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Researcher Sharma, Papori
Advisor Yadav, A. S.
Source KrishiKosh-Indian National Agricultural Research System
Content type Text
Educational Degree Doctor of Philosophy (Ph.D.)
Publisher LUVAS
File Format PDF
Language English
Subject Domain (in DDC) Natural sciences & mathematics ♦ Animals (Zoology) ♦ Specific topics in natural history
Subject Keyword Sodium Butyrate (nabu) ♦ Immunocytochemistry ♦ Qpcr ♦ Hdac ♦ Epigenetic Marker ♦ Animal Genetics and Breeding ♦ Studies On Epigenetic Characteristics of Somatic Cells In Water Buffaloes
Abstract To improve the efficiency of SCNT, knowledge of epigenetic status of donor cells (Somatic cells) are essential, which till date is missing in buffaloes. Keeping this in mind present study was planned to elucidate the molecular characteristics of somatic cells derived from adult and new born fibroblast cells in buffaloes, to assess the epigenetic status of two types of somatic cells epigenetic status after the treatment of chromatin remodeling agent, Sodium Butyrate (NaBu). The cultured adult and new born fibroblast/somatic cells were characterized for fibroblast properties by fibroblast specific marker proteins (Vimentin, Tubulin, Cytokeratin) using immunostaining and by fibroblast specific marker gene (Vimentin, Tubulin, Cytokeratin) expression with qPCR. The chromosomal integrity was also analyzed. Adult as well as new born somatic cells were treated with NaBu; 1mM, 3mM and 5mM and observations were taken after 48hrs.Morphological changes Population doubling, cell proliferation, epigenetic change were observed and analyzed (H3K9ac and H3K27me3). The adult cells took more time to attain confluence as compared to new born cell line. However, both type of cells behaved like typical fibroblast cells in morphology and growth pattern; spindle shaped. The somatic cells of adult as well as new born buffalo cell lines were also characterized with the help of fibroblast specific gene Vimentin . It was found that cytokeratin gene expression was present in the adult cells but not in the new born cells. Chromosomal integrity at different passages, 50 (2n). The morphology of the cells changed after NaBu treatment after 48hrs (1mM, 3mM and 5mM). Doubling time increases with the increase in concentration and decrease in the proliferation of cells. Epigenetic (acetylation) marker, H3K9ac revealed that the acetylation level in the four groups in both adult and new born fibroblast cells increased significantly in dose dependent manner (P 0.05). Acetylation in new born cells were significantly higher (P 0.05) as compared to the adult cells.Epigenetic (methylation) marker H3K27me3 in NaBu treated adult fibroblast cells as indicated by the mean pixel intensity, the methylation level in the four groups of adult cell line didn’t differ significantly (P 0.05). The methylation level in the four groups of new born cell line increased significantly (P 0.05). The methylation status of adult cells were significantly higher (P 0.05) as compared to the new born cells. The study concludes that purity of fibroblast can be ascertained using Vimentin, Chromatin remodeling agent NaBu increases histone acetylation but doesn't reduces methylation. Acetylation was found more in New born cell line as compared to Adult cell lines. However, Adult cell lines are more methylated as compared to new born cell lines.
Educational Use Research
Education Level UG and PG
Learning Resource Type Thesis
Size (in Bytes) 7.16 MB