Cryopreservation and rederivation of mouse strains

Transcription

1 Cryopreservation and rederivation of mouse strains Raija Soininen Biocenter Oulu University of Oulu Finnish INFRAFRONTIER-EMMA node

2 Insurance against loss Accident/Catastrophe (fire/flood) Contamination (facility outbreak) Reproductive failure Disease Economic Advantages of Cryopreservation Significant long-term cost savings Huge space savings Scientific Minimize genetic drift Provides a genetic archive Animal welfare Reduces & Refines (3 R s) Alternative to shipping live animals

3 Cryopreservation and IVF, short history 1789 Frog sperm frozen in snow can fertilize frog eggs (Spallanzani) 1890 Embryo transplantation in rabbits (Heape) 1940 Maintenance of human sperm motility after freezing in -79, - 196, and/or -269 (Shettles) 1952 Fertilizing capacity of bull spermatozoa after freezing at 79 (Polge) 1953 Human sperm frozen by Sherman and Bunge 1957 Foal produced using frozen sperm from stallion (Barker and Gandier ) 1972 Mouse embryo cryopreservation and storage in LN2 (Wilmut) 1972 Live mice from cryopreserved embryos (Whittingham, Leibo & Mazur) 1978 First human baby via IVF (Edwards & Steptoe) 1990 Successful mouse sperm cryopreservation (Tada et al, Okuyama et al, Yokoyama et al.) 2008 Conserving, Distributing and Managing Genetically modified mouse lines by sperm cryopreservation (Ostermeier et al.) Brown family 1978

4 Cryopreservation of GM mouse lines Cryopreservation of morula-stage embryos (natural mating), complex genotype Sperm cryopreservation (haploid genome!) Sperm isolation and in vitro fertilization -> cryopreservation of 2-cell stage embryos Cryopreservation of zygotes Cryopreservation of oocytes (haploid genome!)

5 In vitro fertilization

6 Rescue of poor breeders/old colony Males: Isolation of sperm -> IVF Immotile sperm: Intracytoplasmic Sperm Injection (ICSI) Naomi Nakagata Sperm head is injected directly into the oocyte cytoplasm Young(ish) females: Ovary transplantation Oocyte isolation/cryopreservation -> IVF

7 Public cryoarchive Benefits to the researcher Higher visibility of strains More citations/collaborations No need to maintain the colony for collaborators No need to arrange the paperwork, shipping etc. + Sharing adds value!

8 European Mouse Mutant Archive 3rd largest repository worldwide A unified repository for maintaining mouse mutants Distribution to the scientific community Ownership remains by the submitting researcher Cryopreservation of sperm and embryos Technology development Courses, education

9 Pan-European Research Infrastructure Eumorphia, Empress, Europhenome Eumodic Protocols and services developed in several EC-funded projects (FP5-FP7): EMMA since

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11 Resources and services

12 EMMA cryopreservation service Procedure Strain submissions via INFRAFRONTIER website Submissions are evaluated by an external scientific committee After approval mice (or reprod.organs) are sent to one of the EMMA facilities for embryo or sperm cryopreservation Free of charge!

13 EMMA cryopreservation service Evaluation criteria Described phenotype with clear evidence of heritability Identifiable genotype Difficulty of maintenance relative to scientific value Other sources for the strain Popularity of the strain Importance for current/future research

14 Work flow and quality controls in sperm cryopreservation

15 Saving 14 animals/strain; 14 cage-weeks In vitro QC

16 EMMA database Strain data is stored in the EMMA database EMMA curators check the description and names Nomenclature service (rules and guidelines established by the International Committee on Standardized Genetic Nomenclature for Mice) Increased data quality Weekly feed to International Mouse Strain Resource (IMSR),

17 Advantages and of Cryopreservation Insurance against loss Accident/Catastrophe (fire/flood) Contamination (facility outbreak) Reproductive failure Disease Scientific Minimize genetic drift Provides a genetic archive Reproducibility Economic Significant long-term cost savings Huge space savings Animal welfare Reduces & Refines (3 R s) Alternative to shipping live animals

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20 EMMA distribution service public strain list If you plan to order embryos/sperm, contact the Tg core. (License!) If you plan to order mice, contact the Laboratory Animal Center. (need for rederivation?-> Tg Core)

21 Distribution service exports from EMMA Procedure Colonies on shelf and frozen material are shipped within 4 weeks after paperwork in place Rederivation from frozen stock takes 2-6 months Paperwork (Material Transfer Agreements, Purchase Orders) can take time Animals to be sent are SPF (specific pathogen free, FELASA recommendations) Protocols provided with the shipment

22 Transporting frozen germplasm Dry (vapour) shipper (liquid nitrogen, 5-14 days)

23 Technology development within INFRAFRONTIER Technology development groups for archiving and phenotyping Health monitoring development in archiving group Develop techniques that are More efficient Use fewer mice Cheaper Convenient for users

24 Alternatives to LN 2 or live mice transport Shipment of sperm in dry ice Shipment of embryos at +4 Shipment of epididymides instead of live mice

25 Shipping refrigerated mouse embryos from CNB-CSIC in Madrid Embryo transfer same or following day of arrival Linear distances: Orleans: 945 km Harwell: 1259 km Strasbourg: 1283 km Monterotondo: 1376 km Munich: 1486 km Vienna: 1812 km Oulu: 3308 km Lluis Montoliu

26 Rederivation The most effective method to eliminate pathogens from mice. Surgical transfer of embryos into pathogen-free recipient females. Healthy breeding colonies are established.

27 a) Import of live mice with one mutated allele or transgene insertion Order 2-4 fertile males Mutant males are mated with superovulated SPF wild-type females -> Embryo isolation, washing, and transfer -> SPF recipient females -> +/- pups (father homozygote), or +/- and +/+ (father hetero/hemizygote) (+ sperm cryopreservation)

28 b) Import of live mice with a complex genotype (several mutations/transgenes) Order both males and females i) Breeding -> embryo isolation (females put down!) -> transfer ii) Breeding -> pups to maintain the colony -> new breeding -> embryo isolation -> transfer c) Import of (cryopreserved) embryos/sperm IVF Embryo transfer

29 Rederivation/cleaning Contact the animal facility Animal license Provide a health screening report Contact the Tg Core Facility: Reetta Vuolteenaho/RS Place an order ( If the mouse line to be re-derived carries pathogenic viruses, a health screening is performed for the recipient mothers after the pups are weaned