Melody Clark Roger Worland Gordana Grubor-Lajsic Jelena Pilipovic Sleeping Beauty.

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Presentation transcript:

Melody Clark Roger Worland Gordana Grubor-Lajsic Jelena Pilipovic Sleeping Beauty

cryoprotective dehydration anhydro / cryoprotectant (trehalose) Onychiurus arcticus – freeze avoiding

Fresh sample Sample cooled to –14°C

What has been done until now?

Determination of critical stages in the desiccation process samples cooled from +2 to ‑ 14°C (2°C per week) determined the freezing point and water content (DSC) polyol analysis (GC) the time taken to recover and return to normal water content was measured at each 2 degree interval we:

Going home

Onychiurus arcticus 1.Control live animals from +5°C 2.Desiccatingcooled to - 2°C (rate 2°C/week) 3.Fully desiccatedcooled to - 14°C (rate 2°C/week) 4.Recoveringfrom - 14°C, after 8 hours at RT with moisture 5.Fully recoveredfrom - 14°C, after 24 hours at RT with moisture

enzymes involved in synthesis of trehalose LEA proteins molecular analysis in Berlin (expecting sequences) antioxidative enzymes

-desiccation is not characteristic -rapidly cold hardens, on a daily basis Cryptopygus antarcticus – freeze avoiding removing ice nucleators cryoprotectants (glycerol)

1.Control live animals from +2°C 2.Cooled animals to -5°C (rate 0.5°C/min) 3.Heated animals to +15°C (rate of 1°C/min) 4.“High group” SCP when they were collected was above -15°C 5.“Low group” SCP when they were collected was below -15°C Cryptopygus antarcticus

microarray with cDNA of Cryptopygus antarcticus and Onychiurus arcticus together differential expression of genes in different treatment how closely related are the two species

Constructing 5 cDNA libraries (Stratagene pBluescript  II XR cDNA Library Construction Kit) Total RNA isolation with TRIZOL ( animals, ~0.05mg) Purification of poly A+ mRNA (Qiagen Oligotex mRNA Kit- Midi) Transformation (Stratagene XL10-Gold® Ultracompetent Cells) Sequencing ~ 1000 clones (BIG Dye Terminator)

What are we planning to do…

continue work on final microarray submit these sequences to database microarray just with Cryptopygus antarcticus as a pilot experiment (about 800 clones have already been printed) general primers for HSP and AQP

Collecting samples in the Arctic (August 13 th to 24th)

Thank you!