SERVICES
 

CRISPR-Cas9 mouse model generation services

The Platform provides support for the design and generation of mouse models such as conditional knockout mice and targeted mutations, insertion of knock-in cassettes, using targeting vectors or single-stranded DNA donors. Projects and design are discussed with platform users. Our service includes:

     - DNA donor design (single DNA donor or targeting vector)

     - guide RNAs design

     - Ribonucleprotein complex (Cas9 and guide RNAs) and DNA donor injection into fertilized oocytes

     - transfer of injected oocytes into oviduct of pseudo-pregnant female mice

Platform users take charge of ordering the guide RNAs and single strand DNA donor (Ultramer or Megamer).

Generation of transgenic mice by injection of DNA construct in fertilized oocytes

The platform also provides help in the design of the DNA constructs and shares available plasmids.

Homologous recombination

Platform users take charge of generating DNA constructs. Culture, electroporation and selection of ES cells is performed by platform users under supervision of Dr Y. Achouri. The platform takes charge of injecting the recombined ES cells in blastocysts and reimplanting the injected blastocysts into oviduct of pseudo-pregnant female mice.

Cryopreservation and recovery services

     Cryopreservation

Transgenic lines are stored as frozen sperm. Users provides platform with 3 fertile males (3 to 4 months old). Frozen samples are stored in liquid nitrogen storage vessels at de Duve Institute cryotheque.

     Cryorecovery

Cryorecovery is used for transgenic line revitalization from frozen sperm and vitrified embryos.

- For sperm recovery: straw or cryovials of frozen sperm is thawed and used for in vitro fertilization (IVF) with oocytes. Fertilized oocytes are selected and transferred into oviduct of pseudo-pregnant female mice;

- For embryo recovery: straw or cryovials of frozen embryos is thawed. 40 to 50 embryos are transferred into oviduct of pseudo-pregnant female mice.