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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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Chromosome content of cell cultures during serial cultivation in vitro. Percentages of chromosomally normal cells (% Normal Cells) were assessed for (A) cattle and (B) gaur cell cultures. A decrease in chromosomally normal cells occurred in all cultures with increased passages. Differences in the initial percentage and the rate of decline of chromosomally normal cells were observed in the different treatment groups. Effects of sample collection techniques were clearly evident in the gaur cell cultures.
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Figure 5: Chromosome content of cell cultures during serial cultivation in vitro. Percentages of chromosomally normal cells (% Normal Cells) were assessed for (A) cattle and (B) gaur cell cultures. A decrease in chromosomally normal cells occurred in all cultures with increased passages. Differences in the initial percentage and the rate of decline of chromosomally normal cells were observed in the different treatment groups. Effects of sample collection techniques were clearly evident in the gaur cell cultures.

Mentions: To examine changes in genomic stability during serial cultivation, chromosome content of the cultures was assessed. Analysis of metaphase spreads at P-30 was not successful due to the low numbers of actively dividing cells in the late passage cultures. A decrease in the percentage of chromosomally normal cells was evident in all cultures during serial cultivation (Figure 5A–B). Abnormalities included primarily aneuploidy and polyploidy. The initial percent normal cells, final percent normal cells, and rate of change (slope) varied between cultures, indicating differences due to both sample collection and cell dissociation techniques. Long-lived cultures (cattle SKIN/EAR, Figure 5A; gaur EAR, Figure 5B) showed a gradual decrease (slope range: -0.75 to -2.00, Table 1) in chromosomally normal cells from 82–90% normal down to 42–70% normal by passage 25 (50 PDL). Short-lived cultures (DART and PUNCH, Figure 5B) initially consisted of a lower percentage of chromosomally normal cells and dropped dramatically (slope range: -4.44 to -10.00, Table 1) within the first few passages to <40% normal cells. In general, EXPL cultures resulted in a lower percentage of normal cells compared to COLL cultures. The DART EXPL cell line failed to establish and was not analyzed beyond P-1, which already consisted of only 42% normal cells.


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)

Chromosome content of cell cultures during serial cultivation in vitro. Percentages of chromosomally normal cells (% Normal Cells) were assessed for (A) cattle and (B) gaur cell cultures. A decrease in chromosomally normal cells occurred in all cultures with increased passages. Differences in the initial percentage and the rate of decline of chromosomally normal cells were observed in the different treatment groups. Effects of sample collection techniques were clearly evident in the gaur cell cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Chromosome content of cell cultures during serial cultivation in vitro. Percentages of chromosomally normal cells (% Normal Cells) were assessed for (A) cattle and (B) gaur cell cultures. A decrease in chromosomally normal cells occurred in all cultures with increased passages. Differences in the initial percentage and the rate of decline of chromosomally normal cells were observed in the different treatment groups. Effects of sample collection techniques were clearly evident in the gaur cell cultures.
Mentions: To examine changes in genomic stability during serial cultivation, chromosome content of the cultures was assessed. Analysis of metaphase spreads at P-30 was not successful due to the low numbers of actively dividing cells in the late passage cultures. A decrease in the percentage of chromosomally normal cells was evident in all cultures during serial cultivation (Figure 5A–B). Abnormalities included primarily aneuploidy and polyploidy. The initial percent normal cells, final percent normal cells, and rate of change (slope) varied between cultures, indicating differences due to both sample collection and cell dissociation techniques. Long-lived cultures (cattle SKIN/EAR, Figure 5A; gaur EAR, Figure 5B) showed a gradual decrease (slope range: -0.75 to -2.00, Table 1) in chromosomally normal cells from 82–90% normal down to 42–70% normal by passage 25 (50 PDL). Short-lived cultures (DART and PUNCH, Figure 5B) initially consisted of a lower percentage of chromosomally normal cells and dropped dramatically (slope range: -4.44 to -10.00, Table 1) within the first few passages to <40% normal cells. In general, EXPL cultures resulted in a lower percentage of normal cells compared to COLL cultures. The DART EXPL cell line failed to establish and was not analyzed beyond P-1, which already consisted of only 42% normal cells.

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