Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer.
BACKGROUND
Somatic cell nuclear transfer (SCNT) provides an appealing alternative for the preservation of genetic material in non-domestic and endangered species. An important prerequisite for successful SCNT is the availability of good quality donor cells, as normal embryo development is dependent upon proper reprogramming of the donor genome so that embryonic genes can be appropriately expressed. The characteristics of donor cell lines and their ability to produce embryos by SCNT were evaluated by testing the effects of tissue sample collection (DART biopsy, PUNCH biopsy, post-mortem EAR sample) and culture initiation (explant, collagenase digestion) techniques.


RESULTS
Differences in initial sample size based on sample collection technique had an effect on the amount of time necessary for achieving primary confluence and the number of population doublings (PDL) produced. Thus, DART and PUNCH biopsies resulted in cultures with decreased lifespans (<30 PDL) accompanied by senescence-like morphology and decreased normal chromosome content (<40% normal cells at 20 PDL) compared to the long-lived (>50 PDL) and chromosomally stable (>70% normal cells at 20 PDL) cultures produced by post-mortem EAR samples. Chromosome stability was influenced by sample collection technique and was dependent upon the culture's initial telomere length and its rate of shortening over cell passages. Following SCNT, short-lived cultures resulted in significantly lower blastocyst development (< or = 0.9%) compared to highly proliferative cultures (11.8%). Chromosome stability and sample collection technique were significant factors in determining blastocyst development outcome.


CONCLUSIONS
These data demonstrate the influence of culture establishment techniques on cell culture characteristics, including the viability, longevity and normality of cells. The identification of a quantifiable marker associated with SCNT embryo developmental potential, chromosome stability, provides a means by which cell culture conditions can be monitored and improved.
Mastromonaco
Gabriela F
GF
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada. gmastrom@uoguelph.ca
Perrault
Steve D
SD
Betts
Dean H
DH
King
W Allan
WA
D000818:Animals; D001706:Biopsy; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival; D043171:Chromosomal Instability; D002869:Chromosome Aberrations; D002874:Chromosome Mapping; D004423:Ear; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D008297:Male; D008677:Metaphase; D053652:Nuclear Transfer Techniques; D012867:Skin / Q000166:cytology; D016615:Telomere / Q000235:genetics* / Q000648:ultrastructure