Point Mutant Mice Research Articles

Upregulation of NFE2L1 reduces ROS levels and α-synuclein aggregation caused by GBA1 knockdown

Biallelic mutations in the GBA1 gene result in Gaucher disease (GD), and both patients with GD and carriers of a single GBA1 mutation have an increased susceptibility to Parkinson's disease (PD), but the underlying mechanisms of this association are not yet clear. In previous studies, we established Gba1 F213I point mutation mice and found that homozygous Gba1 F213I mutant mice died shortly after birth, while heterozygous mice could survive normally. In this study, we investigated the transcriptomic changes in the brain tissue of Gba1 F213I heterozygous mice, identifying 138 differentially expressed genes. Among them, Nfe2l1 was the most significantly downregulated gene. Inhibition or knockdown of GBA1 in BE(2)-M17 cells resulted in decreased expression levels of NFE2L1. Knockdown of GBA1 or NFE2L1 could lead to an elevation in intracellular aggregation of α-synuclein (α-syn) and reactive oxygen species (ROS) levels, while upregulation of NFE2L1 effectively mitigated those cellular manifestations induced by GBA1 knockdown. In summary, our in vitro results showed that upregulation of NFE2L1 may provide a therapeutic benefit for cellular phenotypes resulting from GBA1 knockdown, providing new insights for future research on GD and GBA1-associated PD.

CHCHD2 mutant mice display mitochondrial protein accumulation and disrupted energy metabolism.

Mutations in the mitochondrial cristae protein CHCHD2 lead to a late-onset autosomal dominant form of Parkinson's disease (PD) which closely resembles idiopathic PD, providing the opportunity to gain new insights into the mechanisms of mitochondrial dysfunction contributing to PD. To begin to address this, we used CRISPR genome-editing to generate CHCHD2 T61I point mutant mice. CHCHD2 T61I mice had normal viability, and had only subtle motor deficits with no signs of premature dopaminergic (DA) neuron degeneration. Nonetheless, CHCHD2 T61I mice exhibited robust molecular changes in the brain including increased CHCHD2 insolubility, accumulation of CHCHD2 protein preferentially in the substantia nigra (SN), and elevated levels of α-synuclein. Metabolic analyses revealed an increase in glucose metabolism through glycolysis relative to the TCA cycle with increased respiratory exchange ratio, and immune-electron microscopy revelated disrupted mitochondria in DA neurons. Moreover, spatial genomics revealed decreased expression of mitochondrial complex I and III respiratory chain proteins, while proteomics revealed increased respiratory chain and other mitochondrial protein-protein interactions. As such, the CHCHD2 T61I point-mutation mice exhibit robust mitochondrial disruption and a consequent metabolic shift towards glycolysis. These findings thus establish CHCHD2 T61I mice as a new model for mitochondrial-based PD, and implicate disrupted respiratory chain function as a likely causative driver.

A Novel MCT8 point-mutant mouse model shows effectiveness of phenylbutyrate treatment

A Novel MCT8 point-mutant mouse model shows effectiveness of phenylbutyrate treatment

Fine-tuning AMPK in physiology and disease using point-mutant mouse models.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that monitors the cellular energy status to adapt it to the fluctuating nutritional and environmental conditions in an organism. AMPK plays an integral part in a wide array of physiological processes, such as cell growth, autophagy and mitochondrial function, and is implicated in diverse diseases, including cancer, metabolic disorders, cardiovascular diseases and neurodegenerative diseases. AMPK orchestrates many different physiological outcomes by phosphorylating a broad range of downstream substrates. However, the importance of AMPK-mediated regulation of these substrates in vivo remains an ongoing area of investigation to better understand its precise role in cellular and metabolic homeostasis. Here, we provide a comprehensive overview of our understanding of the kinase function of AMPK in vivo, as uncovered from mouse models that harbor phosphorylation mutations in AMPK substrates. We discuss some of the inherent limitations of these mouse models, highlight the broader implications of these studies for understanding human health and disease, and explore the valuable insights gained that could inform future therapeutic strategies for the treatment of metabolic and non-metabolic disorders.

SLC10A5 deficiency causes hypercholanemia.

Solute Carrier Family 10 Member 5 (SLC10A5) is a member of SLC10, comprising transporters of bile acids, steroidal hormones, and other substrates, but its function remains unclear. The aim of the current investigation was to clarify its function in the metabolism of bile acid and hypercholanemia. Whole-exome sequencing and Sanger sequencing were used to identify and confirm the variant in the subjects of hypercholanemia. CRISPR/Cas9-mediated genome engineering was used to establish the knockout and point mutation mice. Primary mouse hepatocytes were isolated, and cell lines were cultured. SLC10A5 was silenced by siRNA and overexpressed by wild-type and mutant plasmids. The fluorescent bile acid derivative was used for the bile acid uptake assay. Bile acids were assessed with ultra-performance liquid chromatography tandem mass spectrometry. A heterozygous variant SLC10A5 : c.994_995del (p.D332X) was identified in subjects with elevated total bile acid or altered bile acid profiles. Bile acids were increased in the serum and liver of knockout and point mutation mice. The expressions of FXR and SHP, regulators involved in the negative feedback of bile acid synthesis, were downregulated, while the bile acid synthesis genes CYP7A1 and CYP8B1 were upregulated in both gene-edited mice. Both the wild and mutant SLC10A5 proteins were localized on the plasma membrane. Knockdown, knockout, or targeted mutation of SLC10A5 led to the inhibition of bile acid uptake by cell lines and primary mouse hepatocytes. SLC10A5 is involved in the uptake of bile acid, and its deficiency causes hypercholanemia.

EphB2 Receptor Promotes Dermal Fibrosis in Systemic Sclerosis.

Erythropoietin-producing hepatocellular (Eph)/Ephrin cell-cell signaling is emerging as a key player in tissue fibrogenesis. The aim of this study was to test the hypothesis that the receptor tyrosine kinase EphB2 mediates dermal fibrosis in systemic sclerosis (SSc). We assessed normal and SSc human skin biopsies for EphB2 expression. The in vivo role of EphB2 in skin fibrosis was investigated by subjecting EphB2-knockout mice to both bleomycin-induced and tight skin (Tsk1/+) genetic mouse models of skin fibrosis. EphB2 kinase-dead and overactive point mutant mice were used to evaluate the role of EphB2 forward signaling in bleomycin-induced dermal fibrosis. In vitro studies were performed on dermal fibroblasts from patients with SSc and healthy controls, which was followed by in vivo analysis of fibroblast-specific Ephb2-deficient mice. Expression of EphB2 is up-regulated in SSc skin tissue and explanted SSc dermal fibroblasts compared with healthy controls. EphB2 expression is elevated in two animal models of dermal fibrosis. In mice, EphB2 drives dermal fibrosis in both the bleomycin and the Tsk1/+ models of skin fibrosis. EphB2 forward signaling is a critical mediator of dermal fibrosis. Transforming growth factor-β (TGF-β) cytokines up-regulate EphB2 in dermal fibroblasts via noncanonical TGF-β/mother against decapentaplegic signaling, and silencing EPHB2 in human dermal fibroblasts is sufficient to dampen TGF-β-induced fibroblast-to-myofibroblast differentiation. Moreover, mice with fibroblast-specific deletion of EphB2 showed impaired fibroblast-to-myofibroblast differentiation and reduced skin fibrosis upon bleomycin challenge. Our data implicate TGF-β regulation of EphB2 overexpression and kinase-mediated forward signaling in the development of dermal fibrosis in SSc. EphB2 thus represents a potential new therapeutic target for SSc.

GEN1 as a risk factor for human congenital anomalies of the kidney and urinary tract

BackgroundCongenital anomalies of the kidney and urinary tract (CAKUT) are prevalent birth defects. Although pathogenic CAKUT genes are known, they are insufficient to reveal the causes for all patients. Our previous studies indicated GEN1 as a pathogenic gene of CAKUT in mice, and this study further investigated the correlation between GEN1 and human CAKUT.MethodsIn this study, DNA from 910 individuals with CAKUT was collected; 26 GEN1 rare variants were identified, and two GEN1 (missense) variants in a non-CAKUT group were found. Mainly due to the stability results of the predicted mutant on the website, in vitro, 10 variants (eight CAKUT, two non-CAKUT) were selected to verify mutant protein stability. In addition, mainly based on the division of the mutation site located in the functional region of the GEN1 protein, 8 variants (six CAKUT, two non-CAKUT) were selected to verify enzymatic hydrolysis, and the splice variant GEN1 (c.1071 + 3(IVS10) A > G) was selected to verify shear ability. Based on the results of in vitro experiments and higher frequency, three sites with the most significant functional change were selected to build mouse models.ResultsProtein stability changed in six variants in the CAKUT group. Based on electrophoretic mobility shift assay of eight variants (six CAKUT, two non-CAKUT), the enzymatic hydrolysis and DNA-binding abilities of mutant proteins were impaired in the CAKUT group. The most serious functional damage was observed in the Gen1 variant that produced a truncated protein. A mini-gene splicing assay showed that the variant GEN1 (c.1071 + 3(IVS10) A > G) in the CAKUT group significantly affected splicing function. An abnormal exon10 was detected in the mini-gene splicing assay. Point-mutant mouse strains were constructed (Gen1: c.1068 + 3 A > G, p.R400X, and p.T105R) based on the variant frequency in the CAKUT group and functional impairment in vitro study and CAKUT phenotypes were replicated in each.ConclusionOverall, our findings indicated GEN1 as a risk factor for human CAKUT.

A missense mutation in Lama3 causes androgen alopecia

Hair loss disorders such as androgenetic alopecia have caused serious disturbances to normal human life. Animal models play an important role in exploring pathogenesis of disease and evaluating new therapies. NIH hairless mice are a spontaneous hairless mouse discovered and bred in our laboratory. In this study, we resequenced the genomes of NIH normal mice and NIH hairless mice and obtained 3,575,560 high-quality, plausible SNP loci and 995,475 InDels. The Euclidean distance algorithm was used to assess the association of SNP loci with the hairless phenotype, at a threshold of 0.62. Two regions of chromosome 18 having the highest association with the phenotype contained 345 genes with a total length of 13.98 Mb. The same algorithm was used to assess the association of InDels with the hairless phenotype at a threshold of 0.54 and revealed a region of 25.45 Mb in length, containing 518 genes. The mutation candidate gene Lama3 (NM_010680.2: c.652C>T; NP_034810.1: p. Arg217Cys) was selected based on the results of functional gene analysis and mutation prediction screening. Lama3 (R217C) mutant mice were further constructed using CRISPR/Cas9 technology, and the relationship between Lama3 point mutations and the hairless phenotype were clarified by phenotypic observation. The results showed that male Lama3 point mutation mice started to lose hair on the 80th day after birth, and the hair loss area gradually expanded over time. H&E staining of skin sections showed that the point mutation mice had increased sebaceous glands in the dermis and missing hair follicle structure (i.e., typical symptoms of androgenetic alopecia). This study is a good extension of the current body of knowledge about the function of Lama3, and the constructed Lama3 (R217C) mutant mice may be a good animal model for studying androgenetic alopecia.

Homozygous missense variant in MEIOSIN causes premature ovarian insufficiency.

Are variants of genes involved in meiosis initiation responsible for premature ovarian insufficiency (POI)? A MEIOSIN variant participates in the pathogenesis of human POI by impairing meiosis due to insufficient transcriptional activation of essential meiotic genes. Meiosis is the key event for the establishment of the ovarian reserve, and several gene defects impairing meiotic homologous recombination have been found to contribute to the pathogenesis of POI. Although STRA8 and MEIOISN variants have been found to associate with POI in a recent study, the condition of other meiosis initiation genes is unknown and direct evidence of variants participating in the pathogenesis of POI is still lacking. This was a retrospective genetic study. An in-house whole exome sequencing (WES) database of 1030 idiopathic POI patients was screened for variations of meiosis initiation genes. Homozygous or compound heterozygous variations of genes involved in meiosis initiation were screened in the in-house WES database. The pathogenicity of the variation was verified by in vitro experiments, including protein structure prediction and dual-luciferase reporter assay. The effect of the variant on ovarian function and meiosis was demonstrated through histological analyses in a point mutation mouse model. One homozygous variant in MEIOSIN (c.1735C>T, p.R579W) and one in STRA8 (c.258 + 1G>A), which initiates meiosis via the retinoic acid-dependent pathway, were identified in a patient with idiopathic POI respectively. The STRA8 variation has been reported in the recently published work. For the MEIOSIN variation, the dual-luciferase reporter assay revealed that the variant adversely affected the transcriptional function of MEIOSIN in upregulating meiotic genes. Furthermore, knock-in mice with the homologous mutation confirmed that the variation impacted the meiotic prophase I program and accelerated oocyte depletion. Moreover, the variant p.R579W localizing in the high-mobility group (HMG) box domain disrupted the nuclear localization of the MEIOSIN protein but was dispensable for the cell-cycle switch of oocytes, suggesting a unique role of the MEIOSIN HMG box domain in meiosis initiation. Further studies are needed to explore the role of other meiosis initiation genes in the pathogenesis of POI. The MEIOSIN variant was verified to cause POI by impaired transcriptional regulation of meiotic genes and was inherited by a recessive mode. The function of HMG box domain in MEIOSIN protein was also expanded by this study. Although causative variations in meiotic initiation genes are rare in POI, our study confirmed the pathogenicity of a MEIOSIN variant and elucidated another mechanism of human infertility. This work was supported by the National Key Research & Developmental Program of China (2022YFC2703800, 2022YFC2703000), National Natural Science Foundation for Distinguished Young Scholars (82125014), National Natural Science Foundation of China (32070847, 32170867, 82071609), Basic Science Center Program of NSFC (31988101), Natural Science Foundation of Shandong Province for Grand Basic Projects (ZR2021ZD33), Natural Science Foundation of Shandong Province for Excellent Young Scholars (ZR2022YQ69), Taishan Scholars Program for Young Experts of Shandong Province (tsqn202211371), and Qilu Young Scholars Program of Shandong University. The authors declare no conflict of interest. N/A.

Multiple Pregnancy Can Induce Preeclampsia-Like Changes in Complement Factor H Point Mutation Mice

Multiple Pregnancy Can Induce Preeclampsia-Like Changes in Complement Factor H Point Mutation Mice

Development of a tau-V337M mouse model using CRISPR/Cas9 system and enhanced ssODN-mediated recombination

The generation of a tau-V337M point mutation mouse model using gene editing technology can provide an animal model with fast disease progression and more severe symptoms, which facilitate the study of pathogenesis and treatment of Alzheimer's disease (AD). In this study, single guide RNAs (sgRNA) and single-stranded oligonucleotides (ssODN) were designed and synthesized in vitro. The mixture of sgRNA, Cas9 protein and ssODN was microinjected into the zygotes of C57BL/6J mice. After DNA cutting and recombination, the site homologous to human 337 valine (GTG) in exon 11 was mutated into methionine (ATG). In order to improve the efficiency of recombination, a Rad51 protein was added. The female mice mated with the nonvasectomy male mice were used as the surrogates. Subsequently, the 2-cell stage gene edited embryos were transferred into the unilateral oviduct, and the F0 tau-V337M mutation mice were obtained. Higher mutation efficiency could be obtained by adding Rad51 protein. The F0 tau-V337M point mutation mice can pass the mutation on to the F1 generation mice. In conclusion, this study successfully established the first tau-V337M mutation mouse by using Cas9, ssODN and Rad51. These results provide a new method for developing AD mice model which can be used in further research on the pathogenesis and treatment of AD.

Importance of glutamine in synaptic vesicles revealed by functional studies of SLC6A17 and its mutations pathogenic for intellectual disability

Human mutations in the gene encoding the solute carrier (SLC) 6A17 caused intellectual disability (ID). The physiological role of SLC6A17 and pathogenesis of SLC6A17-based-ID were both unclear. Here, we report learning deficits in Slc6a17 knockout and point mutant mice. Biochemistry, proteomic, and electron microscopy (EM) support SLC6A17 protein localization in synaptic vesicles (SVs). Chemical analysis of SVs by liquid chromatography coupled to mass spectrometry (LC-MS) revealed glutamine (Gln) in SVs containing SLC6A17. Virally mediated overexpression of SLC6A17 increased Gln in SVs. Either genetic or virally mediated targeting of Slc6a17 reduced Gln in SVs. One ID mutation caused SLC6A17 mislocalization while the other caused defective Gln transport. Multidisciplinary approaches with seven types of genetically modified mice have shown Gln as an endogenous substrate of SLC6A17, uncovered Gln as a new molecule in SVs, established the necessary and sufficient roles of SLC6A17 in Gln transport into SVs, and suggested SV Gln decrease as the key pathogenetic mechanism in human ID.

Importance of glutamine in synaptic vesicles revealed by functional studies of SLC6A17 and its mutations pathogenic for intellectual disability.

Human mutations in the gene encoding the solute carrier (SLC) 6A17 caused intellectual disability (ID). The physiological role of SLC6A17 and pathogenesis of SLC6A17-based-ID were both unclear. Here, we report learning deficits in Slc6a17 knockout and point mutant mice. Biochemistry, proteomic, and electron microscopy (EM) support SLC6A17 protein localization in synaptic vesicles (SVs). Chemical analysis of SVs by liquid chromatography coupled to mass spectrometry (LC-MS) revealed glutamine (Gln) in SVs containing SLC6A17. Virally mediated overexpression of SLC6A17 increased Gln in SVs. Either genetic or virally mediated targeting of Slc6a17 reduced Gln in SVs. One ID mutation caused SLC6A17 mislocalization while the other caused defective Gln transport. Multidisciplinary approaches with seven types of genetically modified mice have shown Gln as an endogenous substrate of SLC6A17, uncovered Gln as a new molecule in SVs, established the necessary and sufficient roles of SLC6A17 in Gln transport into SVs, and suggested SV Gln decrease as the key pathogenetic mechanism in human ID.

P-705 Homozygous missense variant in MEIOSIN causes premature ovarian insufficiency

Abstract Study question Are the gene variants involved in meiosis initiation responsible for premature ovarian insufficiency (POI)? Summary answer MEIOSIN variant participates in the pathogenesis of human POI by impairing meiosis due to insufficient transcriptional activation of essential meiotic genes. What is known already Meiosis is the key event for the establishment of the ovarian reserve, and several gene defects impairing meiotic homologous recombination have been found to contribute to the pathogenesis of POI. However, the genes involved in meiosis initiation have not been associated with POI. Study design, size, duration Retrospective genetic study. An in-house whole exome sequencing (WES) database of 1,030 idiopathic POI patients was screened for variations of meiosis initiation genes. Participants/materials, setting, methods Gene variations involved in meiosis initiation were screened in the in-house WES database of 1,030 POI cases. The pathogenicity of the variation was verified by in vitro experiments, including protein structure prediction and dual-luciferase reporter assay. The effect of the variant on ovarian function and meiosis was demonstrated through histological analyses in a point mutation mouse model. Main results and the role of chance A homozygous variant of MEIOSIN that initiates meiosis via the retinoic acid dependent pathway was identified in a patient with idiopathic POI. The dual-luciferase reporter assay revealed that the variant adversely affected the transcriptional function of MEIOSIN in upregulating meiotic genes. Further knock-in mice with the homologous mutation confirmed that the variation impacted the meiotic prophase I program and accelerated oocyte depletion. Limitations, reasons for caution Further studies are needed to explore the role of other meiosis initiation genes in the pathogenesis of POI. Wider implications of the findings The present study firstly identified pathogenic MEIOSIN variants in patients with POI. Although the causative variation in meiotic initiation genes is rare in POI, our study expanded the spectrum of causative genes and elucidated the different mechanisms in human infertility. Trial registration number Not available

An SNX31 variant underlies dominant familial exudative vitreoretinopathy-like pathogenesis.

Familial exudative vitreoretinopathy (FEVR) is a complex hereditary eye disorder characterized by incomplete development of the retinal vasculature, which thereby affects retinal angiogenesis. But the genetic factors contributing to FEVR's development or pathogenesis remain elusive. In a Chinese family with FEVR with 19 members, by using whole-exome sequencing, we identified a candidate disease-causing DNA variant in sorting nexin 31 (SNX31) (c.963delG; p. Trp321Cys), which results in a frameshift mutation. We studied the biochemical mechanism of this mutation and determined that it is deficient in β1-integrin binding and stability. The SNX31 c.963delG point mutation mouse model (SNX31m/m) was constructed with CRISPR/Cas9 technology. At 2-4 months of age, SNX31m/m mice showed fundus phenotypes similar to FEVR-like changes, including vascular leakage and retinal atrophy. Moreover, we found that VEGF and apoptotic pathways were involved in these ocular phenotypes. Hence, our study extended the FEVR mutation spectrum to include SNX31. These findings expanded our understanding of the molecular pathogenesis of FEVR and may facilitate the development of methods for the diagnosis and prevention of FEVR.

Whole exome sequencing in unexplained recurrent miscarriage families identified novel pathogenic genetic causes of euploid miscarriage.

Can whole exome sequencing (WES) followed by trio bioinformatics analysis identify novel pathogenic genetic causes of first trimester euploid miscarriage? We identified genetic variants in six candidate genes that indicated plausible underlying causes of first-trimester euploid miscarriage. Previous studies have identified several monogenic causes of Mendelian inheritance in euploid miscarriages. However, most of these studies are without trio analyses and lack cellular and animal models to validate the functional effect of putative pathogenic variants. Eight unexplained recurrent miscarriage (URM) couples and corresponding euploid miscarriages were included in our study for whole genome sequencing (WGS) and WES followed by trio bioinformatics analysis. Knock-in mice with Rry2 and Plxnb2 variants and immortalized human trophoblasts were utilized for functional study. Additional 113 unexplained miscarriages were included to identify the mutation prevalence of specific genes by multiplex PCR. Whole blood from URM couples and their <13 weeks gestation miscarriage products were both collected for WES, and all variants in selected genes were verified by Sanger sequencing. Different stage C57BL/6J wild-type mouse embryos were collected for immunofluorescence. Ryr2N1552S/+, Ryr2R137W/+, Plxnb2D1577E/+, and Plxnb2R465Q/+ point mutation mice were generated and backcrossed. Matrigel-coated transwell invasion assays and wound-healing assays were performed using HTR-8/SVneo cells transfected with PLXNB2 small-interfering RNA and negative control. Multiplex PCR was performed focusing on RYR2 and PLXNB2. Six novel candidate genes, including ATP2A2, NAP1L1, RYR2, NRK, PLXNB2, and SSPO, were identified. Immunofluorescence staining showed that ATP2A2, NAP1L1, RyR2, and PLXNB2 were widely expressed from the zygote to the blastocyst stage in mouse embryos. Although compound heterozygous mice with Rry2 and Plxnb2 variants did not show embryonic lethality, the number of pups per litter was significantly reduced when backcrossing Ryr2N1552S/+ ♂ with Ryr2R137W/+ ♀ or Plxnb2D1577E/+ ♂ with Plxnb2R465Q/+ ♀ (P < 0.05), which were in accordance with the sequencing results of Family 2 and Family 3, and the proportion of Ryr2N1552S/+ offspring was significantly lower when Ryr2N1552S/+ female mice were backcrossed with Ryr2R137W/+ male mice (P < 0.05). Moreover, siRNA-mediated PLXNB2 knockdown inhibited the migratory and invasive abilities of immortalized human trophoblasts. Besides, additional 10 variants of RYR2 and PLXNB2 were detected in 113 unexplained euploid miscarriages by multiplex PCR. The relatively small number of samples is a limitation of our study which may result in the identification of variants in unique candidate genes with no definitive although plausible causal effect. Larger cohorts are needed to replicate these findings and additional functional research is needed to confirm the pathogenic effects of these variants. Moreover, the sequencing coverage restricted the detection of low-level parental mosaic variants. For first-trimester euploid miscarriage, variants in unique genes may be underlying genetic etiologies and WES on trio could be an ideal model to identify potential genetic causes, which could facilitate individualized precise diagnostic and therapeutic regimens in the future. This study was supported by grants from the National Key Research and Development Program of China (2021YFC2700604), National Natural Science Foundation of China (31900492, 82101784, 82171648), Basic Science Center Program of the National Natural Science Foundation of China (31988101), Key Research and Development Program of Shandong Province (2021LCZX02), Natural Science Foundation of Shandong Province (ZR2020QH051), Natural Science Foundation of Jiangsu Province (BK20200223), Taishan Scholars Program for Young Experts of Shandong Province (tsqn201812154) and Young Scholars Program of Shandong University. The authors declare no conflicts of interest. N/A.

The role of proteoglycan form of DMP1 in cranial repair

BackgroundThe cranial region is a complex set of blood vessels, cartilage, nerves and soft tissues. The reconstruction of cranial defects caused by trauma, congenital defects and surgical procedures presents clinical challenges. Our previous data showed that deficiency of the proteoglycan (PG) form of dentin matrix protein 1 (DMP1-PG) could lead to abnormal cranial development. In addition, DMP1-PG was highly expressed in the cranial defect areas. The present study aimed to investigate the potential role of DMP1-PG in intramembranous ossification in cranial defect repair.MethodsMouse cranial defect models were established by using wild- type (WT) and DMP1-PG point mutation mice. Microcomputed tomography (micro-CT) and histological staining were performed to assess the extent of repair. Immunofluorescence assays and real-time quantitative polymerase chain reaction (RT‒qPCR) were applied to detect the differentially expressed osteogenic markers. RNA sequencing was performed to probe the molecular mechanism of DMP1-PG in regulating defect healing.ResultsA delayed healing process and an abnormal osteogenic capacity of primary osteoblasts were observed in DMP1-PG point mutation mice. Furthermore, impaired inflammatory signaling pathways were detected by using RNA transcription analysis of this model.ConclusionsOur data indicate that DMP1-PG is an indispensable positive regulator during cranial defect healing.

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice.

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (>1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (>1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment.

Sphingosine-1-phosphate lyase is encoded by the Sgpl1 gene, degrades S1P, and is crucial for S1P homeostasis in animal models and humans. S1P lyase deficient patients suffer from adrenal insufficiency, severe lymphopenia, and skin disorders. In this study, we used random mutagenesis screening to identify a mouse line carrying a missense mutation of Sgpl1 (M467K). This mutation caused similar pathologies as Sgpl1 knock-out mice in multiple organs, but greatly preserved its lifespan, which M467K mutation mice look normal under SPF conditions for over 40 weeks, in contrast, the knock-out mice live no more than 6 weeks. When treated with Imiquimod, Sgpl1M467K mice experienced exacerbated skin inflammation, as revealed by aggravated acanthosis and orthokeratotic hyperkeratosis. We also demonstrated that the IL17a producing Vγ6+ cell was enriched in Sgpl1M467K skin and caused severe pathology after imiquimod treatment. Interestingly, hyperchromic plaque occurred in the mutant mice one month after Imiquimod treatment but not in the controls, which resembled the skin disorder found in Sgpl1 deficient patients. Therefore, our results demonstrate that Sgpl1M467K point mutation mice successfully modeled a human disease after being treated with Imiquimod. We also revealed a major subset of γδT cells in the skin, IL17 secreting Vγ6 T cells were augmented by Sgpl1 deficiency and led to skin pathology. Therefore, we have, for the first time, linked the IL17a and γδT cells to SPL insufficiency.