Fish PGC cryopreservation: is it a realistic tool for fish - - PowerPoint PPT Presentation

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

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

Contents:

Why and what for?

Genetic modification and selection Transplant

Biotechnological applications Surrogate production Conservation of endangered species

PGC cryopreservation PGC xenotransplantation

Host sterilization improves efficiency

PGC cryopreservation: tools for PGC visualization

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

PGC cryopreservation: protocols

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

• AFP 20mg/mL)

Riesco, Martínez‐Pastor, Chereguini and Robles 2012. Theriogenology 77:122‐130

PGC cryopreservation: protocols

PGC cryopreservation: evaluation after thawing

Viability Pseudopodial emission DNA integrity (Comet Assay) 90% survival ‹10% (≈ control)

Riesco, Martínez‐Pastor, Chereguini and Robles 2012. Theriogenology 77:122‐130

PGC cryopreservation: evaluation after thawing

Aitken et al. 2008 Lewis and Aitken 2005

Rothfuss et al. Nucleic Acids Research, 2010

Riesco and Robles 2012 J. Appl. Ichthyol. 28 (2012), 925–929

PGC cryopreservation: evaluation after thawing

Quantification of lessions in specific genes and genome regions

Riesco and Robles 2012 PLOS ONE (2013)

Lesions per 10 Kb

mRNAs

Riesco and Robles PLOS ONE (2013)

PGC cryopreservation: evaluation after thawing

PGC cryopreservation: evaluation after thawing

Promoter methylation

Patterson et al. J Vis Exp. (2011)

mRNAs

Riesco and Robles 2012 PLOS ONE (2013)

PGC cryopreservation: evaluation after thawing

Promoter methylation

Limitations and future perspectives: PGC in vitro generation

Limitations and future perspectives: PGC in vitro generation

Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

Limitations and future perspectives: PGC in vitro generation

Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

Limitations and future perspectives: PGC in vitro generation

Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

Limitations and future perspectives: PGC in vitro generation

Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

Limitations and future perspectives: PGC in vitro generation

Dnd MO (3months) Control (3months)

Germline contribution

Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

Limitations and future perspectives: PGC in vitro generation

in vivo

Hypermethylated promoter vasa vasa expression

A

CONFIRMED

in vitro

Cells non committed to be PGCs Cells non committed to be PGCs

vasa translation

HYPOTHESIS

Demethylated promoter vasa vasa expression dnd-1 translation DND-1 binding

A B

miRNA

B

miRNA Transcripts degradation dnd-1 expression vasa transcripts stabilization vasa translation dnd-1 expression

`

Limitations and future perspectives: PGC in vitro generation

Riesco, Valcarce, Alfonso, Herráez, Robles (2014), Biology of Reproduction 91 (5):114, 1‐11

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

Conclusions

Thank you!

vanesa.robles@st.ieo.es