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Embryo implantation failures are a major challenge in reproductive medicine, but the underlying mechanism remains poorly understood. Successful implantation requires dynamic remodeling of the endometrium through integrated proliferation and differentiation of endometrial cells including luminal epithelial, glandular epithelial, and stromal cells. Conversely, their disruption causes infertility. Spatiotemporal control of transcription is required for these processes; however, the underlying epigenetic regulation is largely unknown. In this study, we examined expression data from the human endometrium during implantation and discovered that expression of the histone lysine methyltransferase KMT2D was significantly suppressed in patients with recurrent implantation failure. Further study revealed that uterine deletion of Kmt2d in mice caused infertility due to implantation failure. Morphological analysis discovered a reduction in the number of uterine glands and aberrant differentiation of the luminal and glandular epithelium into stratified phenotypes in Kmt2d knockout uteri. Administration of leukemia inhibitory factor protein, which is expressed in uterine glands and is essential for implantation, did not rescue implantation failure in Kmt2d knockout mice, suggesting that infertility was not solely due to uterine gland dysfunction. RNA sequencing analysis revealed that Kmt2d knockout uteri displayed suppressed expression of genes involved in ion homeostasis, which may affect the uterine luminal morphology. Our study suggests that KMT2D plays an essential role in facilitating successful embryo implantation by regulating the coordinated differentiation of endometrial cells, providing valuable insights into unexplained implantation failures in women.
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DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are considered worthwhile and many epigenome editing technologies have been developed, which were based on ZF, TALE, and CRISPR-dCas9. Here, we describe a protocol for the application of a modified dCas9-SunTag system, which increased the efficiency of targeted demethylation and gene activation at specific DNA loci. The original SunTag system consists of 10 copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve more efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids. Moreover, we describe the co-recruitment of TET1 and VP64 for efficient gene activation. Since we showed the manipulation of DNA methylation at specific loci and gene activation, its application could lead to its future use in the clinic.
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Sistemas CRISPR-Cas , Metilación de ADN , Humanos , Edición Génica/métodos , Regulación de la Expresión Génica , Epigénesis Genética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
Most studies on fat appetite have focused on long-chain triglycerides (LCTs) due to their obesogenic properties. Medium-chain triglycerides (MCTs), conversely, exhibit antiobesogenic effects; however, the regulation of MCT intake remains elusive. Here, we demonstrate that mice can distinguish between MCTs and LCTs, and the specific appetite for MCTs is governed by hepatic ß-oxidation. We generated liver-specific medium-chain acyl-CoA dehydrogenase (MCAD)-deficient (MCADL-/-) mice and analyzed their preference for MCT and LCT solutions using glyceryl trioctanoate (C8-TG), glyceryl tridecanoate (C10-TG), corn oil, and lard oil in two-bottle choice tests conducted over 8 days. In addition, we used lick microstructure analyses to evaluate the palatability and appetite for MCT and LCT solutions. Finally, we measured the expression levels of genes associated with fat ingestion (Galanin, Qrfp, and Nmu) in the hypothalamus 2 h after oral gavage of fat. Compared with control mice, MCADL-/- mice exhibited a significantly reduced preference for MCT solutions, with no alteration in the preference for LCTs. Lick analysis revealed that MCADL-/- mice displayed a significantly decreased appetite for MCT solutions only while the palatability of both MCT and LCT solutions remained unaffected. Hypothalamic Galanin expression in control mice was elevated by oral gavage of C8-TG but not by LCTs, and this response was abrogated in MCADL-/- mice. In summary, our data suggest that hepatic ß-oxidation is required for MCT-specific appetite but not for LCT-specific appetite. The induction of hypothalamic galanin upon MCT ingestion, dependent on hepatic ß-oxidation, could be involved in the regulation of MCT-specific appetite.NEW & NOTEWORTHY Whether and how medium-chain triglyceride (MCT) intake is regulated remains unknown. Here, we showed that mice can discriminate between MCTs and LCTs. Hepatic ß-oxidation participates in MCT-specific appetite, and hypothalamic galanin may be one of the factors that regulate MCT intake. Because of the antiobesity effects of MCTs, studying MCT-specific appetite may help combat obesity by promoting the intake of MCTs instead of LCTs.
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Acil-CoA Deshidrogenasa , Apetito , Ácidos Grasos , Hígado , Ratones Noqueados , Oxidación-Reducción , Triglicéridos , Animales , Triglicéridos/metabolismo , Ratones , Oxidación-Reducción/efectos de los fármacos , Hígado/metabolismo , Hígado/efectos de los fármacos , Masculino , Ácidos Grasos/metabolismo , Apetito/efectos de los fármacos , Apetito/fisiología , Acil-CoA Deshidrogenasa/metabolismo , Acil-CoA Deshidrogenasa/genética , Ratones Endogámicos C57BL , Hipotálamo/metabolismo , Hipotálamo/efectos de los fármacosRESUMEN
Strabismus, a neuro-ophthalmological condition characterized by misalignment of the eyes, is a common ophthalmic disorder affecting both children and adults. In our previous study, we identified the microsomal glutathione S-transferase 2 (MGST2) gene as one of the potential candidates for comitant strabismus susceptibility in a Japanese population. The MGST2 gene belongs to the membrane-associated protein involved in the generation of pro-inflammatory mediators, and it is also found in the protection against oxidative stress by decreasing the reactivity of oxidized lipids. To look for the roles of the MGST2 gene in the development, eye alignment, and overall morphology of the eye as the possible background of strabismus, MGST2 gene knockout (KO) mice were generated by CRISPR/Cas9-mediated gene editing with guide RNAs targeting the MGST2 exon 2. The ocular morphology of the KO mice was analyzed through high-resolution images obtained by a magnetic resonance imaging (MRI) machine for small animals. The morphometric analyses showed that the height, width, and volume of the eyeballs in MGST2 KO homozygous mice were significantly greater than those of wild-type mice, indicating that the eyes of MGST2 KO homozygous mice were significantly enlarged. There were no significant differences in the axis length and axis angle. These morphological changes may potentially contribute to the development of a subgroup of strabismus.
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The production of cell-type- and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)-mediated cell-type- and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier-penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed nonspecific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in nonastrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre-DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driven AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target-flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO-dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice.
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Knockout mice are useful tools that can provide information about the normal function of genes, including their biochemical, developmental, and physiological roles. One problem associated with the generation of knockout mice is that the loss of some genes of interest produces a lethal phenotype. Therefore, the use of conditioned knockout mice, in which genes are disrupted in specific organs, is essential for the elucidation of disease pathogenesis and the verification of drug targets. In general, conditional knockout mice are produced using the Cre/loxP system; however, the production of the large numbers of Cre/flox knockout and control mice required for analysis requires substantial time and effort. Here, we describe the generation of liver-specific conditional knockout mice via the introduction of lipid nanoparticles encapsulating Cre mRNA into the liver of floxed mice. This technique does not require the production of offspring by mating floxed mice and is therefore more convenient than the conventional method. The results presented here demonstrate that the LNP-based method enables liver-specific gene knockout in a short period of time.
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The precise control of endometrial receptivity is crucial for successful embryo implantation, which is strictly regulated by the ovarian steroid hormones estrogen and progesterone. Despite our improved understanding of the genetic regulation of implantation downstream of the action of hormones, we do not know much about the epigenetic regulation that occurs during early pregnancy. To investigate the role of the N6-methyladenosine (m6A) RNA modification in embryo implantation, we generated mice with conditional deletion of Mettl14, a core component of the m6A writer complex, in the uterus. These mice were infertile due to implantation failure. We showed that Mettl14-deficient uteri had aberrant upregulation of estrogen receptor α (ERα) signaling and ERα phosphorylation, but progesterone receptor (PGR) signaling was largely unaffected. Additionally, Mettl14 deletion led to abnormal activation of the innate immune pathway in Mettl14-deficient uteri. This effect was accompanied by the infiltration of immune cells, such as macrophages and dendritic cells, into the basal region of the endometrial epithelium. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that genes involved in the innate immune response had decreased m6A peaks in Mettl14-deficient mice. These results suggest that Mettl14 plays a crucial role in successful implantation by precisely regulating both ERα signaling and innate immunity in the uterus.
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Receptor alfa de Estrógeno , Receptores de Estrógenos , Embarazo , Femenino , Ratones , Animales , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptores de Estrógenos/metabolismo , Epigénesis Genética , Implantación del Embrión/fisiología , Útero/metabolismo , Progesterona/metabolismo , ARN/metabolismoRESUMEN
CRISPR/Cas9 is the genome-editing technology that is most widely used around the world. Its widespread adoption is largely due to its simplicity and ease of use. Here, we introduce the construction of vectors and genome editing of the target gene in cells using the CRISPR/Cas9 system.
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Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genéticaRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) gene editing enables rapid production of genome-edited animals. The Cas9/guide RNA (gRNA) component can be introduced into zygotes in several ways. Here, we provide an instructional guide for the generation of knockout mice using cytoplasmic injection of in vitro transcribed Cas9 RNA and gRNA.
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Sistemas CRISPR-Cas , Edición Génica , Ratones , Animales , Sistemas CRISPR-Cas/genética , Proteína 9 Asociada a CRISPR/genética , Citoplasma , Ratones Noqueados , ARN Guía de Sistemas CRISPR-CasRESUMEN
Generation of conditional knockout mice using the Cre-loxP system is essential for the analysis of gene functions. The use of CRISPR-Cas9 in combination with two sets of guide RNAs and single-stranded oligonucleotides including loxP sites enables simultaneous insertion of two loxP sequences. Unfortunately, this method induces double-strand breaks at two sites in the same chromosome, which causes an undesirable large chromosomal deletion and reduces the flanked loxP (flox) rate. To overcome this problem, we have developed a method that sequentially introduces each loxP sequence by electroporation at the one- and two-cell embryonic stages, respectively. This sequential electroporation method improves the floxing efficiency compared with the conventional simultaneous method, leading to a high yield of offspring with floxed alleles.
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Terapia de Electroporación , Electroporación , Ratones , Animales , Ratones Noqueados , Integrasas/genética , Sistemas CRISPR-Cas , Ratones TransgénicosRESUMEN
Advances in CRISPR/Cas9 genome editing technologies have allowed for the rapid generation of Cre-loxP conditional knockout mice. However, current strategies for genotyping flox mice, typically based on Sanger sequencing following cloning of target sequences from dozens of pups, are time-consuming. Here, we describe a rapid screening method for flox mice, using in vitro Cre recombination that can be performed using simple enzymatic reactions and enables detection of functional flox mouse within 1 day. In addition, we introduce an efficient strategy for subsequent sequence analysis by cloning of floxed regions using the In-Fusion system. Our genotyping pipeline reduces laborious tasks and thus contributes to the rapid selection of accurately edited flox mice.
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Edición Génica , Integrasas , Ratones , Animales , Ratones Noqueados , Integrasas/genética , Integrasas/metabolismo , Recombinación Genética , Sistemas CRISPR-CasRESUMEN
RAD2lL and REC8, meiosis-specific paralogs of the canonical cohesin subunit RAD21, are essential for proper formation of axial/lateral elements of the synaptonemal complex, synapsis of homologous chromosomes, and crossover recombination in mammalian meiosis. However, how many meiotic cohesins are present in germ cells has not been investigated because of the lack of an appropriate method of analysis. In the present study, to examine the intracellular amount of meiotic cohesins, we generated two strains of knock-in (KI) mice that expressed a 3×FLAG-tag at the C-terminus of RAD21L or REC8 protein using the CRISPR/Cas9 genome editing system. Both KI mice were fertile. Western blot analyses and immunocytochemical studies revealed that expression levels and localization patterns of both RAD21L-3×FLAG and REC8-3×FLAG in KI mice were similar to those in wild-type mice. After confirming that tagging of endogenous RAD21L and REC8 with 3×FLAG did not affect their expression profiles, we evaluated the levels of RAD21L-3×FLAG and REC8-3×FLAG in the testes of 2-week-old mice in which only RAD21L and REC8 but little RAD21 are expressed in the meiocytes. By comparing the band intensities of testicular RAD21L-3×FLAG and REC8-3×FLAG with 3×FLAG-tagged recombinant proteins of known concentrations in western blot analysis, we found that there were approximately 413,000 RAD21L and 453,000 REC8 molecules per spermatocyte in the early stages of prophase I. These findings provide new insights into the role played by cohesins in the process of meiotic chromosome organization in mammalian germ cells.
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Proteínas Nucleares , Espermatocitos , Animales , Masculino , Ratones , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/química , Meiosis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Espermatocitos/metabolismo , CohesinasRESUMEN
Regulating gene expression is important for basic research and therapeutic applications. The epigenome is a record of genetic modifications such as DNA methylation and histone modifications, and epigenetic changes can play a key role in modifying gene expression. With the advent of genome editing technologies, it has become possible to manipulate the epigenome of specific genomic regions to control gene expression. In particular, CRISPR-Cas9 systems have been used widely for epigenome editing due to their high efficiency, versatility, specificity, and ease of use. Here, we describe a protocol for the upregulation of specific genes using the dCas9-SunTag system.
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Sistemas CRISPR-Cas , Edición Génica , Metilación de ADN , Epigénesis Genética , Edición Génica/métodos , Regulación de la Expresión Génica , Activación TranscripcionalRESUMEN
Epigenetic regulatory mechanisms play an important role in gene silencing and genome stability; therefore, epigenetic mutations cause a variety of diseases. Analysis of the epigenome by next-generation sequencers has revealed many epigenetic mutations in various diseases such as cancer, obesity, diabetes, autism, allergies, immune diseases, and imprinting diseases. Unfortunately, it has been difficult to identify the causative epigenetic mutations because there has been no method to generate animals with target-specific epigenetic mutations. However, it has become possible to generate such animals due to the recent development of epigenome editing technology. Here, we introduce the generation of epigenome-edited mice by target-specific DNA demethylation.
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Sistemas CRISPR-Cas , Edición Génica , Animales , Desmetilación del ADN , Metilación de ADN , Epigénesis Genética , Epigenoma , Edición Génica/métodos , RatonesRESUMEN
Synergistic effects among multiple gene mutations are involved in cancer development and progression. However, developing genetically modified mouse models to analyze various combinations of mutations is extremely labor-intensive and time-consuming. To address these problems, we developed a novel method for in vivo multiplexed genome editing of the murine uterus to model human endometrial carcinoma (EMC). To do this, we injected a CRISPR-Cas9 ribonucleoprotein complex into the uterine cavity of adult female mice, followed by electroporation. Evaluation of reporter mice demonstrated that genome editing occurred specifically in uterine epithelial cells, which are the origin of EMCs. Simultaneous targeting of Pten/Trp53/Lkb1, or targeting of Pten/Lkb1 along with the Ctnnb1ΔEx3 mutation, resulted in efficient generation of invasive tumors in wild-type females within 3 months. This novel method will enable rapid and easy validation of many combinations of gene mutations that lead to endometrial carcinogenesis.
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Neoplasias Endometriales , Edición Génica , Ratones , Femenino , Humanos , Animales , Edición Génica/métodos , Sistemas CRISPR-Cas , Ribonucleoproteínas/genética , Electroporación/métodos , Neoplasias Endometriales/genéticaRESUMEN
BACKGROUND: Epigenome-edited animal models enable direct demonstration of disease causing epigenetic mutations. Transgenic (TG) mice stably expressing epigenome-editing factors exhibit dramatic and stable changes in target epigenome modifications. Successful germline transmission of a transgene from founder mice to offspring will yield a sufficient number of epigenome-edited mice for phenotypic analysis; however, if the epigenetic mutation has a detrimental phenotypic effect, it can become difficult to obtain the next generation of animals. In this case, the phenotype of founder mice must be analyzed directly. Unfortunately, current TG mouse production efficiency (TG founders per pups born) is relatively low, and improvements would increase the versatility of this technology. RESULTS: In the current study, we describe an approach to generate epigenome-edited TG mice using a combination of both the dCas9-SunTag and piggyBac (PB) transposon systems. Using this system, we successfully generated mice with demethylation of the differential methylated region of the H19 gene (H19-DMR), as a model for Silver-Russell syndrome (SRS). SRS is a disorder leading to growth retardation, resulting from low insulin-like growth factor 2 (IGF2) gene expression, often caused by epimutations at the H19-IGF2 locus. Under optimized conditions, the efficiency of TG mice production using the PB system was approximately threefold higher than that using the conventional method. TG mice generated by this system showed demethylation of the targeted DNA region and associated changes in gene expression. In addition, these mice exhibited some features of SRS, including intrauterine and postnatal growth retardation, due to demethylation of H19-DMR. CONCLUSIONS: The dCas9-SunTag and PB systems serve as a simple and reliable platform for conducting direct experiments using epigenome-edited founder mice.
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Epigenoma , ARN Largo no Codificante , Ratones , Animales , Metilación de ADN , ARN Largo no Codificante/genética , Ratones Transgénicos , Epigénesis Genética , Trastornos del Crecimiento/genéticaRESUMEN
During erythroid differentiation, the maintenance of genome integrity is key for the success of multiple rounds of cell division. However, molecular mechanisms coordinating the expression of DNA repair machinery in erythroid progenitors are poorly understood. Here, we discover that an RNA N6-methyladenosine (m6A) methyltransferase, METTL16, plays an essential role in proper erythropoiesis by safeguarding genome integrity via the control of DNA-repair-related genes. METTL16-deficient erythroblasts exhibit defective differentiation capacity, DNA damage and activation of the apoptotic program. Mechanistically, METTL16 controls m6A deposition at the structured motifs in DNA-repair-related transcripts including Brca2 and Fancm mRNAs, thereby upregulating their expression. Furthermore, a pairwise CRISPRi screen revealed that the MTR4-nuclear RNA exosome complex is involved in the regulation of METTL16 substrate mRNAs in erythroblasts. Collectively, our study uncovers that METTL16 and the MTR4-nuclear RNA exosome act as essential regulatory machinery to maintain genome integrity and erythropoiesis.
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Eritropoyesis , Metiltransferasas , Metiltransferasas/metabolismo , Metilación , Eritropoyesis/genética , Adenosina/metabolismo , ARN Mensajero/metabolismo , Eritroblastos/metabolismo , ADN/metabolismoRESUMEN
The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ-knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature Prkcgfl/fl mice was impaired by the Cre-dependent removal of PKCγ from PCs. Notably, the rescue or removal of PKCγ from mature PKCγ-KO or Prkcgfl/fl mice, respectively, did not affect the CF innervation profile of PCs, suggesting the presence of a mechanism distinct from multiple CF innervation of PCs for the motor defects in PKCγ-deficient mice. We found marked potentiation of Ca2+-activated large-conductance K+ (BK) channel currents in PKCγ-deficient mice, as compared to wild-type mice, which decreased the membrane resistance, resulting in attenuation of the electrical signal during the propagation and significant alterations of the complex spike waveform. These changes in PKCγ-deficient mice were restored by the rescue of PKCγ or pharmacological suppression of BK channels. Our results suggest that PKCγ is a critical regulator that negatively modulates BK currents in PCs, which significantly influences PC output from the cerebellar cortex and, eventually, motor coordination.
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Terapia Genética , Actividad Motora/genética , Canales de Potasio Calcio-Activados/metabolismo , Proteína Quinasa C/metabolismo , Células de Purkinje/enzimología , Animales , Señalización del Calcio , Eliminación de Gen , Ratones , Ratones Noqueados , Actividad Motora/fisiología , Canales de Potasio Calcio-Activados/genética , Proteína Quinasa C/genética , Potenciales SinápticosRESUMEN
Congenital malformations cause life-threatening diseases in pediatrics, yet the molecular mechanism of organogenesis is poorly understood. Here we show that Dyrk2-deficient mice display congenital malformations in multiple organs. Transcriptome analysis reveals molecular pathology of Dyrk2-deficient mice, particularly with respect to Foxf1 reduction. Mutant pups exhibit sudden death soon after birth due to respiratory failure. Detailed analyses of primordial lungs at the early developmental stage demonstrate that Dyrk2 deficiency leads to altered airway branching and insufficient alveolar development. Furthermore, the Foxf1 expression gradient in mutant lung mesenchyme is disrupted, reducing Foxf1 target genes, which are necessary for proper airway and alveolar development. In ex vivo lung culture system, we rescue the expression of Foxf1 and its target genes in Dyrk2-deficient lung by restoring Shh signaling activity. Taken together, we demonstrate that Dyrk2 is essential for embryogenesis and its disruption results in congenital malformation.
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Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Expresión Génica , Enfermedades Pulmonares/genética , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Tirosina Quinasas/deficiencia , Animales , Factores de Transcripción Forkhead/metabolismo , Enfermedades Pulmonares/congénito , Ratones , Quinasas DyrKRESUMEN
The HapMap Project is a major international research effort to construct a resource to facilitate the discovery of relationships between human genetic variations and health and disease. The Ser19Stop single nucleotide polymorphism (SNP) of human phytanoyl-CoA hydroxylase-interacting protein-like (PHYHIPL) gene was detected in HapMap project and registered in the dbSNP. PHYHIPL gene expression is altered in global ischemia and glioblastoma multiforme. However, the function of PHYHIPL is unknown. We generated PHYHIPL Ser19Stop knock-in mice and found that PHYHIPL impacts the morphology of cerebellar Purkinje cells (PCs), the innervation of climbing fibers to PCs, the inhibitory inputs to PCs from molecular layer interneurons, and motor learning ability. Thus, the Ser19Stop SNP of the PHYHIPL gene may be associated with cerebellum-related diseases.