Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci.

BACKGROUND: The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e. two loxP sites flanking an exon or a critical DNA sequence of interest) are amongst the most widely requested project type that are challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. An approach, using embryo sequential electroporation has been reported in the literature to successfully introduce loxP sites on the same allele. Here, we describe a modification of this sequential electroporation procedure that demonstrated the production of conditional allele mouse models for eight different genes via one of two possible strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). This latest strategy originated from using the by-product produced when using consecutive sequential electroporation (i.e. mice with a single targeted loxP site) to complete the project. RESULTS: By using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 and 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 and 25%. CONCLUSION: Our modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.

Absence of Mutation Enrichment for Genes Phylogenetically Conserved in the Olivocerebellar Motor Circuitry in a Cohort of Canadian Essential Tremor Cases.

Essential Tremor is a prevalent neurological disorder of unknown etiology. Studies suggest that genetic factors contribute to this pathology. To date, no causative mutations in a gene have been reproducibly reported. All three structures of the olivocerebellar motor circuitry have been linked to Essential Tremor. We postulated that genes enriched for their expression in the olivocerebellar circuitry would be more susceptible to harbor mutations in Essential Tremor patients. A list of 11 candidate genes, enriched for their expression in the olivocerebellar circuitry, was assessed for their variation spectrum and frequency in a cohort of Canadian Essential Tremor cases. Our results from this list of 11 candidate genes do not support an association for Essential Tremor in our cohort of Canadian cases. The heterogenic nature of ET and modest size of the cohort used in this study are two confounding factors that could explain these results.

Teneurin transmembrane protein 4 is not a cause for essential tremor in a Canadian population.

INTRODUCTION: Mutations in teneurin transmembrane protein 4 were reported to be a risk factor for essential tremor, but the relevance of this across different population remains to be examined. The aim of this study was to determine the frequency and spectrum of variations in teneurin transmembrane protein 4 in a cohort of Canadian essential tremor cases. METHODS: The coding portion of teneurin transmembrane protein 4 was sequenced in 269 unrelated essential tremor cases and 288 matched control individuals using a targeted and high-throughput sequencing approach. RESULTS: A total of 157 single nucleotide variations were identified, and from these 99 were a missense or nonsense mutation. A total of 68 cases were carriers of ≥1 rare missense or nonsense mutations, and 39 control individuals were carriers of the same types of variations. Gene-based association tests were used to jointly analyze the single nucleotide variations. CONCLUSIONS: Our results do not support a positive association between teneurin transmembrane protein 4 and the Canadian population. © 2017 International Parkinson and Movement Disorder Society.

No rare deleterious variants from STK32B, PPARGC1A, and CTNNA3 are associated with essential tremor.

OBJECTIVE: To assess the contribution of variants in STK32B, PPARGC1A, and CTNNA3 as essential tremor (ET) predisposing factors following their association in a 2-stage genome-wide association study (GWAS). METHODS: The coding regions of these genes was examined for the presence of rare variants using two approaches: (1) Looking at whole-exome and whole-genome sequencing data of 14 autosomal dominant multiplex ET families. (2) Conducting a targeted massive parallel sequencing to examine the three genes in cohorts of 269 ET cases and 287 control individuals. The cumulative impact of rare variants was assessed using SKAT-O analyses using (1) all variants, (2) only rare variants, and (3) only the rare variants altering the mRNA. RESULTS: Thirty-four variants were identified. No difference emerged regarding the distributions of individual variants (or gene) between cases and controls. CONCLUSION: No rare exonic variants further validated one of these genes as a risk factor for ET. The recent GWAS offers promising avenues, but the genetic heterogeneity of ET is nonetheless challenging for the validation of risk factors, and ultimately larger cohorts of cases should help to overcome this task.

Inhibition of the kinase WNK1/HSN2 ameliorates neuropathic pain by restoring GABA inhibition.

HSN2is a nervous system predominant exon of the gene encoding the kinase WNK1 and is mutated in an autosomal recessive, inherited form of congenital pain insensitivity. The HSN2-containing splice variant is referred to as WNK1/HSN2. We created a knockout mouse specifically lacking theHsn2exon ofWnk1 Although these mice had normal spinal neuron and peripheral sensory neuron morphology and distribution, the mice were less susceptible to hypersensitivity to cold and mechanical stimuli after peripheral nerve injury. In contrast, thermal and mechanical nociceptive responses were similar to control mice in an inflammation-induced pain model. In the nerve injury model of neuropathic pain, WNK1/HSN2 contributed to a maladaptive decrease in the activity of the K(+)-Cl(-)cotransporter KCC2 by increasing its inhibitory phosphorylation at Thr(906)and Thr(1007), resulting in an associated loss of GABA (γ-aminobutyric acid)-mediated inhibition of spinal pain-transmitting nerves. Electrophysiological analysis showed that WNK1/HSN2 shifted the concentration of Cl(-)such that GABA signaling resulted in a less hyperpolarized state (increased neuronal activity) rather than a more hyperpolarized state (decreased neuronal activity) in mouse spinal nerves. Pharmacologically antagonizing WNK activity reduced cold allodynia and mechanical hyperalgesia, decreased KCC2 Thr(906)and Thr(1007)phosphorylation, and restored GABA-mediated inhibition (hyperpolarization) of injured spinal cord lamina II neurons. These data provide mechanistic insight into, and a compelling therapeutic target for treating, neuropathic pain after nerve injury.

Combined serial analysis of gene expression and transcription factor binding site prediction identifies novel-candidate-target genes of Nr2e1 in neocortex development.

BACKGROUND: Nr2e1 (nuclear receptor subfamily 2, group e, member 1) encodes a transcription factor important in neocortex development. Previous work has shown that nuclear receptors can have hundreds of target genes, and bind more than 300 co-interacting proteins. However, recognition of the critical role of Nr2e1 in neural stem cells and neocortex development is relatively recent, thus the molecular mechanisms involved for this nuclear receptor are only beginning to be understood. Serial analysis of gene expression (SAGE), has given researchers both qualitative and quantitative information pertaining to biological processes. Thus, in this work, six LongSAGE mouse libraries were generated from laser microdissected tissue samples of dorsal VZ/SVZ (ventricular zone and subventricular zone) from the telencephalon of wild-type (Wt) and Nr2e1-null embryos at the critical development ages E13.5, E15.5, and E17.5. We then used a novel approach, implementing multiple computational methods followed by biological validation to further our understanding of Nr2e1 in neocortex development. RESULTS: In this work, we have generated a list of 1279 genes that are differentially expressed in response to altered Nr2e1 expression during in vivo neocortex development. We have refined this list to 64 candidate direct-targets of NR2E1. Our data suggested distinct roles for Nr2e1 during different neocortex developmental stages. Most importantly, our results suggest a possible novel pathway by which Nr2e1 regulates neurogenesis, which includes Lhx2 as one of the candidate direct-target genes, and SOX9 as a co-interactor. CONCLUSIONS: In conclusion, we have provided new candidate interacting partners and numerous well-developed testable hypotheses for understanding the pathways by which Nr2e1 functions to regulate neocortex development.

Genetics of essential tremor: from phenotype to genes, insights from both human and mouse studies.

Essential tremor (ET) is a prevalent neurological disorder of unknown etiology characterized by the presence of an action tremor that occurs during voluntary motion and affects primarily the upper limbs. The worldwide prevalence of the disease in the general population is 0.9%, increasing to 4.6% in individuals ≥65 years old. Standard pharmaceutical treatments are only moderately effective, reducing tremor amplitudes in ∼50% of patients, a phenomenon partly explained by the fact that the diagnosis of ET is based solely on clinical findings rather than biological markers. Furthermore, the pathophysiological origin of ET remains controversial, leading to heated debates as to whether it should be considered a neurodegenerative disorder or as a dynamic oscillatory disturbances of neurologic origin. Progress has been made in the understanding of its etiology as it is now accepted that genetic components must at least explain the familial cases of ET, and the evidence implicating the olivocerebellar and cerebello-thalamo-cortical pathways is strong. However, a strong disconnection between human genetics, pathophysiological, and mouse genetics studies exists in the field of ET, with little use of all the knowledge gathered from the different research disciplines. This review highlights our current knowledge on ET from both a human population and mouse genetics perspective hoping to reconcile evidence from both fields and leading to novel clues guiding future studies. We argue that better communication between researchers of different fields is warranted to define the biological origin of ET in the hope of leading to the development of better treatments.

Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice.

BACKGROUND: The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome') strategy to expand our understanding of human gene regulation in vivo. RESULTS: In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear. CONCLUSIONS: We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression.

Modelling human regulatory variation in mouse: finding the function in genome-wide association studies and whole-genome sequencing.

An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and ß-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant-harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation.

Loss of Smad5 leads to the disassembly of the apical junctional complex and increased susceptibility to experimental colitis.

The regulation of intestinal epithelial cell adhesion and migratory properties is often compromised in inflammatory bowel disease (IBD). Despite an increasing interest in bone morphogenetic protein (Bmp) signaling in gut pathologies, little is known of the specific roles played by individual Smads in intestinal epithelial functions. In the present study, we generated a mouse model with deletion of Smad5 transcriptional effector of the Bmp signaling pathway exclusively in the intestinal epithelium. Proliferation, migration, and apical junctional complex (AJC) protein expression were analyzed by immunofluorescence and Western blot. Human intestinal biopsies from control and IBD patients were analyzed for SMAD5 gene transcript expression by quantitative PCR (qPCR). Smad5(ΔIEC) and control mice were subjected to dextran sulfate sodium (DSS)-induced experimental colitis, and their clinical and histological symptoms were assessed. Loss of Smad5 led to intestinal epithelial hypermigration and deregulation of the expression of claudin-1 and claudin-2. E-cadherin was found to be equally expressed but displaced from the AJC to the cytoplasm in Smad5(ΔIEC) mice. Analysis of SMAD5 gene expression in human IBD patient samples revealed a significant downregulation of the gene transcript in Crohn's disease and ulcerative colitis samples. Smad5(ΔIEC) mice exposed to experimental DSS colitis were significantly more susceptible to the disease and had impaired wound healing during the recovery phase. Our results support that Smad5 is partly responsible for mediating Bmp signals in intestinal epithelial cells. In addition, deficiency in epithelial Smad5 leads to the deregulation of cell migration by disassembling the AJC with increasing susceptibility to experimental colitis and impairment in wound healing.

A regulatory toolbox of MiniPromoters to drive selective expression in the brain.

The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination "knockins" in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5' of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type-specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies.