Cynomolgus macaque model of neuronal ceroid lipofuscinosis type 2 disease.

Neuronal ceroid lipofuscinoses (NCLs) are autosomal-recessive fatal neurodegenerative diseases that occur in children and young adults, with symptoms including ataxia, seizures and visual impairment. We report the discovery of cynomolgus macaques carrying the CLN2/TPP1 variant and our analysis of whether the macaques could be a new non-human primate model for NCL type 2 (CLN2) disease. Three cynomolgus macaques presented progressive neuronal clinical symptoms such as limb tremors and gait disturbance after about 2 years of age. Morphological analyses using brain MRI at the endpoint of approximately 3 years of age revealed marked cerebellar and cerebral atrophy of the gray matter, with sulcus dilation, gyrus thinning, and ventricular enlargement. Histopathological analyses of three affected macaques revealed severe neuronal loss and degeneration in the cerebellar and cerebral cortices, accompanied by glial activation and/or changes in axonal morphology. Neurons observed throughout the central nervous system contained autofluorescent cytoplasmic pigments, which were identified as ceroid-lipofuscin based on staining properties, and the cerebral cortex examined by transmission electron microscopy had curvilinear profiles, the typical ultrastructural pattern of CLN2. These findings are commonly observed in all forms of NCL. DNA sequencing analysis identified a homozygous single-base deletion (c.42delC) of the CLN2/TPP1 gene, resulting in a frameshifted premature stop codon. Immunohistochemical analysis showed that tissue from the affected macaques lacked a detectable signal against TPP1, the product of the CLN2/TPP1 gene. Analysis for transmission of the CLN2/TPP1 mutated gene revealed that 47 (49.5%) and 48 (50.5%) of the 95 individuals genotyped in the CLN2-affected macaque family were heterozygous carriers and homozygous wild-type individuals, respectively. Thus, we identified cynomolgus macaques as a non-human primate model of CLN2 disease. The CLN2 macaques reported here could become a useful resource for research and the development of drugs and methods for treating CLN2 disease, which involves severe symptoms in humans.

EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice.

The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway.

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.

Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.

Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.

Generation of B6-Ddx4em1(CreERT2)Utr , a novel CreERT2 knock-in line, for germ cell lineage by CRISPR/Cas9.

Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock-out mouse studies. In this study, we established a novel knock-in mouse line, B6-Ddx4 em1(CreERT2)Utr , which expresses CreERT2 recombinase under the control of the endogenous DEAD-box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen-treated B6-Ddx4 em1(CreERT2)Utr ::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6-Ddx4 em1(CreERT2)Utr is beneficial for studying female and male germ cell development.

Simple generation of hairless mice for in vivo imaging.

The in vivo imaging of mice makes it possible to analyze disease progress non-invasively through reporter gene expression. As the removal of hair improves the accuracy of in vivo imaging, gene-modified mice with a reporter gene are often crossed with Hos:HR-1 mutant mice homozygous for the spontaneous Hrhr mutation that exhibit a hair loss phenotype. However, it is time consuming to produce mice carrying both the reporter gene and mutant Hrhr gene by mating. In addition, there is a risk that genetic background of the gene-modified mice would be altered by mating. To resolve these issues, we established a simple method to generate hairless mice maintaining the original genetic background by CRISPR technology. First, we constructed the pX330 vector, which targets exon 3 of Hr. This DNA vector (5 ng/µl) was microinjected into the pronuclei of C57BL/6J mice. Induced Hr gene mutations were found in many founders (76.1%) and these mutations were heritable. Next, we performed in vivo imaging using these gene-modified hairless mice. As expected, luminescent objects in their body were detected by in vivo imaging. This study clearly showed that hairless mice could be simply generated by the CRISPR/Cas9 system, and this method may be useful for in vivo imaging studies with various gene-modified mice.

Angiodysplasia in embryo lacking protein arginine methyltransferase 1 in vascular endothelial cells.

Protein arginine methyltransferase 1 (PRMT1) is involved in multiple cellular functions including proliferation and differentiation. Although PRMT1 is expressed in vascular endothelial cells (ECs), which are responsible for angiogenesis during embryonic development, its role has remained elusive. In this study, we generated endothelial-specific prmt1-knockout (Prmt1-ECKO) mice, and found that they died before embryonic day 15. The superficial temporal arteries in these embryos were poorly perfused with blood, and whole-mount 3D imaging revealed dilated and segmentalized luminal structures in Prmt1-ECKO fetuses in comparison with those of controls. Our findings provide evidence that PRMT1 is important for embryonic vascular formation.

H1foo Has a Pivotal Role in Qualifying Induced Pluripotent Stem Cells.

Embryonic stem cells (ESCs) are a hallmark of ideal pluripotent stem cells. Epigenetic reprogramming of induced pluripotent stem cells (iPSCs) has not been fully accomplished. iPSC generation is similar to somatic cell nuclear transfer (SCNT) in oocytes, and this procedure can be used to generate ESCs (SCNT-ESCs), which suggests the contribution of oocyte-specific constituents. Here, we show that the mammalian oocyte-specific linker histone H1foo has beneficial effects on iPSC generation. Induction of H1foo with Oct4, Sox2, and Klf4 significantly enhanced the efficiency of iPSC generation. H1foo promoted in vitro differentiation characteristics with low heterogeneity in iPSCs. H1foo enhanced the generation of germline-competent chimeric mice from iPSCs in a manner similar to that for ESCs. These findings indicate that H1foo contributes to the generation of higher-quality iPSCs.

Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice.

In the present study, we generated novel cre driver mice for gene manipulation in pancreatic ß cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1(em1 (cre) Utr) strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1(em1 (cre) Utr)) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting ß cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1(em1 (cre) Utr) is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.

Ground-based assessment of JAXA mouse habitat cage unit by mouse phenotypic studies.

The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module ("Kibo") on the International Space Station. The CBEF provides "space-based controls" by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments.

Combined Overexpression of JARID2, PRDM14, ESRRB, and SALL4A Dramatically Improves Efficiency and Kinetics of Reprogramming to Induced Pluripotent Stem Cells.

Identification of a gene set capable of driving rapid and proper reprogramming to induced pluripotent stem cells (iPSCs) is an important issue. Here we show that the efficiency and kinetics of iPSC reprogramming are dramatically improved by the combined expression of Jarid2 and genes encoding its associated proteins. We demonstrate that forced expression of JARID2 promotes iPSC reprogramming by suppressing the expression of Arf, a known reprogramming barrier, and that the N-terminal half of JARID2 is sufficient for such promotion. Moreover, JARID2 accelerated silencing of the retroviral Klf4 transgene and demethylation of the Nanog promoter, underpinning the potentiating activity of JARID2 in iPSC reprogramming. We further show that JARID2 physically interacts with ESRRB, SALL4A, and PRDM14, and that these JARID2-associated proteins synergistically and robustly facilitate iPSC reprogramming in a JARID2-dependent manner. Our findings provide an insight into the important roles of JARID2 during reprogramming and suggest that the JARID2-associated protein network contributes to overcoming reprogramming barriers.

Peri-implantation lethality in mice carrying megabase-scale deletion on 5qc3.3 is caused by Exoc1 null mutation.

We found a novel spontaneous mouse mutant with depigmentation in the ventral body, which we called White Spotting (WS) mouse. Genetic investigation revealed deletion of a > 1.2-Mb genomic region containing nine genes (Kit, Kdr, Srd5a3, Tmeme165, Clock, Pdcl2, Nmu, Exoc1, and Cep135). We designated this mutant allele Kit(WS). Interestingly, homozygous mutants (Kit(WS/WS)) showed a peri-implantation lethal phenotype. Expression analyses of these nine genes in blastocysts suggested that Exoc1 was a prime candidate for this phenotype. We produced Exoc1 knockout mice, and the same peri-implantation lethal phenotype was seen in Exoc1(-/-) embryos. In addition, the polygenic effect without Exoc1 was investigated in genome-edited Kit(WE) mice carrying the Mb-scale deletion induced by the CRISPR/Cas9 system. As Kit(WE/WE) embryos did not exhibit the abnormal phenotype, which was seen in Kit(WS/WS). We concluded that peri-implantation lethality in Kit(WS/WS) was caused by a monogenic defect of Exoc1.

Erythropoiesis and Blood Pressure Are Regulated via AT1 Receptor by Distinctive Pathways.

The renin-angiotensin system (RAS) plays a central role in blood pressure regulation. Although clinical and experimental studies have suggested that inhibition of RAS is associated with progression of anemia, little evidence is available to support this claim. Here we report that knockout mice that lack angiotensin II, including angiotensinogen and renin knockout mice, exhibit anemia. The anemia of angiotensinogen knockout mice was rescued by angiotensin II infusion, and rescue was completely blocked by simultaneous administration of AT1 receptor blocker. To genetically determine the responsible receptor subtype, we examined AT1a, AT1b, and AT2 knockout mice, but did not observe anemia in any of them. To investigate whether pharmacological AT1 receptor inhibition recapitulates the anemic phenotype, we administered AT1 receptor antagonist in hypotensive AT1a receptor knockout mice to inhibit the remaining AT1b receptor. In these animals, hematocrit levels barely decreased, but blood pressure further decreased to the level observed in angiotensinogen knockout mice. We then generated AT1a and AT1b double-knockout mice to completely ablate the AT1 receptors; the mice finally exhibited the anemic phenotype. These results provide clear evidence that although erythropoiesis and blood pressure are negatively controlled through the AT1 receptor inhibition in vivo, the pathways involved are complex and distinct, because erythropoiesis is more resistant to AT1 receptor inhibition than blood pressure control.

Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system.

Single nucleotide mutations (SNMs) are associated with a variety of human diseases. The CRISPR/Cas9 genome-editing system is expected to be useful as a genetic modification method for production of SNM-induced mice. To investigate whether SNM-induced mice can be generated by zygote microinjection of CRISPR/Cas9 vector and single-stranded DNA (ssDNA) donor, we attempted to produce albino C57BL/6J mice carrying the Tyr gene SNM (G291T) from pigmented C57BL/6J zygotes. We first designed and constructed a CRISPR/Cas9 expression vector for the Tyr gene (px330-Tyr-M). DNA cleavage activity of px330-Tyr-M at the target site of the Tyr gene was confirmed by the EGxxFP system. We also designed an ssDNA donor for homology-directed repair (HDR)-mediated gene modification. The px330-Tyr-M vector and ssDNA donor were co-microinjected into the pronuclei of 224 one-cell-stage embryos derived from C57BL/6J mice. We obtained 60 neonates, 28 of which showed the ocular albinism and absence of coat pigmentation. Genomic sequencing analysis of the albino mice revealed that the target of SNM, G291T in the Tyr gene, occurred in 11 mice and one founder was homozygously mutated. The remaining albino founders without Tyr G291T mutation also possessed biallelic deletion and insertion mutants adjacent to the target site in the Tyr locus. Simple production of albino C57BL/6J mice was provided by C57BL/6J zygote microinjection with px330-Tyr-M DNA vector and mutant ssDNA (G291T in Tyr) donor. A combination of CRISPR/Cas9 vector and optional mutant ssDNA could be expected to efficiently produce novel SNM-induced mouse models for investigating human diseases.

Generation and characterization of Ins1-cre-driver C57BL/6N for exclusive pancreatic beta cell-specific Cre-loxP recombination.

Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic ß cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F1 mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F1 mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F1 fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F1 mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic ß cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15.

Role of retinoic acid and fibroblast growth factor 2 in neural differentiation from cynomolgus monkey (Macaca fascicularis) embryonic stem cells.

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.

Truncated Cables1 causes agenesis of the corpus callosum in mice.

Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. More than 60 genes are known to be involved in corpus callosum development. However, the molecular mechanisms underlying ACC are not fully understood. Previously, we produced a novel transgenic mouse strain, TAS, carrying genes of the tetracycline-inducible expression system that are not involved in brain development, and inherited ACC was observed in the brains of all homozygous TAS mice. Although ACC was probably induced by transgene insertion mutation, the causative gene and the molecular mechanism of its pathogenesis remain unclear. Here, we first performed interphase three-color fluorescence in situ hybridization (FISH) analysis to determine the genomic insertion site. Transgenes were inserted into chromosome 18 ∼12.0 Mb from the centromere. Gene expression analysis and genomic PCR walking showed that the genomic region containing exon 4 of Cables1 was deleted by transgene insertion and the other exons of Cables1 were intact. The mutant allele was designated as Cables1(TAS). Interestingly, Cables1(TAS) mRNA consisted of exons 1-3 of Cables1 and part of the transgene that encoded a novel truncated Cables1 protein. Homozygous TAS mice exhibited mRNA expression of Cables1(TAS) in the fetal cerebrum, but not that of wild-type Cables1. To investigate whether a dominant negative effect of Cables1(TAS) or complete loss of function of Cables1 gives rise to ACC, we produced Cables1-null mutant mice. ACC was not observed in Cables1-null mutant mice, suggesting that a dominant negative effect of Cables1(TAS) impairs callosal formation. Moreover, ACC frequency in Cables1(+/TAS) mice was significantly lower than that in Cables1(-/TAS) mice, indicating that wild-type Cables1 interfered with the dominant negative effect of Cables1(TAS). This study indicated that truncated Cables1 causes ACC and wild-type Cables1 contributes to callosal formation.

Novel ROSA26 Cre-reporter knock-in C57BL/6N mice exhibiting green emission before and red emission after Cre-mediated recombination.

The Cre/loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. As successful Cre/loxP genome alteration depends on Cre-driver mice, Cre-reporter mice are essential for validation of Cre gene expression in vivo. In most Cre-reporter mouse strains, although the presence of reporter product indicates the expression of Cre recombinase, it has remained unclear whether a lack of reporter signal indicates either no Cre recombinase expression or insufficient reporter gene promoter activity. We produced a novel ROSA26 knock-in Cre-reporter C57BL/6N strain exhibiting green emission before and red after Cre-mediated recombination, designated as strain R26GRR. Ubiquitous green fluorescence and no red fluorescence were observed in R26GRR mice. To investigate the activation of tdsRed, EGFP-excised R26GRR, R26RR, mice were produced through the crossing of C57BL/6N mice with R26GRR/Ayu1-Cre F1 mice. R26RR mice showed extraordinarily strong red fluorescence in almost all tissues examined, suggesting ubiquitous activation of the second reporter in all tissues after Cre/loxP recombination. Moreover, endothelial cell lineage and pancreatic islet-specific expression of red fluorescence were detected in R26GRR/Tie2-Cre F1 mice and R26GRR /Ins1-Cre F1 mice, respectively. These results indicated that R26GRR mice are a useful novel Cre-reporter mouse strain. In addition, R26GRR mice with a pure C57BL/6N background represent a valuable source of green-to-red photoconvertible cells following Cre/loxP recombination for application in transplantation studies. The R26GRR mouse strain will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/animal/en/).

Effect of lactation on postpartum cardiac function of pregnancy-associated hypertensive mice.

Preeclampsia is a serious complication during pregnancy, and recent epidemiological studies indicate the association between preeclampsia and cardiac morbidity and mortality during the postpartum period. Although the risk of cardiovascular diseases in the postpartum period is affected by lactation, its role in maternal heart with a history of preeclampsia remains unclear. In this study, we investigated postpartum change in cardiac remodeling and function of pregnancy-associated hypertensive (PAH) mice with and without lactation. The systolic blood pressure was increased in PAH mice at day 19 of gestation (E19) and was reduced to normal levels in both lactating and nonlactating (NL) groups in the postpartum period. Histological analyses revealed that cardiac hypertrophy and macrophage infiltration in PAH mice at E19 were improved in both lactating and NL groups at 4 weeks postpartum (4W-PP), while marked fibrosis remained. Increased mRNA expression of profibrotic genes and proinflammatory cytokines in PAH mice at E19 was significantly reduced in both lactating and NL groups at 4W-PP. Echocardiographic analysis found no significant differences in fractional shortening between PAH mice and C57BL/6J mice at E19. On the other hand, at 4W-PP, NL PAH mice showed normal fractional shortening, but lactating PAH mice exhibited significant decreases in cardiac contractility compared with NL PAH mice. These results show that cardiac remodeling induced by hypertension during pregnancy are improved in the postpartum period except fibrosis, whereas lactation induces cardiac contractile dysfunction in mice with a history of pregnancy-associated hypertension.

Nrf2 in bone marrow-derived cells positively contributes to the advanced stage of atherosclerotic plaque formation.

Atherosclerosis is the major etiology underlying myocardial infarction and stroke, and strategies for preventing atherosclerosis are urgently needed. In the context of atherosclerosis, the deletion of the Nrf2 gene, which encodes a master regulator of the oxidative stress response in mammals, reportedly attenuates atherosclerosis formation. However, the precise mechanisms of protection against atherosclerosis are largely unknown. To further clarify the role of Nrf2 in atherosclerosis in vivo, we performed a time course analysis of atherosclerosis development utilizing an ApoE knockout (KO) mouse model. The results demonstrate that oil red O-stainable lesions were similar in size 5 weeks after the initiation of an HFC (high fat and high cholesterol) diet, but the lesions were markedly attenuated in the Nrf2 and ApoE double KO mice (A0N0 mice) compared with the lesions in the ApoE KO mice (A0N2 mice) at 12 weeks. Consistent with these results, the immunohistochemical analysis revealed that Nrf2 activation is observed in late-stage atherosclerotic plaques but not in earlier lesions. The RT-qPCR analysis of 12-week atherosclerotic plaques revealed that Nrf2 target genes, such as Ho-1 and SLPI, are expressed at significantly lower levels in the A0N0 mice compared with the A0N2 mice, and this change was associated with a decreased expression of macrophage M1-subtype genes Arginase II and inducible NO synthase in the A0N0 mice. Furthermore, the bone marrow (BM) transplantation (BMT) analysis revealed that the Nrf2 activity in the BM-derived cells contributed to lesion formation. Therefore, our study has characterized the positive role of Nrf2 in the BM-derived cells during the development of atherosclerosis, which suggests that Nrf2 may influence the inflammatory reactions in the plaques.