Establishment of a galactocerebrosidase-deficient twitcher mouse cell line that expresses galactocerebrosidase activity in hybrids with control human fibroblasts |
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Authors: | Jukka T Konola Timothy A Lyerla Mark C Skiba Srinivasa Raghavan |
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Institution: | (1) Department of Biology, Clark University, 01610 Worcester, Massachusetts;(2) Biochemistry Division, Kennedy Shriver Center for Mental Retardation, 02254 Waltham, Massachusetts |
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Abstract: | Summary Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation
of skin obtained from 3-d-old littermates of atwi+/twi×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.
Partial support for these studies was provided by a National Institutes of Health AREA grant (HD21222-01) and a NIH subcontract
to Clark University from the Shriver Center for Mental Retardation This research forms a portion of studies performed to fulfill
the requirements for the Ph.D. degree in biology at Clark University for J. T. K. |
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Keywords: | twitcher mouse Krabbe disease galactocerebrosidase galactosylceramide β -galactosidase somatic cell hybrids |
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