Distinct roles of α- and ß-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration.

Tubulin polyglutamylation is a post-translational modification of the microtubule cytoskeleton, which is generated by a variety of enzymes with different specificities. The "tubulin code" hypothesis predicts that modifications generated by specific enzymes selectively control microtubule functions. Our recent finding that excessive accumulation of polyglutamylation in neurons causes their degeneration and perturbs axonal transport provides an opportunity for testing this hypothesis. By developing novel mouse models and a new glutamylation-specific antibody, we demonstrate here that the glutamylases TTLL1 and TTLL7 generate unique and distinct glutamylation patterns on neuronal microtubules. We find that under physiological conditions, TTLL1 polyglutamylates α-tubulin, while TTLL7 modifies ß-tubulin. TTLL1, but not TTLL7, catalyses the excessive hyperglutamylation found in mice lacking the deglutamylase CCP1. Consequently, deletion of TTLL1, but not of TTLL7, prevents degeneration of Purkinje cells and of myelinated axons in peripheral nerves in these mice. Moreover, loss of TTLL1 leads to increased mitochondria motility in neurons, while loss of TTLL7 has no such effect. By revealing how specific patterns of tubulin glutamylation, generated by distinct enzymes, translate into specific physiological and pathological readouts, we demonstrate the relevance of the tubulin code for homeostasis.

A conditional null allele of Dync1h1 enables targeted analyses of dynein roles in neuronal length sensing.

Size homeostasis is a fundamental process in biology and is particularly important for large cells such as neurons. We previously proposed a motor-dependent length-sensing mechanism wherein reductions in microtubule motor levels would be expected to accelerate neuronal growth, and validated this prediction in dynein heavy chain 1 Loa mutant (Dync1h1Loa) sensory neurons. Here, we describe a new mouse model with a conditional deletion allele of exons 24 and 25 in Dync1h1. Homozygous Islet1-Cre-mediated deletion of Dync1h1 (Isl1-Dync1h1-/-), which deletes protein from the motor and sensory neurons, is embryonic lethal, but heterozygous animals (Isl1-Dync1h1+/-) survive to adulthood with ∼50% dynein expression in targeted cells. Isl1-Dync1h1+/- sensory neurons reveal accelerated growth, as previously reported in Dync1h1Loa neurons. Moreover, Isl1-Dync1h1+/- mice show mild impairments in gait, proprioception and tactile sensation, similar to what is seen in Dync1h1Loa mice, confirming that specific aspects of the Loa phenotype are due to reduced dynein levels. Isl1-Dync1h1+/- mice also show delayed recovery from peripheral nerve injury, likely due to reduced injury signal delivery from axonal lesion sites. Thus, conditional deletion of Dync1h1 exons 24 and 25 enables targeted studies of the role of dynein in neuronal growth.

The COL6A5-p.Glu2272* mutation induces chronic itch in mice.

Pruritus is a common irritating sensation that provokes the desire to scratch. Environmental and genetic factors contribute to the onset of pruritus. Moreover, itch can become a major burden when it becomes chronic. Interestingly, the rare Collagen VI alpha 5 (COL6A5) gene variant p.Glu2272* has been identified in two families and an independent patient with chronic neuropathic itch. These patients showed reduced COL6A5 expression in skin and normal skin morphology. However, little progress has been made until now toward understanding the relationships between this mutation and chronic itch. Therefore, we developed the first mouse model that recapitulates COL6A5-p.Glu2272* mutation using the CRISPR-Cas technology and characterized this new mouse model. The mutant mRNA, measured by RT-ddPCR, was expressed at normal levels in dorsal root ganglia and was decreased in skin. The functional exploration showed effects of the mutation with some sex dysmorphology. Mutant mice had increased skin permeability. Elevated spontaneous scratching and grooming was detected in male and female mutants, with increased anxiety-like behavior in female mutants. These results suggest that the COL6A5-p.Glu2272* mutation found in patients contributes to chronic itch and induces in mice additional behavioral changes. The COL6A5-p.Glu2272* mouse model could elucidate the pathophysiological mechanisms underlying COL6A5 role in itch and help identify potential new therapeutic targets.

Dyrk1a gene dosage in glutamatergic neurons has key effects in cognitive deficits observed in mouse models of MRD7 and Down syndrome.

Perturbation of the excitation/inhibition (E/I) balance leads to neurodevelopmental diseases including to autism spectrum disorders, intellectual disability, and epilepsy. Loss-of-function mutations in the DYRK1A gene, located on human chromosome 21 (Hsa21,) lead to an intellectual disability syndrome associated with microcephaly, epilepsy, and autistic troubles. Overexpression of DYRK1A, on the other hand, has been linked with learning and memory defects observed in people with Down syndrome (DS). Dyrk1a is expressed in both glutamatergic and GABAergic neurons, but its impact on each neuronal population has not yet been elucidated. Here we investigated the impact of Dyrk1a gene copy number variation in glutamatergic neurons using a conditional knockout allele of Dyrk1a crossed with the Tg(Camk2-Cre)4Gsc transgenic mouse. We explored this genetic modification in homozygotes, heterozygotes and combined with the Dp(16Lipi-Zbtb21)1Yey trisomic mouse model to unravel the consequence of Dyrk1a dosage from 0 to 3, to understand its role in normal physiology, and in MRD7 and DS. Overall, Dyrk1a dosage in postnatal glutamatergic neurons did not impact locomotor activity, working memory or epileptic susceptibility, but revealed that Dyrk1a is involved in long-term explicit memory. Molecular analyses pointed at a deregulation of transcriptional activity through immediate early genes and a role of DYRK1A at the glutamatergic post-synapse by deregulating and interacting with key post-synaptic proteins implicated in mechanism leading to long-term enhanced synaptic plasticity. Altogether, our work gives important information to understand the action of DYRK1A inhibitors and have a better therapeutic approach.

A human dynein heavy chain mutation impacts cortical progenitor cells causing developmental defects, reduced brain size and altered brain architecture.

Dynein heavy chain (DYNC1H1) mutations can either lead to severe cerebral cortical malformations, or alternatively may be associated with the development of spinal muscular atrophy with lower extremity predominance (SMA-LED). To assess the origin of such differences, we studied a new Dync1h1 knock-in mouse carrying the cortical malformation p.Lys3334Asn mutation. Comparing with an existing neurodegenerative Dync1h1 mutant (Legs at odd angles, Loa, p.Phe580Tyr/+), we assessed Dync1h1's roles in cortical progenitor and especially radial glia functions during embryogenesis, and assessed neuronal differentiation. p.Lys3334Asn /+ mice exhibit reduced brain and body size. Embryonic brains show increased and disorganized radial glia: interkinetic nuclear migration occurs in mutants, however there are increased basally positioned cells and abventricular mitoses. The ventricular boundary is disorganized potentially contributing to progenitor mislocalization and death. Morphologies of mitochondria and Golgi apparatus are perturbed in vitro, with different effects also in Loa mice. Perturbations of neuronal migration and layering are also observed in p.Lys3334Asn /+ mutants. Overall, we identify specific developmental effects due to a severe cortical malformation mutation in Dync1h1, highlighting the differences with a mutation known instead to primarily affect motor function.

Conditional switching of KIF2A mutation provides new insights into cortical malformation pathogeny.

By using the Cre-mediated genetic switch technology, we were able to successfully generate a conditional knock-in mouse, bearing the KIF2A p.His321Asp missense point variant, identified in a subject with malformations of cortical development. These mice present with neuroanatomical anomalies and microcephaly associated with behavioral deficiencies and susceptibility to epilepsy, correlating with the described human phenotype. Using the flexibility of this model, we investigated RosaCre-, NestinCre- and NexCre-driven expression of the mutation to dissect the pathophysiological mechanisms underlying neurodevelopmental cortical abnormalities. We show that the expression of the p.His321Asp pathogenic variant increases apoptosis and causes abnormal multipolar to bipolar transition in newborn neurons, providing therefore insights to better understand cortical organization and brain growth defects that characterize KIF2A-related human disorders. We further demonstrate that the observed cellular phenotypes are likely to be linked to deficiency in the microtubule depolymerizing function of KIF2A.

Reliable and robust droplet digital PCR (ddPCR) and RT-ddPCR protocols for mouse studies.

Droplet digital PCR (ddPCR) is a recent method developed for the quantification of nucleic acids sequences. It is an evolution of PCR methodology incorporating two principal differences: a PCR reaction is performed in thousands of water-oil emulsion droplets and fluorescence is measured at the end of PCR amplification. It leads to the precise and reproducible quantification of DNA and RNA sequences. Here, we present quantitative methods for DNA and RNA analysis using Bio-Rad QX100 or QX200 systems, respectively. The aim of these methods is to provide useful molecular tools for validating genetically altered animal models such as those subject to CRISPR/Cas9 genome editing, as well for expression or CNV studies. A standard procedure for simultaneous DNA and RNA extraction adapted for mouse organs is also described. These methods were initially designed for mouse studies but also work for samples from other species like rat or human. In our lab, thousands of samples and hundreds of target genes from genetically altered lines were examined using these methods. This large dataset was analyzed to evaluate technical optimizations and limitations. Finally, we propose additional recommendations to be included in dMIQE (Minimum information for publication of quantitative digital PCR experiments) guidelines when using ddPCR instruments.

Droplet digital PCR or quantitative PCR for in-depth genomic and functional validation of genetically altered rodents.

Gene targeting and additive (random) transgenesis have proven to be powerful technologies with which to decipher the mammalian genome. With the advent of CRISPR/Cas9 genome editing, the ability to inactivate or modify the function of a gene has become even more accessible. However, the impact of each generated modification may be different from what was initially desired. Minimal validation of mutant alleles from genetically altered (GA) rodents remains essential to guarantee the interpretation of experimental results. The protocol described here combines design strategies for genomic and functional validation of genetically modified alleles with droplet digital PCR (ddPCR) or quantitative PCR (qPCR) for target DNA or mRNA quantification. In-depth analysis of the results obtained with GA models through the analysis of target DNA and mRNA quantification is also provided, to evaluate which pitfalls can be detected using these two methods, and we propose recommendations for the characterization of different type of mutant allele (knock-out, knock-in, conditional knock-out, FLEx, IKMC model or transgenic). Our results also highlight the possibility that mRNA expression of any mutated allele can be different from what might be expected in theory or according to common assumptions. For example, mRNA analyses on knock-out lines showed that nonsense-mediated mRNA decay is generally not achieved with a critical-exon approach. Likewise, comparison of multiple conditional lines crossed with the same CreERT2 deleter showed that the inactivation outcome was very different for each conditional model. DNA quantification by ddPCR of G0 to G2 generations of transgenic rodents generated by pronuclear injection showed an unexpected variability, demonstrating that G1 generation rodents cannot be considered as established lines.

Universal Southern blot protocol with cold or radioactive probes for the validation of alleles obtained by homologous recombination.

The widespread availability of recombineered vectors and gene targeted embryonic stem cells from large-scale repositories facilitates the generation of mouse models for functional genetic studies. Southern blotting validates the structure of these targeted alleles produced by homologous recombination, as well as indicating any additional integrations of the vector into the genome. Traditionally this technique employs radioactively-labelled probes; however, there are many laboratories that are restricted in their use of radioactivity. Here, we present a widely applicable protocol for Southern blot analysis using cold probes and alternative procedures employing radioactive probes. Furthermore, the probes are designed to recognise standardised regions of gene-targeting cassettes and so represent universally applicable reagents for assessing allelic integrity.

Cbs overdosage is necessary and sufficient to induce cognitive phenotypes in mouse models of Down syndrome and interacts genetically with Dyrk1a.

Identifying dosage-sensitive genes is a key to understand the mechanisms underlying intellectual disability in Down syndrome (DS). The Dp(17Abcg1-Cbs)1Yah DS mouse model (Dp1Yah) shows cognitive phenotypes that need to be investigated to identify the main genetic driver. Here, we report that three copies of the cystathionine-beta-synthase gene (Cbs) in the Dp1Yah mice are necessary to observe a deficit in the novel object recognition (NOR) paradigm. Moreover, the overexpression of Cbs alone is sufficient to induce deficits in the NOR test. Accordingly, overexpressing human CBS specifically in Camk2a-expressing neurons leads to impaired objects discrimination. Altogether, this shows that Cbs overdosage is involved in DS learning and memory phenotypes. To go further, we identified compounds that interfere with the phenotypical consequence of CBS overdosage in yeast. Pharmacological intervention in Tg(CBS) mice with one selected compound restored memory in the NOR test. In addition, using a genetic approach, we demonstrated an epistatic interaction between Cbs and Dyrk1a, another human chromosome 21-located gene (which encodes the dual-specificity tyrosine phosphorylation-regulated kinase 1a) and an already known target for DS therapeutic intervention. Further analysis using proteomic approaches highlighted several molecular pathways, including synaptic transmission, cell projection morphogenesis and actin cytoskeleton, that are affected by DYRK1A and CBS overexpression. Overall, we demonstrated that CBS overdosage underpins the DS-related recognition memory deficit and that both CBS and DYRK1A interact to control accurate memory processes in DS. In addition, our study establishes CBS as an intervention point for treating intellectual deficiencies linked to DS.

A new mouse model of ARX dup24 recapitulates the patients' behavioral and fine motor alterations.

The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment.

Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss.

FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons.

Mouse models of 17q21.31 microdeletion and microduplication syndromes highlight the importance of Kansl1 for cognition.

Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches.

The neuroanatomy of Eml1 knockout mice, a model of subcortical heterotopia.

The cerebral cortex is a highly organized structure responsible for advanced cognitive functions. Its development relies on a series of steps including neural progenitor cell proliferation, neuronal migration, axonal outgrowth and brain wiring. Disruption of these steps leads to cortical malformations, often associated with intellectual disability and epilepsy. We have generated a new resource to shed further light on subcortical heterotopia, a malformation characterized by abnormal neuronal position. We describe here the generation and characterization of a knockout (KO) mouse model for Eml1, a microtubule-associated protein showing mutations in human ribbon-like subcortical heterotopia. As previously reported for a spontaneous mouse mutant showing a mutation in Eml1, we observe severe cortical heterotopia in the KO. We also observe abnormal progenitor cells in early corticogenesis, likely to be the origin of the defects. EML1 KO mice on the C57BL/6N genetic background also appear to present a wider phenotype than the original mouse mutant, showing additional brain anomalies, such as corpus callosum abnormalities. We compare the anatomy of male and female mice and also study heterozygote animals. This new resource will help unravel roles for Eml1 in brain development and tissue architecture, as well as the mechanisms leading to severe subcortical heterotopia.

Reciprocal Effects on Neurocognitive and Metabolic Phenotypes in Mouse Models of 16p11.2 Deletion and Duplication Syndromes.

The 16p11.2 600 kb BP4-BP5 deletion and duplication syndromes have been associated with developmental delay; autism spectrum disorders; and reciprocal effects on the body mass index, head circumference and brain volumes. Here, we explored these relationships using novel engineered mouse models carrying a deletion (Del/+) or a duplication (Dup/+) of the Sult1a1-Spn region homologous to the human 16p11.2 BP4-BP5 locus. On a C57BL/6N inbred genetic background, Del/+ mice exhibited reduced weight and impaired adipogenesis, hyperactivity, repetitive behaviors, and recognition memory deficits. In contrast, Dup/+ mice showed largely opposite phenotypes. On a F1 C57BL/6N × C3B hybrid genetic background, we also observed alterations in social interaction in the Del/+ and the Dup/+ animals, with other robust phenotypes affecting recognition memory and weight. To explore the dosage effect of the 16p11.2 genes on metabolism, Del/+ and Dup/+ models were challenged with high fat and high sugar diet, which revealed opposite energy imbalance. Transcriptomic analysis revealed that the majority of the genes located in the Sult1a1-Spn region were sensitive to dosage with a major effect on several pathways associated with neurocognitive and metabolic phenotypes. Whereas the behavioral consequence of the 16p11 region genetic dosage was similar in mice and humans with activity and memory alterations, the metabolic defects were opposite: adult Del/+ mice are lean in comparison to the human obese phenotype and the Dup/+ mice are overweight in comparison to the human underweight phenotype. Together, these data indicate that the dosage imbalance at the 16p11.2 locus perturbs the expression of modifiers outside the CNV that can modulate the penetrance, expressivity and direction of effects in both humans and mice.

Conditional depletion of intellectual disability and Parkinsonism candidate gene ATP6AP2 in fly and mouse induces cognitive impairment and neurodegeneration.

ATP6AP2, an essential accessory component of the vacuolar H+ ATPase (V-ATPase), has been associated with intellectual disability (ID) and Parkinsonism. ATP6AP2 has been implicated in several signalling pathways; however, little is known regarding its role in the nervous system. To decipher its function in behaviour and cognition, we generated and characterized conditional knockdowns of ATP6AP2 in the nervous system of Drosophila and mouse models. In Drosophila, ATP6AP2 knockdown induced defective phototaxis and vacuolated photoreceptor neurons and pigment cells when depleted in eyes and altered short- and long-term memory when depleted in the mushroom body. In mouse, conditional Atp6ap2 deletion in glutamatergic neurons (Atp6ap2(Camk2aCre/0) mice) caused increased spontaneous locomotor activity and altered fear memory. Both Drosophila ATP6AP2 knockdown and Atp6ap2(Camk2aCre/0) mice presented with presynaptic transmission defects, and with an abnormal number and morphology of synapses. In addition, Atp6ap2(Camk2aCre/0) mice showed autophagy defects that led to axonal and neuronal degeneration in the cortex and hippocampus. Surprisingly, axon myelination was affected in our mutant mice, and axonal transport alterations were observed in Drosophila. In accordance with the identified phenotypes across species, genome-wide transcriptome profiling of Atp6ap2(Camk2aCre/0) mouse hippocampi revealed dysregulation of genes involved in myelination, action potential, membrane-bound vesicles and motor behaviour. In summary, ATP6AP2 disruption in mouse and fly leads to cognitive impairment and neurodegeneration, mimicking aspects of the neuropathology associated with ATP6AP2 mutations in humans. Our results identify ATP6AP2 as an essential gene for the nervous system.

Modeling human disease in rodents by CRISPR/Cas9 genome editing.

Modeling human disease has proven to be a challenge for the scientific community. For years, generating an animal model was complicated and restricted to very few species. With the rise of CRISPR/Cas9, it is now possible to generate more or less any animal model. In this review, we will show how this technology is and will change our way to obtain relevant disease animal models and how it should impact human health.

Counterregulation between thymic stromal lymphopoietin- and IL-23-driven immune axes shapes skin inflammation in mice with epidermal barrier defects.

BACKGROUND: Epidermal barrier dysfunction has been recognized as a critical factor in the initiation and exacerbation of skin inflammation, particularly in patients with atopic dermatitis (AD) and AD-like congenital disorders, including peeling skin syndrome type B. However, inflammatory responses developed in barrier-defective skin, as well as the underlying mechanisms, remained incompletely understood. OBJECTIVE: We aimed to decipher inflammatory axes and the cytokine network in mouse skin on breakdown of epidermal stratum corneum barrier. METHODS: We generated Cdsn(iep-/-) mice with corneodesmosin ablation in keratinocytes selectively in an inducible manner. We characterized inflammatory responses and cytokine expression by using histology, immunohistochemistry, ELISA, and quantitative PCR. We combined mouse genetic tools, antibody-mediated neutralization, signal-blocking reagents, and topical antibiotic treatment to explore the inflammatory axes. RESULTS: We show that on breakdown of the epidermal stratum corneum barrier, type 2 and type 17 inflammatory responses are developed simultaneously, driven by thymic stromal lymphopoietin (TSLP) and IL-23, respectively. Importantly, we reveal a counterregulation between these 2 inflammatory axes. Furthermore, we show that protease-activated receptor 2 signaling is involved in mediating the TSLP/type 2 axis, whereas skin bacteria are engaged in induction of the IL-23/type 17 axis. Moreover, we find that IL-1ß is induced in skin of Cdsn(iep-/-) mice and that blockade of IL-1 signaling suppresses both TSLP and IL-23 expression and ameliorates skin inflammation. CONCLUSION: The inflammatory phenotype in barrier-defective skin is shaped by counterregulation between the TSLP/type 2 and IL-23/type 17 axes. Targeting IL-1 signaling could be a promising therapeutic option for controlling skin inflammation in patients with peeling skin syndrome type B and other diseases related to epidermal barrier dysfunction, including AD.

Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction.

BACKGROUND: Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes. RESULTS: As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost. CONCLUSION: We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).