Search

BackgroundThe 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) remain the most challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. In the current manuscript, we describe a modification of the sequential electroporation procedure described by Horii et al (2017). We demonstrate production of conditional allele mouse models for eight different genes via one of two alternative strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B).ResultsBy 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 to 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 to 25%.ConclusionOur 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.

Background Aims Hirschsprung disease (HSCR) is a life-threatening birth defect in which the distal colon is devoid of enteric neural ganglia. HSCR is treated by surgical removal of aganglionic bowel, but many children continue to have severe problems after surgery. We studied whether administration of glial cell derived neurotrophic factor (GDNF) induces enteric nervous system regeneration in mouse models of HSCR. Methods We performed studies with four mouse models of HSCR: Holstein (HolTg/Tg, a model for trisomy 21-associated HSCR), TashT (TashTTg/Tg, a model for male-biased HSCR), Piebald-lethal (Ednrbs-l//s-l, a model for EDNRB mutation-associated HSCR), and Ret9/- (with aganglionosis induced by mycophenolate). Mice were given rectal enemas containing GDNF or saline (control) from postnatal days 4 through 8. We measured survival times of mice, and colon tissues were analyzed by histology, immunofluorescence, and immunoblots. Neural ganglia regeneration and structure, bowel motility, epithelial permeability, muscle thickness, and neutrophil infiltration were studied in colon tissues and in mice. Stool samples were collected, and microbiomes were analyzed by 16S rRNA gene sequencing. Time-lapse imaging and genetic cell-lineage tracing were used to identify a source of GDNF-targeted neural progenitors. Human aganglionic colon explants from children with HSCR were cultured with GDNF and evaluated for neurogenesis. Results GDNF significantly prolonged mean survival times of HolTg/Tg mice, Ednrbs-l//s-l mice, and male TashTTg/Tg mice, compared with control mice, but not Ret9/- mice (which had mycophenolate toxicity). Mice given GDNF developed neurons and glia in distal bowel tissues that were aganglionic in control mice, had a significant increase in colon motility, and had significant decreases in epithelial permeability, muscle thickness, and neutrophil density. We observed dysbiosis in fecal samples from HolTg/Tg mice compared with feces from wild-type mice; fecal microbiomes of mice given GDNF were similar to those of wild-type mice except for Bacteroides. Exogenous luminal GDNF penetrated aganglionic colon epithelium of HolTg/Tg mice, inducing production of endogenous GDNF, and new enteric neurons and glia appeared to arise from Schwann cells within extrinsic nerves. GDNF application to cultured explants of human aganglionic bowel induced proliferation of Schwann cells and formation of new neurons. Conclusions GDNF prolonged survival, induced enteric neurogenesis, and improved colon structure and function in 3 mouse models of HSCR. Application of GDNF to cultured explants of aganglionic bowel from children with HSCR induced proliferation of Schwann cells and formation of new neurons. GDNF might be developed for treatment of HSCR.

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (

CHARGE syndrome—which stands for coloboma of the eye, heart defects, atresia of choanae, retardation of growth/development, genital abnormalities, and ear anomalies—is a severe developmental disorder with wide phenotypic variability, caused mainly by mutations in CHD7 (chromodomain helicase DNA-binding protein 7), known to encode a chromatin remodeler. The genetic lesions responsible for CHD7 mutation-negative cases are unknown, at least in part because the pathogenic mechanisms underlying CHARGE syndrome remain poorly defined. Here, we report the characterization of a mouse model for CHD7 mutation-negative cases of CHARGE syndrome generated by insertional mutagenesis of Fam172a (family with sequence similarity 172, member A). We show that Fam172a plays a key role in the regulation of cotranscriptional alternative splicing, notably by interacting with Ago2 (Argonaute-2) and Chd7. Validation studies in a human cohort allow us to propose that dysregulation of cotranscriptional alternative splicing is a unifying pathogenic mechanism for both CHD7 mutation-positive and CHD7 mutation-negative cases. We also present evidence that such splicing defects can be corrected in vitro by acute rapamycin treatment.

Pax3 encodes a paired-box transcription factor with key roles in neural crest and neural tube ontogenesis. Robust control of Pax3 neural expression is ensured by two redundant sets of cis-regulatory modules (CRMs) that integrate anterior-posterior (such as Wnt-βCatenin signaling) as well as dorsal-ventral (such as Shh-Gli signaling) instructive cues. In previous work, we sought to characterize the Wnt-mediated regulation of Pax3 expression and identified the Cdx transcription factors (Cdx1/2/4) as critical intermediates in this process. We identified the neural crest enhancer-2 (NCE2) from the 5'-flanking region of Pax3 as a Cdx-dependent CRM that recapitulates the restricted expression of Pax3 in the mouse caudal neuroectoderm. While this is consistent with a key role in relaying the inductive signal from posteriorizing Wnt ligands, the broad expression of Cdx proteins in the tailbud region is not consistent with the restricted activity of NCE2. This implies that other positive and/or negative inputs are required and, here, we report a novel role for the transcription factor Zic2 in this regulation. Our data strongly suggests that Zic2 is involved in the induction (as a direct Pax3NCE2 activator and Cdx neural cofactor) as well as the maintenance of Pax3 dorsal restriction (as a target of the ventral Shh repressive input). We also provide evidence that the inductive Cdx-Zic2 interaction is integrated on NCE2 with a positive input from the neural-specific transcription factor Sox2. Altogether, our data provide important mechanistic insights into the coordinated integration of different signaling pathways on a short Pax3 CRM.

Numerous studies in chordates and arthropods currently indicate that Cdx proteins have a major ancestral role in the organization of post-head tissues. In urochordate embryos, Cdx loss-of-function has notably been shown to impair axial elongation, neural tube (NT) closure and pigment cell development. Intriguingly, in contrast to axial elongation and NT closure, a Cdx role in neural crest (NC)-derived melanocyte/pigment cell development has not been reported in any other chordate species. To address this, we generated a new conditional pan-Cdx functional knockdown mouse model that allows circumventing Cdx functional redundancy as well as the early embryonic lethality of Cdx mutants. Through directed inhibition in the neuroectoderm, we here provide in vivo evidence that murine Cdx proteins impact melanocyte and enteric nervous system development via, at least in part, directly controlling the expression of the key early regulators of NC ontogenesis Pax3, Msx1 and FoxD3. Our work thus reveals a novel role for the Cdx proteins atop of the trunk NC-gene regulatory network in the mouse, which appears to be inherited from their ancestral orthologue.