Homologous Recombination in ES cells

Homologous recombination (HR) in ES cells is a reference technology to generate high-value targeted mouse models allowing to achieve highly relevant animal models for most applications, from R&D to preclinical studies.

ES cells transfection with targeting vector		Targeted ES cells selection	ES cells validation (PCR, Southern blot, Karyotyping)	Injection into blastocyst
		Chimeric new born mice		Implantation into pseudo pregnant female
m/+ and +/+ (WT) animals	Sequencing and selection of GLT animals

Advantages

• Optimized timelines for your project.
• Precise modification of genes (gold standard method).
• Correct targetting is checked by PCR and southern blotting using both internal and external probes. In order to increase the GLT rate, only clones without gross karyotypic abnormalities are injected (Codner et al., 2016). This approach allows a perfect validation of the targeted alleles at the ES stage and is in accordance with the principles of the 3Rs (Replacement, Reduction and Refinement).
• Easier genotyping as genome modifications are known. 
• Numerous applications: humanization, monitoring of biomarker expression, labelling cell trafficking, conditional knock-out, complex model …

Drawbacks

• Time consuming and more expensive method compared to CRISPR/Cas9 genome editing technologies.
• Requires good germline-competent ES cell lines (only restricted to few genetic backgrounds i.e. C57BL/6N or BALB/cN).

Note: for generation of complex mutations, ES route remains (yet!) superior to CRISPR/Cas9.

PHENOMIN expertise

• More than 25 years of experience and more than 3,000 models successfully created through this technology.
• Personalized genome engineering service to fit our customer needs.
• PHENOMIN uses its experience to determine the most appropriate technology (nuclease-based or ES cell-based) for each project.
• PHENOMIN meticulously validates all selected ES clones to ensure proper model establishement ; this allows to speed up the genotyping step to discriminate mutant from wild type animals.
• PHENOMIN delivers selection cassette-free animals.
• Compared to all members from the IMPC international consortium, PHENOMIN has the highest germ line transmission (GLT) rate from in house ES cells and from IKMC ES cells.
• Increased efficiency by combining the HR with CRISPR/Cas9  in ES cells (coelectroporation of standard plasmid HR with Cas9 and gRNA plasmid) and generate large replacements that were not (easily) feasible previously.
• Leading scientific insight & expertise in the field.
• Continuous R&D activity to improve our gene targeting success rate and to propose the best approach to our customers.
• Member of international consortia (near 20 different consortia) to benefit from the latest developments.

Publications

Non exhaustive list of publications from PHENOMIN scientists or collaborators and customers.

• Bonet et al., 2017, Deciphering the Role of Oncogenic MITFE318K in Senescence Delay and Melanoma Progression.
• Arbogast et al., 2017, Mouse models of 17q21.31 microdeletion and microduplication syndromes highlight the importance of Kansl1 for cognition.
• Karp et al., 2017, Prevalence of sexual dimorphism in mammalian phenotypic traits.
• Ung et al., Ptchd1 deficiency induces excitatory synaptic and cognitive dysfunctions in mouse.
• Sellier et al., 2017, Translation of Expanded CGG Repeats into FMRpolyG Is Pathogenic and May Contribute to Fragile X Tremor Ataxia Syndrome.
• Codner et al., 2016, Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction.
• Dickinson et al., 2016, High-throughput discovery of novel developmental phenotypes.
• Li et al., 2016, Counterregulation between thymic stromal lymphopoietin- and IL-23-driven immune axes shapes skin inflammation in mice with epidermal barrier defects.
• Scekic-Zahirovic et al., 2016, Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss.
• Arbogast et al., 2016, Reciprocal Effects on Neurocognitive and Metabolic Phenotypes in Mouse Models of 16p11.2 Deletion and Duplication Syndromes.
• Dubos et al., 2015, Conditional depletion of intellectual disability and Parkinsonism candidate gene ATP6AP2 in fly and mouse induces cognitive impairment and neurodegeneration.
• de Angelis et al., 2015, Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.
• Piccand et al., 2014, Rfx6 maintains the functional identity of adult pancreatic β cells.
• Lopez-Delisle et al., 2014, Hyperactivation of Alk induces neonatal lethality in knock-in AlkF1178L mice.
• Clar et al., 2014, Targeted deletion of kidney glucose-6 phosphatase leads to nephropathy.
• Erbs et al., 2014, A mu-delta opioid receptor brain atlas reveals neuronal co-occurrence in subcortical networks.
• Gallet et al., 2013, Repression of osteoblast maturation by ERRα accounts for bone loss induced by estrogen deficiency.
• Bradley et al., 2012, The mammalian gene function resource: the International Knockout Mouse Consortium.
• Ayadi et al., 2012, Mouse large-scale phenotyping initiatives: overview of the European Mouse Disease Clinic (EUMODIC) and of the Wellcome Trust Sanger Institute Mouse Genetics Project.
• Danglot et al., 2012, Absence of TI-VAMP/Vamp7 leads to increased anxiety in mice.
• Birling et al., 2012, Highly-efficient, fluorescent, locus directed cre and FlpO deleter mice on a pure C57BL/6N genetic background.
• Marty et al., 2010, Myeloproliferative neoplasm induced by constitutive expression of JAK2V617F in knock-in mice.