O-14: In vitro TERT mRNA Expression in Relation to Chromosomal Stability, Senescence and Telomere Length and SCNT Cloned Embryo Production in Bubalus Bubalis

Background: Somatic cell nuclear transfer (SCNT) provides an appealing alternative for the conservation and improvement of genetic material of unique 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. This study was conducted to improve SCNT success rates in buffaloes through viable and chromosomally normal cells. The degree of cellular senescence, chromosomal abnormality and telomere shortening of skin fibroblast cells in cultures were analysed in relation to telomerase reverse transcriptase (TERT) mRNA expression with real time PCR and competent donor cells for SCNT of water buffaloes (Bubalus bubalis) were selected. Materials and Methods: Skin tissues (1 cm x 1 cm) of ear pinna of adult Bhadawari buffaloes were prepared upto 24th passage. The cell lines were tested for cell proliferation assay, growth curve analysis, in vitro cell senescence, telomere length assay, Karyological studies through metaphase stage analysis, aging genes expression analysis for every 3rd passage cell lines. Results: Primary cells isolated from buffalo ear biopsy were serially cultured up to 24th passage. Cell proliferation rate (CPR) increased up to 9th passage and decreased later. Growth curve analysis showed sigmoid pattern in 8 day of culture (P5) and till mid (P15) passage cells only. Replicative senescence (β gal+ blue stained) also increased due to contact inhibition of cells in later passages. Chromosomal abnormalities (polyploidy and aneuploidy) increased up to 17.50% in later passage group cells. Mean telomere repeat fragment (TRF) loss increased (p≤0.5) from 6th to 24th passages. TERT mRNA expression was detected from 9th passage and increased up to 24th passage. The TERT mRNA expression was positively correlated with chromosomal abnormality and mean TRF loss. Skin fibroblast cells showed increased lifespan in culture and expressed TERT mRNA. SCNT embryo production rate was highest from 9th-15th passages nuclear donor cells as these cells showed relatively stable genome amenable for genetic reprogramming after attaining totipotency in SCNT embryos. In our study, the cleavage rate was significantly lower (48.60%) in zygote derived from early passage (3-6) cultures than in zygote derived from mid passaged (9-15) cultures (55.50%). Further, cleavage rate (42.40%) in zygote developed from cells of later passages (16-24) was again lower. SCNT embryos derived from mid passage cultures showed higher (p≤0.05) blastocyst production rate than the blastocyst derived from early and late passage cultures. Conclusion: This study on effect of passaging on aging and genome stability of donor cells in culture, will help to understand influence of multiple passaging on in vitro growth characteristics, replicative senescence, chromosome content, telomere dynamics, TERT expression of somatic cells and the subsequent developmental potential of SCNT embryos. Furthermore, various markers can be assessed in the initial phases of culture to evaluate the proliferative potential of a donor cell for SCNT. The significant correlation between chromosome stability, TERT expression and SCNT embryo development will provide a means for assessing the developmental potential of a donor cell. This will further help for somatic cell banking of unique and endangered valuable germplasm of livestock species for conservation and improvement