Model Creation with Charles River
Charles River Europe has joined forces with PHENOMIN-ICS, a scientific leader in functional genomics, to deliver a complete solution for mouse and rat model creation. Our combined in vitro and in vivo expertise provides an optimal environment for creating, validating, characterizing, preserving and distributing your transgenic and mutant rodent lines. PHENOMIN-ICS is considered a gold standard for successful germline transmission by the IMPC.
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Homologous Recombination Models (Mice)
- Design of an optimized strategy including prediction of off-target sites
- Preparation of the construct(s) (targeted construct +/- CRISPR/Cas9* construct)
- Electroporation in ES cell (C57BL/N, C57BL/6J** and BALB/cN
- Microinjection of validated ES cells into defined blastocysts
- Germline-transmitted F1 animals
- Excision of the selection cassette (if required)
** Europe only -
CRISPR/Cas9*- Generated Models
- Design of an optimized strategy including prediction of off-target sites
- Preparation of all materials for injection/electroporation
- Injection/ electroporation of genetic materials to generate F0 mutated animals
- Genotyping of F0 animals and delivery of F0 candidates
- Off-target analysis in F0 founder animals (optional)
- Germline breeding of F0 founders to achieve germ line transmission (optional)
- Off-target analysis in germline transmitted F1 animals (optional)
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Models That Can be Generated
Mouse
- Knockout: constitutive, conditional (tissue specific, inducible)
- Knockin: reporter/tag, point mutation, humanization and Rosa26, Hprt
- Structural variants including CNV
- Transgenic: gene-overexpression
Rat- Knockout: constitutive
- Knockin: tag, point mutation
- Structural variants including CNV
Other options including 2-in-1 models (e.g., conditional humanization and knockout) are available.
Contact us to discuss your project -
Core Competencies
- CRISPR/Cas9* validation: e.g. quantification of NHEJ or HDR events
- Cre or creERT2 mediated tissue/cell deletion
- Selection of transgenic line
- mRNA expression validation e.g., validation of impact of complex mutations
- Gene regulatory network evaluation
- Viral transduction - in vivo
- Biomarkers detection e.g., cancer cells
- Validated mRNA panels e.g., cytokines and chemokines
- Sequence of mutant allele
- Precise CNV quantification by ddPCR
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PHENOMIN-ICS Publications
Variability in Genome Editing Outcomes: Challenges for Research Reproducibility and Clinical Safety
Teboul L (1), Herault Y (2), Wells S (3), Qasim W(4), Pavlovic G(5)
Molecular Therapy Vol. 28 No. 6 June 2020Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High-Throughput Genotyping Protocol for Any Type of Mutation
Jacquot S(1), Chartoire N(1), Piguet F(2), Herault Y(1)(3), Pavlovic G(1).
Curr Protoc Mouse Biol 2019 Dec;9(4):e65Efficient and Rapid Generation of Large Genomic Variants in Rats and Mice Using CRISMERE
Birling MC(1), Schaeffer L(1), Andre P(1), Lindner L(1), Marechal D(1), Ayadi A(1), Sorg T(1), Pavlovic G(1), Herault Y(1,)(2,)(3,)(4,)(5). Sci Rep 2017 Mar 7;7():43331View a complete list of PHENOMIN-ICS publications
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Genotyping
PHENOMIN-ICS has more than 10 years of experience in providing robust, accurate, and fast results.
- Genotype your transgenic line by a customized protocol PCR or qPCR on complex alleles or multiple regions of a locus for more in depth validation of a mutant model
- Genotype your cohort by a high throughput protocol. Simplified genotyping with one qPCR on specific region to minimize costs for established and verified lines
- Genotype your cryopreserved line (blastocysts and sperm genotyping)
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Phenotyping
Systemic phenotyping of mutant mice is crucial in order to understand the relations between genotype, phenotype and environment. With a comprehensive panel of phenotyping tests, we provide standardized and customized protocols. We have developed a battery of tests to address most of the biological functions and cover the main therapeutic areas:
- Neurobiology and behavior platform: analysis of sensory responses, motor abilities, cognitive functions as well as behaviors related to neuropsychiatric disorders, such as anxiety, depression or schizophrenia.
- Metabolic exploration platform: detection of phenotypes in energy metabolism, glucose homeostasis, bone metabolism, renal and intestinal function.
- Cardiovascular platform: detection of congenital heart disease, myocardial infarction, valve disease, cardiac hypertrophy, arrhythmias, hypertension and hypotension.
- Oncology: various of engraftment, monitoring of tumor progression by in vivo imaging Clinical Chemistry platform: blood and urine analysis (clinical chemistry, hematology, immunology, endocrinology).
- Histopathology and embryology: histopathological analyses of adult tissues and mouse developmental biology
About PHENOMIN-ICS
PHENOMIN-ICS, also known as the "Institut Clinique de la Souris," is a scientific leader in the field of phenogenomics. Based in Strasbourg, France, its mission is to support the academic community and commercial organizations by creating highly relevant, validated and robust animal models for research programs and drug development. With a unique set of core capabilities, they have developed over 2000 unique rodent models for leading universities, pharma and biotech companies around the globe.
PHENOMIN-ICS is an active member of the International Mouse Phenotyping Consortium (IMPC), which is dedicated to identifying the function of every protein-coding gene in the mouse genome.
Frequently Asked Questions (FAQs)
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What is the best approach to obtain a conditional point mutation/deletion/KI?
First check some important criteria of the protocol:
- Comparable melting temperature (TM) for both primers (within 5°C of each other)
- If the sequence of the amplicon contains more than 60% G-C, perform the PCR with additives (DMSO, BSA..)
- More details in the publication:
Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High‐Throughput Genotyping Protocol for Any Type of Mutation. Sylvie Jacquot, Nathalie Chartoire, Françoise Piguet, Yann Hérault, Guillaume Pavlovic
Current Protocols in Mouse Biology 2019
Check also if the line imported in your lab is the good model. Indeed 15% of lines deposited to public repositories, do not carry the mutation specified by the depositor (Lloyd et al. 2015)
- Validate your cryopreserved line by direct blastocyst genotyping
Blastocyst genotyping for quality control of mouse mutant archives: An ethical and economical approach. Scavizzi F, Ryder E, Raspa M, Gleeson D, Wardle-Jones H, Montoliu L, Fernandez A, Dessain M-L, Larrigaldie V , Khorshidi Z, Vuolteenaho R, Soininen R, André P, Jacquot S, Hong Y, Hrabe de Angelis M, Ramirez-Solis R, Newman S, Doe B.
Transgenic Res 2015
- Comparable melting temperature (TM) for both primers (within 5°C of each other)
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What can you do if the genotyping protocol sent with the mouse line doesn't work in your lab?
It will all depend on the position of the mutation to introduce in your gene of interest. A conditional allele means a normal expression of the wild type allele before the start of the expression of the modified allele (with the expression of a Cre recombinase in most cases). In many papers, the proper expression of the wild type allele is not verified before the action of the Cre and can lead to a knock-out allele instead of a wild type allele (and thus can lead to lethality if the phenotype is strong). It will all depend on the position of the mutation to introduce in your gene of interest.
- Paper illustrating a successful approach for a conditional deletion of a nuclear localization signal 3' of a gene:
Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss. Scekic-Zahirovic J, Sendscheid O, El Oussini H, Jambeau M, Sun Y, Mersmann S, Wagner M, Dieterlé S, Sinniger J, Dirrig-Grosch S, Drenner K, Birling MC, Qiu J, Zhou Y, Li H, Fu XD, Rouaux C, Shelkovnikova T, Witting A, Ludolph AC, Kiefer F, Storkebaum E, Lagier-Tourenne C, Dupuis L
EMBO J 2016 May 17;35(10):1077-97. doi: 10.15252/embj.201592559
- Paper illustrating a successful ameliorated FLeX2 approach (the conditional mutation is located in the middle of a gene containing 20 exons):
Conditional switching of KIF2A mutation provides new insights into cortical malformation pathogeny.
Gilet JG, Ivanova EL, Trofimova D, Rudolf G, Meziane H, Broix L, Drouot N, Courraud J, Skory V, Voulleminot P, Osipenko M, Bahi-Buisson N, Yalcin B, Birling MC, Hinckelmann MV, Kwok BH, Allingham JS, Chelly J.
Human Molecular Genetics, Volume 29, Issue 5, 1 March 2020, Pages 766–784
- Paper illustrating a successful approach for a conditional deletion of a nuclear localization signal 3' of a gene:
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Should I worry about CRISPR/Ca9* off-targets?
Off-targets are nonspecific and unintended genetic modifications not located at the target site. Off-target are sometimes believed to be the main concern with genome editing. However, with a good experimental design that also favours nuclease transient expression, the frequency of off-target mutation can be minimized. At the opposite, on-target unwanted mutations are less known and rather underestimated. You will find more detailed about variability in genome editing outcomes in
Variability in Genome Editing Outcomes: Challenges for Research Reproducibility and Clinical Safety, L. Teboul, Y. Herault, S. Wells, W. Qasim, and G. Pavlovic, Molecular Therapy, Mar. 2020, doi: 10.1016/j.ymthe.2020.03.015. -
Which technology is the most appropriate to create my mouse line?
CRISPR/Cas9* genome editing is a very powerful technology to develop animal models. However, some complex mutations can remain challenging to achieve with this approach. In mice, the use of embryonic stem cells (sometimes in combination with CRISPR/Cas9*) is sometimes the best technology. You will find more detailed about Modeling disease in rodents by CRISPR/Cas9* genome editing in
Modeling human disease in rodents by CRISPR/Cas9* genome editing, M.-C. Birling, Y. Herault, and G. Pavlovic, Mamm. Genome, Jul. 2017, doi: 10.1007/s00335-017-9703-x.