Services
Assisted Reproductive Technologies
Sperm cryopreservation
Frozen sperm from two proven males will be frozen. As part of our quality control (QC), IVF will be performed. A project is considered complete if the fertilization rate equals or exceeds 20% for each male.
Embryos cryopreservation
Embryos will be frozen at the two-cell stage. As part of our quality control (QC), the GEMF will thaw one straw of frozen embryos and culture them in vitro to the blastocyst stage to ensure future reviving of the embryos.
In Vitro Fertilization (IVF)
In Vitro Fertilization (IVF) will be performed from fresh, cold or frozen sperm to either generate live pups or to freeze embryos. A minimum of two recipient females will be implanted for reviving, otherwise the embryos will be frozen for archiving.
Embryos implants
Frozen embryos will be implanted into recipient females to revive a Genetically Modified (GM) mouse line. A minimum of two recipient females will be implanted.
Blastocysts injection
Injection of embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) into blastocysts to generate chimeric mice or study the contribution of cells to the developing embryos. A minimum of two recipient females will be implanted to generate chimeras.
Rederivation
Production of embryos and subsequent implantation into recipient females to eliminate pathogens from genetically modified (GM) mice. A minimum of two recipient females will be implanted to generate pups.
Genome Editing
Random integration (Transgenic)
A highly concentrated aliquot of the plasmid needs to be provided to the GEMF for purification. Ideally, an endotoxin-free MidiPrep or a MaxiPrep should be provided to the GEMF. Restriction enzyme(s) to isolate the linearized transgene also need(s) to be provided.
Transgenic mice will be generated by pronuclear injection of C57BL/6J zygotes.
Should another background be required, additional costs will be added. Agistments costs beyond the weaning date will be charged to the principal investigator.
Knock out (KO)
Small or large deletions will be performed in C57BL/6J zygotes.
Should another background be required, additional costs will be added. Agistments costs beyond the weaning date will be charged to the principal investigator.
Conditional knock-out (cKO)
Insertion of LoxP sites encompassing critical exon(s) will be attempted in C57BL/6J zygotes. Note that this type of genetic modification is the most difficult to generate, and success rate is typically low. Excision of the critical exon(s) upon Cre recombination is not guaranteed.
Should a background other than C57BL/6J be required, additional costs will be added. Agistments costs passed the weaning date will be charged to the principal investigator.
Knock in (KI) by electroporation
KI mice will be generated by AAV-driven electroporation of C57BL/6J zygotes using engineered endonucleases.
Should another background be required, additional costs will be added. Agistments costs beyond the weaning date will be charged to the principal investigator.
Knock in (KI) by pronuclear injection
The donor plasmid used for Homologous Directed Repair (HDR) needs to be provided to the GEMF at high concentration for purification. Ideally, an endotoxin-free MidiPrep or a MaxiPrep should be provided to the GEMF.
KI mice will be generated by pronuclear injection of C57BL/6J zygotes using engineered endonucleases. Note that this procedure is less efficient than KI by electroporation.
Should another background be required, additional costs will be added. Agistments costs beyond the weaning date will be charged to the principal investigator.
Small genomic modifications
Genetic modifications up to 100 nucleotides will be performed in C57BL/6J zygotes.
Should another background be required, additional costs will be added. Agistments costs beyond the weaning date will be charged to the principal investigator.
All included - Random integration (Transgenic)
A highly concentrated aliquot of the plasmid needs to be provided to the GEMF for purification. Ideally, an endotoxin-free MidiPrep or a MaxiPrep should be provided to the GEMF. Restriction enzyme(s) to isolate the linearised transgene also need(s) to be provided.
Transgenic mice will be generated by pronuclear injection of C57BL/6J zygotes. PCR genotyping using transgene-specific primers will also be carried to identify potential Founders.
Should another background be required, additional costs will be added. Agistments costs passed the weaning date will be charged to the principal investigator.
All included - Knock out (KO)
This service includes:
- Small or large deletions in C57BL/6 zygotes.
- Gel electrophoresis of PCR amplicons (if applicable).
- Confirmation of the deletion will be done by either Sanger sequencing, targeted Next Generation Sequencing (NGS) or targeted Long Read Sequencing (LRS).
Should a different background than C57BL/6J be required, additional costs will be added. Agistments costs beyond the weaning date will be charged to the principal investigator.
All included - conditional knock-out (cKO)
This service includes:
- Insertion of LoxP sites encompassing critical exon(s). Note that this type of genetic modification is the most difficult to generate, and success rate is typically low. Excision of the critical exon(s) upon Cre recombination is not guaranteed.
- Gel electrophoresis of PCR amplicons (if applicable).
- Confirmation of the LoxP insertions will be done by targeted Long Read Sequencing (LRS).
Should a different background than C57BL/6J be required, additional costs will be added. Agistments costs beyond the weaning date will be charged to the principal investigator.
All included - Small genomic modifications
This service includes:
- Genetic modifications up to 100 nucleotides in C57BL/6J zygotes.
- Confirmation of the genetic modification will be done by either Sanger sequencing, targeted Next Generation Sequencing (NGS) or targeted Long Read Sequencing (LRS).
Should a different background than C57BL/6J be required, additional costs will be added. Agistments costs beyond the weaning date will be charged to the principal investigator.
All included - Knock in (KI) by electroporation
This service includes:
- AAV-driven electroporation of C57BL/6J zygotes with engineered endonucleases.
- Transgene-specific PCR to identify potential Founders
- Targeted Long Read Sequencing (LRS) to confirm proper integration. Note that LRS may be performed on selected mice from the F1 progeny.
Should a different background than C57BL/6J be required, additional costs will be added. Agistments costs beyond the weaning date will be charged to the principal investigator.
All included - Knock in (KI) by pronuclear injection
This service includes:
- Pronuclear injection of C57BL/6J zygotes with engineered endonucleases.
- Transgene-specific PCR to identify potential Founders
- Targeted Long Read Sequencing (LRS) to confirm proper integration. Note that LRS may be performed on selected mice from the F1 progeny.
Should a different background than C57BL/6J be required, additional costs will be added. Agistments costs beyond the weaning date will be charged to the principal investigator.
Molecular Biology
PCR Genotyping
DNA extraction and PCR genotyping to identify potential founders.
Sanger Sequencing
DNA extraction and Sanger sequencing to identify potential founders.
Targeted Next Generation Sequencing (NGS)
DNA extraction and next generation sequencing (NGS) for quality control of the editing events.
Targeted Long Read Sequencing (PCR based)
DNA extraction, Long Range PCR and Long Read Sequencing (LRS) are performed as part of our quality control (QC) of the editing events. Note that this type of LRS can be performed at any generation, including to identify potential F0 Founders.
Targeted Long Read Sequencing (Cas9 enrichment)
DNA extraction, Cas9 enrichment and Long Read Sequencing (LRS) are performed as part of our quality control (QC) of the editing events. Note that this type of LRS can be performed at any generation, including to identify potential F0 Founders. Up to 12 samples maximum.
Targeted Long Read Sequencing (Hybridization Capture)
DNA extraction, hybridization capture and Long Read Sequencing (LRS) are performed as part of our quality control (QC) of the editing events. Note that this type of LRS can be performed at any generation, including to identify potential F0 Founders. Up to 12 samples maximum.
Gene Targeting (ES Cells)
For all ES-Cells related projects, the principal investigator will need to provide their competent ES-cell lines. Please contact Fabien Delerue to discuss your project.