Abstract
BackgroundSomatic 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.ResultsDifferences 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 (≤ 0.9%) compared to highly proliferative cultures (11.8%). Chromosome stability and sample collection technique were significant factors in determining blastocyst development outcome.ConclusionThese 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.
Highlights
Somatic cell nuclear transfer (SCNT) provides an appealing alternative for the preservation of genetic material in non-domestic and endangered species
Following the determination that chromosome stability was highly dependent on a combination of initial Relative telomere lengths (RTL) and RTL rate of change, we examined the possibility that the blastocyst development rate of cell lines used for SCNT was dependent on the chromosome stability of the donor cell line
This study was performed to gain a better understanding of the effects of culture initiation techniques on culture characteristics and the subsequent developmental potential of SCNT embryos
Summary
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 development of novel embryo production technologies including somatic cell nuclear transfer (SCNT) has created the potential for the restoration and propagation of valuable genetic lines, and the preservation of endangered species/breeds. Genome resource banks have been established for the collection and storage of somatic/ gonadal tissues, cells, gametes and embryos from genetically valuable individuals and lineages. To ensure that cells are of the utmost quality for possible use as donors for SCNT, a need exists for the identification of quantifiable markers with a direct relationship to developmental potential
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