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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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Morphology of gaur cell cultures during serial cultivation in vitro. Phase contrast micrographs of gaur EAR fibroblast cultures from early and late passages demonstrate the changes in cell morphology that occurred with increased passages. Note the morphology (enlarged and flattened cells) characteristic of senescent cultures in (I) and (J). No major effects of cell dissociation techniques were observed in these long-lived cell cultures.
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Figure 3: Morphology of gaur cell cultures during serial cultivation in vitro. Phase contrast micrographs of gaur EAR fibroblast cultures from early and late passages demonstrate the changes in cell morphology that occurred with increased passages. Note the morphology (enlarged and flattened cells) characteristic of senescent cultures in (I) and (J). No major effects of cell dissociation techniques were observed in these long-lived cell cultures.

Mentions: To examine phenotypic changes during serial cultivation, culture morphology was assessed. Long-lived cultures (gaur EAR, Figure 3; cattle SKIN and EAR not shown) exhibited a typical trend from the tightly compact spindle-shaped cells in early culture (Figure 3A–D) to a gradual enlarging of cells until the spread out, star-shaped phenotype of senescence was observed (Figure 3I–J). In contrast, within the first few passages, short-lived cultures (DART and PUNCH, Figure 4) displayed the phenotypic changes (enlarging and spreading) characteristic of the late passage cells of long-lived cultures approaching senescence.


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)

Morphology of gaur cell cultures during serial cultivation in vitro. Phase contrast micrographs of gaur EAR fibroblast cultures from early and late passages demonstrate the changes in cell morphology that occurred with increased passages. Note the morphology (enlarged and flattened cells) characteristic of senescent cultures in (I) and (J). No major effects of cell dissociation techniques were observed in these long-lived cell cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Morphology of gaur cell cultures during serial cultivation in vitro. Phase contrast micrographs of gaur EAR fibroblast cultures from early and late passages demonstrate the changes in cell morphology that occurred with increased passages. Note the morphology (enlarged and flattened cells) characteristic of senescent cultures in (I) and (J). No major effects of cell dissociation techniques were observed in these long-lived cell cultures.
Mentions: To examine phenotypic changes during serial cultivation, culture morphology was assessed. Long-lived cultures (gaur EAR, Figure 3; cattle SKIN and EAR not shown) exhibited a typical trend from the tightly compact spindle-shaped cells in early culture (Figure 3A–D) to a gradual enlarging of cells until the spread out, star-shaped phenotype of senescence was observed (Figure 3I–J). In contrast, within the first few passages, short-lived cultures (DART and PUNCH, Figure 4) displayed the phenotypic changes (enlarging and spreading) characteristic of the late passage cells of long-lived cultures approaching senescence.

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