• Isolation and culturing of zebrafish pluripotent cells

      Sana, Salma (University of Bedfordshire, 2012-10)
      Zebrafish (Dania rerio) is an important model organIsm for the studies of vertebrate development and gene expression in the field of molecular biology and biomedicine. Its embryonic stem cells provide a unique tool linking in vitra and in vivo genetic manipulations of animal genomes. The aim of the project was to determine the most suitable embryo stage for the isolation and culturing of pluripotent cells of zebrafish embryos. Studies were carried out to investigate the expression of two pluripotency markers i.e. Oct4 and Sa1l4 at certain embryonic stages employing Immunohistochemistry. The protein expression studies indicated maximum expression of Oct4 and Sa1l4 at high stage. Primary cultures were initiated from zebrafish high stage embryos in basal nutrient medium; supplemented with insulin, selenite, epidermal growth factor and Foetal Bovine serum. Experiments were conducted for the determination of optimum concentrations of FBS. The growing cultures identified the signs of differentiation i.e. melanocytes and neurite formation. Basic fibroblast growth factor (bFGF) was found to inhibit the differentiation in cultures. The developed pluripotency markers were tested on cultured embryonic cells. Results indicated that these markers work well for the identification ofphenotype of embryonic cell colonies in zebrafish. The establishment of pluripotent cell line will enable knock out and knock in technologies to be used in this species and have important applications in functional genomics research.