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Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer.

Mastromonaco GF, Perrault SD, Betts DH, King WA - BMC Dev. Biol. (2006)

Bottom Line: 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.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.Following SCNT, short-lived cultures resulted in significantly lower blastocyst development (< or = 0.9%) compared to highly proliferative cultures (11.8%).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada. gmastrom@uoguelph.ca

ABSTRACT

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.

Conclusion: 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.

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Length of time to reach primary confluence for cattle and gaur cell cultures. The numbers of days necessary for the dissociated cells to become confluent monolayers are shown for the sample collection (DART, PUNCH, EAR/SKIN) and cell dissociation (EXPL, COLL) treatment groups. Time to reach confluency varied according to the sample collection and cell dissociation techniques used.
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Figure 1: Length of time to reach primary confluence for cattle and gaur cell cultures. The numbers of days necessary for the dissociated cells to become confluent monolayers are shown for the sample collection (DART, PUNCH, EAR/SKIN) and cell dissociation (EXPL, COLL) treatment groups. Time to reach confluency varied according to the sample collection and cell dissociation techniques used.

Mentions: Culture characteristics were examined for DART, PUNCH and EAR/SKIN samples established using EXPL and COLL techniques. The time to primary confluence varied according to the sample collection technique used (Figure 1). Small initial samples, as in DART, required >20 days to achieve confluence compared to larger samples, as in EAR, which required only 5–6 days (COLL) and 11–12 days (EXPL). In all cases, EXPL treatments required more time than COLL to achieve confluence.


Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer.

Mastromonaco GF, Perrault SD, Betts DH, King WA - BMC Dev. Biol. (2006)

Length of time to reach primary confluence for cattle and gaur cell cultures. The numbers of days necessary for the dissociated cells to become confluent monolayers are shown for the sample collection (DART, PUNCH, EAR/SKIN) and cell dissociation (EXPL, COLL) treatment groups. Time to reach confluency varied according to the sample collection and cell dissociation techniques used.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1590017&req=5

Figure 1: Length of time to reach primary confluence for cattle and gaur cell cultures. The numbers of days necessary for the dissociated cells to become confluent monolayers are shown for the sample collection (DART, PUNCH, EAR/SKIN) and cell dissociation (EXPL, COLL) treatment groups. Time to reach confluency varied according to the sample collection and cell dissociation techniques used.
Mentions: Culture characteristics were examined for DART, PUNCH and EAR/SKIN samples established using EXPL and COLL techniques. The time to primary confluence varied according to the sample collection technique used (Figure 1). Small initial samples, as in DART, required >20 days to achieve confluence compared to larger samples, as in EAR, which required only 5–6 days (COLL) and 11–12 days (EXPL). In all cases, EXPL treatments required more time than COLL to achieve confluence.

Bottom Line: 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.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.Following SCNT, short-lived cultures resulted in significantly lower blastocyst development (< or = 0.9%) compared to highly proliferative cultures (11.8%).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada. gmastrom@uoguelph.ca

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus