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Fish PGC cryopreservation: is it a realistic tool for fish conservation biology? V. Robles, D.G. Valcarce and M.F. Riesco 5th Training School, Valencia Contents: For what purpose should we cryopreserve PGCs? PGC cryopreservation PGC

  1. Fish PGC cryopreservation: is it a realistic tool for fish conservation biology? V. Robles, D.G. Valcarce and M.F. Riesco 5th Training School, Valencia

  2. Contents: For what purpose should we cryopreserve PGCs? PGC cryopreservation ‐ PGC visualization ‐Cryopreservation protocols ‐How to evaluate the success of a PGC cryopreservation method? Limitations and future perspectives

  3. Why and what for? endangered species Conservation of Host sterilization improves efficiency PGC cryopreservation PGC xenotransplantation production Surrogate Biotechnological applications Genetic modification and Transplant selection

  4. PGC cryopreservation: tools for PGC visualization

  5. PGC cryopreservation: tools for PGC visualization There are alternative non‐transgenic methods  GFP‐nos 3′UTR mRNA  FITC (for sturgeon) Saito et al. 2014 Riesco and Robles 2015

  6. PGC cryopreservation: protocols

  7. PGC cryopreservation: protocols DMSO 2 M, EG 0,5 M (10 min) DMSO 5 M, EG 1M (2min) DMSO 5 M, EG 1 M, PVP 4 % (2min) (AFP 10mg/mL o AFP 20mg/mL)

  8. PGC cryopreservation: protocols Riesco, Martínez‐Pastor, Chereguini and Robles 2012. Theriogenology 77:122‐130

  9. PGC cryopreservation: evaluation after thawing Viability Pseudopodial emission 90% survival DNA integrity (Comet Assay) ‹10% (≈ control) Riesco, Martínez‐Pastor, Chereguini and Robles 2012. Theriogenology 77:122‐130

  10. PGC cryopreservation: evaluation after thawing Aitken et al. 2008 Lewis and Aitken 2005

  11. PGC cryopreservation: evaluation after thawing Quantification of lessions in specific genes and genome regions Lesions per 10 Kb Rothfuss et al. Nucleic Acids Research, 2010 Riesco and Robles 2012 J. Appl. Ichthyol. 28 (2012), 925–929 Riesco and Robles 2012 PLOS ONE (2013)

  12. PGC cryopreservation: evaluation after thawing mRNAs Riesco and Robles PLOS ONE (2013)

  13. PGC cryopreservation: evaluation after thawing Promoter methylation Patterson et al. J Vis Exp. (2011)

  14. PGC cryopreservation: evaluation after thawing Promoter methylation mRNAs Riesco and Robles 2012 PLOS ONE (2013)

  15. Limitations and future perspectives: PGC in vitro generation

  16. Limitations and future perspectives: PGC in vitro generation Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

  17. Limitations and future perspectives: PGC in vitro generation Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

  18. Limitations and future perspectives: PGC in vitro generation Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

  19. Limitations and future perspectives: PGC in vitro generation Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

  20. Limitations and future perspectives: PGC in vitro generation Germline contribution Control (3months) Dnd MO (3months) Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

  21. Limitations and future perspectives: PGC in vitro generation

  22. Limitations and future perspectives: PGC in vitro generation A CONFIRMED in vivo Hypermethylated vasa vasa expression promoter Cells non committed to be PGCs Cells non committed to be PGCs B Transcripts degradation miRNA A Demethylated vasa promoter vasa expression B HYPOTHESIS in vitro miRNA dnd-1 expression ` dnd-1 expression vasa transcripts stabilization dnd-1 DND-1 binding vasa translation translation vasa translation Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

  23. Conclusions PGCs can be successfully cryopreserved and successfully transplanted into host sterile embryos Molecular analysis after cryopreservation is crucial to guarantee the success of a cryopreservation protocol and avoid undesirable effects in fertilization and early embryo development. PGCs can be generated in vitro from embryonic cells

  24. Thank you! vanesa.robles@st.ieo.es

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