 01610.
Lyerla
T A
TA
Skiba
M C
MC
Raghavan
S
S
D000630:Aminopterin / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology; D002455:Cell Division; D002460:Cell Line; D005347:Fibroblasts / Q000201:enzymology*; D005696:Galactosidases / Q000172:deficiency*; D005698:Galactosylceramidase / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis; D006801:Humans; D006822:Hybrid Cells / Q000201:enzymology*; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis; D007621:Karyotyping; D007668:Kidney / Q000201:enzymology; D007965:Leukodystrophy, Globoid Cell / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008818:Mice, Neurologic Mutants; D010042:Ouabain / Q000494:pharmacology; D011003:Ploidies; D013936:Thymidine / Q000494:pharmacology; D014960:X Chromosome
D007042:Hypoxanthines; D007527:Isoenzymes; D019271:Hypoxanthine; D010042:Ouabain; D005954:Glucosephosphate Dehydrogenase; D005696:Galactosidases; D005698:Galactosylceramidase; D000630:Aminopterin; D013936:Thymidine