The Centre for Phenogenomics
The Centre for Phenogenomics

Model Production Services

We offer both à la carte services for the experienced mouse researcher as well as full-service model design, production and quality control.

CRISPR/Cas9 Mouse Model Generation

Mouse models, including null and point mutant alleles, are generated by microinjection of Cas9 RNA-guided nuclease (Cas9-RGN) and guide RNAs (gRNAs) that specify the target site into zygotes.

  • Founders can be generated in as few as 3-4 months, depending on allele complexity
  • Any strain background with injectable zygotes can be used
  • Current protocols include efficient production of indel (85% success), exon deletion (85% success), point mutant (75% success) and short sequence tagged (75% success) alleles.

Read More about what services include

You or we can do the gene annotation and allele design, including gRNA identification and selection (design is free of charge when mouse is generated at TCP). The gRNAs are synthesized and prepared for injection with other necessary components at TCP. After microinjection, several à la carte services are available to assist in the identification of founders, their breeding, and the identification and QC of N1 mice

Service includes:

  • Design and synthesis of reagents for Cas9-RGN genome editing
  • Microinjection of Cas9 mRNA and gRNA(s), with or without ssOligo or plasmid templates, into zygotes and transfer of injected zygotes into pseudopregnant recipients
  • Screening founders (P's) for the mutation of interest
  • Breeding founders to test for germline transmission of the desired allele(s)
  • Quality control assays to determine allele sequence and assess repair vector copy number and targeting accuracy
  • Development and validation of genotyping assays
Fee Details for mouse model production Back To Top

Gene Targeting

Gene targeting utilizes homologous recombination to replace targeted regions of endogenous DNA with engineered DNA in validated embryonic stem (ES) cell lines to generate knock-out, knock-in, point mutant or conditional alleles.

  • Gene targeted ES cell clones can be delivered to client for in vitro experimentation or used to generate chimeras at TCP
  • Clones can be generated in ~5-10 weeks from the electroporation stage

Read More about what services include

Service includes:

  • Vector design, construction and preparation for electroporation for the production of knock-out, conditional, reporter or other types of knock-in alleles in ES cells. (Alternatively the investigator can provide purified DNA for electroporation.)
  • Electroporation of the targeting vector into validated parental ES cell lines (C57BL/6NTac-C2, 129S6B6NF1-G4, 129S1 x 129X1-R1), isolation of antibiotic-resistant colonies in maximum two 96-well plates per attempt and their cryopreservation. Replica plates for DNA isolation can be provided to the investigator or screened for the mutation of interest by TCP.
  • Expansion and cryopreservation of confirmed ES cell clones
  • Chromosome copy number assessment by chromosome-specific real-time PCR
Fee Details Gene targeting in ES cell lines Back To Top

Generation of Chimeras

Chimeras are generated using genetically modified mouse ES cells, induced pluripotent stem (iPS) cells or different combinations of mutant and wild type embryos including tetraploid.

  • Chimeras can be used directly for experimentation or to establish mouse lines by breeding to transmit the ES or iPS cell genome.
  • Methods include morula aggregation, morula or blastocyst microinjection, and tetraploid complementation assay
  • Heterozygotes can typically be obtained in ~5 months from generation of chimeras experiment using validated ES cell clones

Read More about what services include

Method used depends on research goals, stem cell type, embryonic stage and strain background of ES cells and host embryos. Genetically modified ES cells can be produced by TCP or by the investigator or obtained from the International Mouse Phenotyping Consortium .

Service includes:

  • Generation of chimeras (Embryos from ~15-20 CD-1 or C57BL/6J females are transferred to ~6-10 pseudopregnant recipients. Any offspring are evaluated for coat colour chimerism and chimeras are weaned.)
  • Transfer of manipulated embryos to pseudopregnant recipients
  • Breeding of chimeras to test germline transmission of the ES or iPS cell genome
  • Screening of offspring for the mutation of interest
  • Quality control assays to assess target vector copy number, targeting accuracy, and allele structure
Fee Details Generation of Chimeras Back To Top

Transgenic Mouse Production

Transgenic mice are produced by pronuclear microinjection of DNA constructs into zygotes.

  • Transgenic lines are established upon random integration of the DNA construct into the host zygote genome and subsequent breeding of founders for germline transmission.
  • Transgenic founder mice can be delivered in ~7-8 weeks from time of microinjection

Read More about what services include

Service includes:

  • Design and construction of transgene vector
  • Preparation and purification of vector for injection. (Alternatively the investigator can provide purified DNA for microinjection.)
  • Pronuclear microinjection into zygotes from ~15-20 embryo donors and transfer to ~8 pseudopregnant recipients, tail clipping of offspring and weaning
  • Screening of founder mice for the presence of the transgene
  • Breeding selected founders to test for germline transmission of the transgene
  • Quality control of transgenic progeny to assess transgene copy number
Fee Details Transgenic mouse production Back To Top

Derivation of Novel ES Cell Lines

ES cell lines are important tools for analysis of mutations, particularly those that result in an embryonic lethal phenotype.

  • ES cell lines can be derived from genetically engineered mice housed at TCP or other local facilities
  • Resulting ES cell lines can be used for in vitro studies or to generate chimeras

Read More about what ES cell derivation includes

Service includes:

  • Superovulation, mating and embryo collection
  • Plating the embryos and disaggregating outgrowths
  • Expanding clones with ES cell morphology and initial cryopreservation
  • Providing cell pellets for genotyping and chromosome copy number assessment. Screening can be performed at TCP.
  • Cryopreserving expanded ES cell lines after genotype confirmation
Fee Details Derivation of novel ES cell lines Back To Top

Rederivation of Mouse Strains

Mice can be rederived into specific pathogen-free (SPF) status to eliminate contamination by specified pathogens.

  • All imported mouse lines, except those from approved commercial suppliers, must be rederived into TCP animal facility
  • Rederived mouse lines can be exported

Read More about what rederivation includes

Rederivation includes:

  • Housing and technical services in containment (Note: rederivation can also be done using dissected oviducts from timed pregnant animals coordinated with local facilities or live embryos shipped by courier to TCP for embryo transfer on arrival)
  • Harvesting pre-implantation stage embryos after mating superovulated wild-type donors to mutant males housed in TCP containment.
  • Transferring embryos into SPF pseudopregnant recipients
  • Providing tissue samples from rederived offspring for genotyping
  • Weaning rederived offspring
  • Health testing to verify health status before release into general TCP colony or export
Fee Details Re-derivation of mouse strains Back To Top