Assessing the effects of temperature and culture medium on the culture of monotreme fibroblasts

  Below are information to guide you.I have also attached the marking rubrics so you understand the expectation Platypus papers : McMillan (2007) Why do we need to optimise culture of platypus fibroblasts? What can you use dibroblass for Platypus conservation iPS cells and cloning? Storage of material in gene banks (can we use standard mouse culture conditions?) Focus of current research Platypus genome (evolutionary history of monotremes Chromosome composition and sex determination genes Fibroblasts are easily accessible tissue from which DNA can be extracted for genetic studies and analysis. Cell culture 32 degrees in 50% (DMEM + 10% FCS) and 50% Aminiomax Karyotype Wrigley (1983) Early culture of platypus fibroblast cell lines Karyotype Cell culture: 23 degrees in DMEM with 10% or 20% FCS Cell counting daily intervals (Haemocytometer and Trypan Blue viability stain) Imaged under microscope Passaging (contact inhibition) Cells did not survive at 37 degrees - failed to attach or lifted of surface if temperature changed Grunchuk (2014) Cultivation of chinese hamster fibroblasts under elevated temperature Themoresistence phenotype or heat shock response - effect on cell type Reduced mitochondrial number, necrosis response Activity dividing cells are more sensitive to heat shock stress Cells related after 48 - 72 hrs Effect on karyotype Expression of HSP proteins (using PCR)