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In JoVE (1)
Other Publications (30)
- Biochemical and Biophysical Research Communications
- Experimental and Molecular Pathology
- Mammalian Genome : Official Journal of the International Mammalian Genome Society
- Japanese Journal of Cancer Research : Gann
- Genetics
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Nature
- Genome Research
- Human Cell : Official Journal of Human Cell Research Society
- Blood
- Journal of Molecular Medicine (Berlin, Germany)
- Molecular and Cellular Biology
- Human Molecular Genetics
- Human Mutation
- ILAR Journal / National Research Council, Institute of Laboratory Animal Resources
- Comparative Medicine
- Genomics
- The Journal of Cell Biology
- Mammalian Genome : Official Journal of the International Mammalian Genome Society
- Genesis (New York, N.Y. : 2000)
- PloS One
- Experimental Animals / Japanese Association for Laboratory Animal Science
- Experimental Animals / Japanese Association for Laboratory Animal Science
- Experimental Animals / Japanese Association for Laboratory Animal Science
- The Journal of Reproduction and Development
- Experimental Animals / Japanese Association for Laboratory Animal Science
- Nucleic Acids Research
- Proceedings of the National Academy of Sciences of the United States of America
- Nucleic Acids Research
- Journal of Biomedical Science
Articles by Atsushi Yoshiki in JoVE
JoVE General
Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification
An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.
Other articles by Atsushi Yoshiki on PubMed
PDGF Receptor-alpha Deficiency in Glomerular Mesangial Cells of Tenascin-C Knockout Mice
Tenascin-C (TNC) knockout (TNKO) mice showed reduced proliferation of mesangial cells and abnormal restoration after habu-snake venom (HSV)-induced glomerulonephritis. In this study, we examined the relationship of TNC and platelet-derived growth factor receptor (PDGFR) in glomerular mesangial cells. TNC and PDGFR-alpha and -beta transcriptions were up-regulated in wild type (WT) mice after HSV injection, but in TNKO mice PDGFR-alpha transcription was not up-regulated. Immunohistochemistry showed that PDGFR-alpha was found in mesangial areas of colocalized alpha-smooth muscle actin, but in TNKO mice it was not detectable. In vitro studies showed that the expressions of PDGFR-alpha and -beta mRNA and protein in cultured glomerular mesangial cells (GMC) of TNKO mice were lower than those in WT GMC. These results suggest that failures of both TNC and PDGFR-alpha are a candidate for abnormal restoration of TNKO mice.
Tenascin-C Expression and Splice Variant in Habu Snake Venom-induced Glomerulonephritis
Extracellular matrix glycoprotein tenascin-C (TNC) expression is up-regulated in tissue remodeling processes such as tumorigenesis and wound healing. Mouse tenascin-C contains six alternatively spliced domains (A1, A2, A4, B, C, and D) between the fifth and the sixth type III fibronectin domains, which generate large numbers of TNC isoforms. To study TNC isoform variability of wound healing in mice, we induced glomerulonephritis by using Habu snake venom (HSV) and examined alternatively spliced regions by the reverse transcription polymerase chain reaction (RT-PCR) technique. RT-PCR products were separated into seven bands in both healthy and diseased kidneys. Among the seven bands, those containing one or five alternatively spliced domains were mainly up-regulated from 2 days to 1 week after HSV injection. Southern blotting revealed that only domain-D detected all six bands in both healthy and diseased kidneys. Furthermore, only the domain-C transcriptional level did not show an obvious change in progress following HSV injection. These results suggested that (a) the isoforms containing one or five alternatively spliced domains play important roles in the healing process of glomerulonephritis, (b) domain-D is particularly significant in kidney, and (c) domain-C may not play an important role in the healing process of HSV-induced glomerulonephritis.
Identification of Seven Loci for Static Glucokinesis and Dynamic Glucokinesis in Mice
Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by a breakdown of glucose homeostasis and is responsible for serious complications in various organs and vessels. Most of the genetic factors of NIDDM are yet unknown. Here, we identified two types of genetic factors that regulate homeostasis of blood glucose by measuring various pharmacokinetic parameters, some of which are used in the non-compartment analysis of drug metabolism in 340 F(2) progeny from the NIDDM model KK-A(y)/Ta Jcl mouse strain, and in non-diabetic PWK strain. We define "static glucokinesis" as the regulation of homeostasis that occurs during glucose deprivation, and "dynamic glucokinesis" as that during glucose stress; for instance, glucose tolerance test. Quantitative trait locus analysis revealed eight loci involved in the regulation of glucose homeostasis on chromosomes 7 ( Nidd1k), 2 ( Nidd2k), 1 ( Nidd3k, Nidd4k, and Nidd5k), 11 ( Nidd6k), 5 ( Nidd7k) (named Nidd1k through Nidd7k), and 4 ( Bwt1k). Nidd1k, Nidd4k, and Nidd7k were novel loci associated with NIDDM in mice. Nidd1k, Nidd2k, Nidd3k, and Nidd4k had linkage to factors characteristic of both static and dynamic glucokinesis. Nidd5k and Nidd6k showed linkage specific to markers of dynamic glucokinesis, and Nidd7k had linkage specific to static glucokinesis. Bwt1k was linked to obesity. Thus, the genetic factors for static glucokinesis and those for dynamic glucokinesis partially overlapped.
Invasion of Melanoma in Double Knockout Mice Lacking Tenascin-X and Tenascin-C
The roles of extracellular matrix glycoproteins belonging to the tenascin family in the regulation of tumor cell proliferation, invasion, and metastasis are not known. To address this issue, we generated tenascin-X (TNX) and tenascin-C (TNC) double knockout mice and compared findings in these mice with those in single knockout (TNX + / + TNC - / - and TNX - / - TNC + / +) mice. We investigated the proliferation and invasion of B16-BL6 melanoma cells after these cells had been injected into the footpads of mice in each group. The primary tumor size and invasion to the ankle adjacent to the primary tumor site were examined at 35 days after injection of the melanoma cells. The primary tumor size in TNX - / - TNC + / + mice was significantly larger than that in wild-type mice, but those of TNX + / + TNC - / - and double knockout mice were comparable to that in the wild-type mice. On the other hand, invasion to the ankle was obviously promoted in TNX - / - TNC + / + and double knockout mice compared with that in the wild-type mice, but invasion to the ankle in TNX + / + TNC - / - mice was only slightly promoted. Gelatin zymography confirmed increased matrix metalloproteinase (MMP)-9 activity in the dorsal skin of TNX - / - TNC + / +, TNX + / + TNC - / - and double knockout mice. However, the amounts of MMP-9 mRNA in the dorsal skins of all mice were almost the same, indicating that the increased activity of MMP-9 in the single and double knockout mice is regulated at the MMP-9 processing level. These results indicate that MMP-9 is activated in all TN-deficient mice, but that TNX exerts a greater effect on tumor invasion than does TNC.
Hague (Hag). A New Mouse Hair Mutation with an Unstable Semidominant Allele
A spontaneous mouse hair mutation was identified in a C3H/HeN colony. The mode of inheritance of the mutation was semidominant, with incomplete penetrance when heterozygous. The trait is controlled by a single locus hague (Hag), which was mapped to the telomeric region of chromosome 15. This mutation was shown to be unstable, since its transmission could be switched from semidominant to recessive. To identify the causative gene and the nature of the mutation, hague was introduced into a high-resolution and high-density molecular genetic map. Over 2000 meioses were analyzed and the mutation was mapped to the keratin 2 complex genes. A YAC and BAC physical map of the critical region was then constructed and the gene involved was located in a 600- to 800-kb-long segment. Fourteen genes were mapped to this region; of these, 11 were expressed in the skin (5 epidermic cytokeratin and 6 hard keratin genes), but none were mutated in hague mice.
Heat Shock Protein 70 Chaperone Overexpression Ameliorates Phenotypes of the Spinal and Bulbar Muscular Atrophy Transgenic Mouse Model by Reducing Nuclear-localized Mutant Androgen Receptor Protein
Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR). The nuclear inclusions consisting of the mutant AR protein are characteristic and combine with many components of ubiquitin-proteasome and molecular chaperone pathways, raising the possibility that misfolding and altered degradation of mutant AR may be involved in the pathogenesis. We have reported that the overexpression of heat shock protein (HSP) chaperones reduces mutant AR aggregation and cell death in a neuronal cell model (Kobayashi et al., 2000). To determine whether increasing the expression level of chaperone improves the phenotype in a mouse model, we cross-bred SBMA transgenic mice with mice overexpressing the inducible form of human HSP70. We demonstrated that high expression of HSP70 markedly ameliorated the motor function of the SBMA model mice. In double-transgenic mice, the nuclear-localized mutant AR protein, particularly that of the large complex form, was significantly reduced. Monomeric mutant AR was also reduced in amount by HSP70 overexpression, suggesting the enhanced degradation of mutant AR. These findings suggest that HSP70 overexpression ameliorates SBMA phenotypes in mice by reducing nuclear-localized mutant AR, probably caused by enhanced mutant AR degradation. Our study may provide the basis for the development of an HSP70-related therapy for SBMA and other polyQ diseases.
An Expressed Pseudogene Regulates the Messenger-RNA Stability of Its Homologous Coding Gene
A pseudogene is a gene copy that does not produce a functional, full-length protein. The human genome is estimated to contain up to 20,000 pseudogenes. Although much effort has been devoted to understanding the function of pseudogenes, their biological roles remain largely unknown. Here we report the role of an expressed pseudogene-regulation of messenger-RNA stability-in a transgene-insertion mouse mutant exhibiting polycystic kidneys and bone deformity. The transgene was integrated into the vicinity of the expressing pseudogene of Makorin1, called Makorin1-p1. This insertion reduced transcription of Makorin1-p1, resulting in destabilization of Makorin1 mRNA in trans by way of a cis-acting RNA decay element within the 5' region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. Either Makorin1 or Makorin1-p1 transgenes could rescue these phenotypes. Our findings demonstrate a specific regulatory role of an expressed pseudogene, and point to the functional significance of non-coding RNAs.
Targeting a Complex Transcriptome: the Construction of the Mouse Full-length CDNA Encyclopedia
We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.
Identification of a New Target Molecule for a Cascade Therapy of Polycystic Kidney
Autosomal dominant polycystic kidney disease is a systemic disorder that primary affects the kidney which is characterized by the formation of fluid-filled cysts in both kidneys that leads to progressive renal failure. Mutated genes, polycystin-1 and polycystin-2, are identified, and evidence has emerged that polycystins are ion channels or regulators of ion channels. In spite of extensive characterization of polycystins, how polycystin channel signaling may be involved in cyst formation in ADPKD is still unclear. We found a mutant mouse which exhibits polycystic kidney and bone deformity in the course of making a transgenic mouse carrying the Drosophila sex-lethal gene. We identified a mutated gene Makorin1 by positional cloning. Makorin1 carries a typical RING-finger motif, suggesting that Makorin1 belongs to ubiquitinase E3 family. Makorin1 would open a new avenue to understand pathogenesis of polycystic kidney, and become a new therapeutic target of polycystic kidney.
Retinoic Acid Controls Blood Vessel Formation by Modulating Endothelial and Mural Cell Interaction Via Suppression of Tie2 Signaling in Vascular Progenitor Cells
Inhibition by all-trans retinoic acid (atRA) of the microvasculature formation in chicken chorioallantoic membrane (CAM) accompanied remarkably reduced numbers of endothelial cells (ECs) and increased numbers of mural cells (MCs) under the chorionic epithelial layer. Ro41-5253 (retinoid antagonist) exerted the opposite effect. Although atRA did not affect the differentiation of murine embryonic stem cell-derived vascular progenitor cells (VPCs) into ECs or MCs, atRA suppressed EC-MC interaction, leading to impaired branching. In both atRA-treated VPC cultures and CAM tissues underneath the chorionic epithelial layer, the expression of angiopoietin-2 (Ang-2; competitor for Ang-1) was enhanced, whereas that of Tie2 (a receptor for Angs) was reduced. Simultaneous treatment with Ang-1 partially blocked RA induction of EC-MC malinteraction and reduction in blood vessel formation. These results suggest that retinoid(s) may reduce EC-MC interaction by down-regulating Tie2 signaling as well as decreased EC numbers, which lead to impaired vascular remodeling.
A New Role for Expressed Pseudogenes As NcRNA: Regulation of MRNA Stability of Its Homologous Coding Gene
We have earlier generated a mutant mouse in a course of making a transgenic line that exhibited interesting heterozygote phenotypes, which exhibited failure to thrive, severe bone deformities, and polycystic kidneys. This mutant mouse provided a clue to uncover a unique role of expressed pseudogenes. In this mutant the transgene was integrated into the vicinity of the expressing pseudogene of Makorin1 called Makorin1-p1. This insertion reduced transcription of the Makorin1-p1, resulting in destabilization of the Makorin1 mRNA in trans via a cis-acting RNA decay element within the 5' region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. These findings demonstrate a novel and specific regulatory role of an expressed pseudogene as well as functional significance for noncoding RNAs. Next, we developed an original algorithm to determine how many pseudogenes are expressed. Based on our examination 2-3% of human processed pseudogenes are expressed using the most strict criteria. Interestingly, the mouse has a much smaller proportion of expressed pseudogenes (0.5-1%). Pseudogenes are functionally less constrained, and have accumulated more mutations than translated genes. If they have some functions in gene regulation, this property would allow more rapid functional diversification than protein-coding genes. In addition, some genetic phenomena that exhibit incomplete penetrance might be attributed to "mutation" or "variation" of pseudogenes.
Complete Loss of Ndel1 Results in Neuronal Migration Defects and Early Embryonic Lethality
Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1(-/-) mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1(-/-) blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1(+/-) mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1(cko/-)) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1(cko/+)-Ndel1(+/-) mice or Lis1(+/-)-Ndel1(+/-) mice displayed more severe neuronal migration defects than Lis1(cko/+)-Ndel1(+/)(+) mice or Lis1(+/-)-Ndel1(+/+) mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of beta-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.
Recruitment of Katanin P60 by Phosphorylated NDEL1, an LIS1 Interacting Protein, is Essential for Mitotic Cell Division and Neuronal Migration
LIS1 is mutated in the human neuronal migration defect lissencephaly and along with NDEL1 (formerly NUDEL) participates in the regulation of cytoplasmic dynein function during neuronal development. Targeted disruption of Ndel1 suggested that NDEL1 could have other molecular targets that regulate microtubule organization for proper neuronal migration. To further understanding the molecular mechanism of LIS1 and lissencephaly, we identified the katanin p60 microtubule-severing protein as an additional molecular target of NDEL1. We demonstrate that phosphorylation of NDEL1 by Cdk5 facilitates interaction between NDEL1 and p60, suggesting that P-NDEL1 regulates the distribution of katanin p60. Abnormal accumulation of p60 in nucleus of Ndel1 null mutants supports an essential role of NDEL1 in p60 regulation. Complete loss of NDEL1 or expression of dominant negative mutants of p60 in migrating neurons results in defective migration and elongation of nuclear-centrosomal distance. Our results suggest that NDEL1 is essential for mitotic cell division and neuronal migration not only via regulation of cytoplasmic dynein function but also by modulation of katanin p60 localization and function.
Fates of Cdh23/CDH23 with Mutations Affecting the Cytoplasmic Region
BUS/Idr mice carrying a mutant waltzer allele (vbus) are characterized by splayed hair bundles in inner ear sensory cells, providing a mouse homolog of USH1D/DFNB12. RT-PCR-based screening for the presence of mutations in mouse Cdh23, the gene responsible for the waltzer phenotype, has identified a G>A mutation in the donor splice site of intron 67 (Cdh23:c.9633+1G>A: GenBank AF308939.1), indicating that two altered Cdh23 molecules having intron-derived COOH-terminal structures could be generated in BUS mouse tissues. Immunochemical analyses with anti-Cdh23 antibodies showed, however, no clear Cdh23-related proteins in vbus/vbus tissues, while the antibodies immunoreacted with approximately 350 kDa proteins in control mice. Immunofluorescent experiments revealed considerable weakening of Cdh23 signals in sensory hair cell stereocilia and Reissner's membrane in the vbus/vbus inner ear, and transmission electron microscopy demonstrated abundant autophagosome/autolysosome vesicles, suggesting aberrant Cdh23:c.9633+1G>A-derived protein-induced acceleration of lysosomal bulk degradation of proteins. In transfection experiments, signal sequence-preceded FLAG-tagged transmembrane plus cytoplasmic regions (TMCy) of tissue-specific Cdh23(+/-68) isoforms were localized to filamentous actin-rich protrusions and the plasma membrane of cultured cells, whereas FLAG-TMCy:c.9633+1G>A proteins were highly insoluble and retained in the cytoplasm. In contrast, FLAG-tagged TMCy:p.Arg3175His and human TMCy:c.9625_9626insC forms were both localized to the plasma membrane in cultured cells, allowing prediction that USH1D-associated CDH23:p.Arg3175His and CDH23:c.9625_9626insC proteins could be transported to the plasma membrane in vivo. The present results thus suggest different fates of CDH23/Cdh23 with mutations affecting the cytoplasmic region.
Mouse Phenome Research: Implications of Genetic Background
Now that sequencing of the mouse genome has been completed, the function of each gene remains to be elucidated through phenotypic analysis. The "genetic background" (in which each gene functions) is defined as the genotype of all other related genes that may interact with the gene of interest, and therefore potentially influences the specific phenotype. To understand the nature and importance of genetic background on phenotypic expression of specific genes, it is necessary to know the origin and evolutionary history of the laboratory mouse genome. Molecular analysis has indicated that the fancy mice of Japan and Europe contributed significantly to the origin of today's laboratory mice. The genetic background of present-day laboratory mice varies by mouse strain, but is mainly derived from the European domesticus subspecies group and to a lesser degree from Asian mice, probably Japanese fancy mice, which belong to the musculus subspecies group. Inbred laboratory mouse strains are genetically uniform due to extensive inbreeding, and they have greatly contributed to the genetic analysis of many Mendelian traits. Meanwhile, for a variety of practical reasons, many transgenic and targeted mutant mice have been created in mice of mixed genetic backgrounds to elucidate the function of the genes, although efforts have been made to create inbred transgenic mice and targeted mutant mice with coisogenic embryonic stem cell lines. Inbred mouse strains have provided uniform genetic background for accurate evaluation of specific genes phenotypes, thus eliminating the phenotypic variations caused by mixed genetic backgrounds. However, the process of inbreeding and selection of various inbred strain characteristics has resulted in inadvertent selection of other undesirable genetic characteristics and mutations that may influence the genotype and preclude effective phenotypic analysis. Because many of the common inbred mouse stains have been established from relatively small gene pools, common inbred strains have limitations in their genetic polymorphisms and phenotypic variations. Wild-derived mouse strains can complement deficiencies of common inbred mouse strains, providing novel allelic variants and phenotypes. Although wild-derived strains are not as tame as the common laboratory strains, their genetic characteristics are attractive for the future study of gene function.
Lymphocytic Choriomeningitis Infection Undetected by Dirty-bedding Sentinel Monitoring and Revealed After Embryo Transfer of an Inbred Strain Derived from Wild Mice
Persistent LCMV infection in wild-derived MAI/Pas mice housed under conventional conditions remained undetected for a decade, despite periodic health monitoring using dirty-bedding sentinels. When MAI/Pas mice were rederived by embryo transfer, recipient mothers produced antiLCMV antibodies, which first revealed the presence of the virus in the colony. Before this information was obtained, MAI/Pas mice had been shipped to another facility, undergone cesarean rederivation there, and been introduced into the recipient barrier. The foster mothers of rederived pups were LCMV-negative according to enzyme-linked immunosorbent assay, but sera of both cesarean-rederived MAI/Pas mice and their foster mothers were positive for LCMV infection by immunofluorescent assay (IFA). LCMV was isolated from the MAI/Pas mice, and its genomic RNA was sequenced. Examination of animal technicians in contact with LCMV-infected mice and of other mouse samples by IFA or a reverse transcriptase-polymerase chain reaction test (or both) revealed that neither the workers nor other animals had been infected with LCMV. Experimental data showed that LCMV transmission from persistently infected mice to naïve ones occurred only after direct contact of animals housed in the same cage. This experience demonstrates the importance of careful viral monitoring in the transfer of laboratory rodents between institutions, the limitation of dirty-bedding sentinels for detection of LCMV infection, and the inadequacy of cesarean rederivation for elimination of enzootic LCMV infection. 111
Mutations in the Helix Termination Motif of Mouse Type I IRS Keratin Genes Impair the Assembly of Keratin Intermediate Filament
Two classical mouse hair coat mutations, Rex (Re) and Rex wavy coat (Re(wc)), are linked to the type I inner root sheath (IRS) keratin genes of chromosome 11. An N-ethyl-N-nitrosourea-induced mutation, M100573, also maps close to the type I IRS keratin genes. In this study, we demonstrate that Re and M100573 mice bear mutations in the type I IRS gene Krt25; Re(wc) mice bear an additional mutation in the type I IRS gene Krt27. These three mutations are located in the helix termination motif of the 2B alpha-helical rod domain of a type I IRS keratin protein. Immunohistological analysis revealed abnormal foam-like immunoreactivity with an antibody raised to type II IRS keratin K71 in the IRS of Re/+ mice. These results suggest that the helix termination motif is essential for the proper assembly of types I and II IRS keratin protein complexes and the formation of keratin intermediate filaments.
Protein Phosphatase 4 Catalytic Subunit Regulates Cdk1 Activity and Microtubule Organization Via NDEL1 Dephosphorylation
Protein phosphatase 4 catalytic subunit (PP4c) is a PP2A-related protein serine/threonine phosphatase with important functions in a variety of cellular processes, including microtubule (MT) growth/organization, apoptosis, and tumor necrosis factor signaling. In this study, we report that NDEL1 is a substrate of PP4c, and PP4c selectively dephosphorylates NDEL1 at Cdk1 sites. We also demonstrate that PP4c negatively regulates Cdk1 activity at the centrosome. Targeted disruption of PP4c reveals disorganization of MTs and disorganized MT array. Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1. In addition, abnormal NDEL1 phosphorylation facilitates excessive recruitment of katanin p60 to the centrosome, suggesting that MT defects may be attributed to katanin p60 in excess. Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition. Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.
Establishment of Germline-competent Embryonic Stem Cell Lines from the MSM/Ms Strain
MSM/Ms is an inbred mouse strain established from the Japanese wild mouse, Mus musculus molossinus, which has been phylogenetically distinct from common laboratory mouse strains for about 1 million years. The nucleotide substitution rate between MSM/Ms and C57BL/6 is estimated to be 0.96%. MSM/Ms mice display unique characteristics not observed in the commonly used laboratory strains, including an extremely low incidence of tumor development, high locomotor activity, and resistance to high-fat-diet-induced diabetes. Thus, functional genomic analyses using MSM/Ms should provide a powerful tool for the identification of novel phenotypes and gene functions. We report here the derivation of germline-competent embryonic stem (ES) cell lines from MSM/Ms blastocysts, allowing genetic manipulation of the M. m. molossinus genome. Fifteen blastocysts were cultured in ES cell medium and three ES lines, Mol/MSM-1, -2, and -3, were established. They were tested for germline competency by aggregation with ICR morulae and germline chimeras were obtained from all three lines. We also injected Mol/MSM-1 ES cells into blastocysts of ICR or C57BL/6 x BDF1 mice and found that blastocyst injection resulted in a higher production rate of chimeric mice than did aggregation. Furthermore, Mol/MSM-1 subclones electroporated with a gene trap vector were also highly efficient at producing germline chimeras using C57BL/6 x BDF1 blastocyst injection. This Mol/MSM-1 ES line should provide an excellent new tool allowing the genetic manipulation of the MSM/Ms genome.
Efficient Production of Androgenetic Embryos by Round Spermatid Injection
Mammalian androgenetic embryos can be produced by pronuclear exchange of fertilized oocytes or by dispermic in vitro fertilization of enucleated oocytes. Here, we report a new technique for producing mouse androgenetic embryos by injection of two round spermatid nuclei into oocytes, followed by female chromosome removal. We found that injection of round spermatids resulted in high rates of oocyte survival (88%). Androgenetic embryos thus produced developed into mid-gestation fetuses at various rates, depending on the mouse strain used. All the fetuses examined maintained paternally specific genomic imprinting memories. This technique also enabled us to produce complete heterozygous F1 embryos by injecting two spermatids from different strains. The best rate of fetal survival (12% per embryos transferred) was obtained with C57BL/6 x DBA/2 androgenetic embryos. We also generated embryonic stem cell lines efficiently with the genotype of Mus musculus domesticus x M. m. molossinus. Thus, injection of two round spermatid nuclei followed by maternal enucleation is an effective alternative method of producing androgenetic embryos that consistently develop into blastocysts and mid-gestation fetuses.
A High-speed Congenic Strategy Using First-wave Male Germ Cells
In laboratory mice and rats, congenic breeding is essential for analyzing the genes of interest on specific genetic backgrounds and for analyzing quantitative trait loci. However, in theory it takes about 3-4 years to achieve a strain carrying about 99% of the recipient genome at the tenth backcrossing (N10). Even with marker-assisted selection, the so-called 'speed congenic strategy', it takes more than a year at N4 or N5.
The Mouse Resources at the RIKEN BioResource Center
Mice are one of the most important model organisms for studying biological phenomena and diseases processes in life sciences. The biomedical research community has succeeded in launching large scale strategic knockout mouse projects around the world. RIKEN BRC, a comprehensive government funded biological resource center was established in 2001. RIKEN BRC has been acting as the core facility for the mouse resources of the National BioResource Project (NBRP) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan since 2002. RIKEN BRC is a founding member of the Federation of International Mouse Resources (FIMRe) together with the Jackson Laboratory, the European Mouse Mutant Archive, and other centers, and has participated in the International Mouse Strain Resource (IMSR) to distribute mouse strains worldwide. With the support of the scientific community, RIKEN BRC has collected over 3,800 strains including inbred, transgenic, knockout, wild-derived, and ENU-induced mutant strains. Excellent mouse models for human diseases and gene functions from academic organizations and private companies are distributed through RIKEN BRC. To meet research and social needs, our mice will be rederived to a specific pathogen-free state, strictly monitored for their health, and accurately tested for their genetic modifications and backgrounds. Users can easily access our mouse resources through the internet and obtain the mouse strains for a minimal fee. Cryopreservation of embryos and sperm is used for efficient preservation of the increasing number of mouse resources. RIKEN BRC collaborates with FIMRe members to support Japanese scientists in the use of valuable mouse resources from around the world.
Unique Inbred Strain MSM/Ms Established from the Japanese Wild Mouse
Most laboratory mice belong to a species of house mouse, Mus musculus. So far, at least three subspecies groups have been recognized; domesticus subspecies group (DOM) distributed in western Europe, musculus subspecies group (MUS) distributed in eastern Europe and northeast Asia, and castaneus subspecies group (CAS) found in southwest and southeast Asia including southern China. These subspecies are estimated to have branched off roughly one million years ago. Genetic comparison between subspecies' groups and common inbred strains (CIS) have revealed that the genetic background of CIS is derived mainly from DOM. This shows the importance of non-DOM wild mice as valuable genetic resources. We started to establish our unique strain, MSM/Ms, from MUS in Japan in 1978. In the beginning, we kept wild mice trapped in Mishima in large plastic buckets. In 1979, breeding by sister-brother mating started. The MSM/Ms inbred strain was established in 1986 and 21 years later it reached F(100). During breeding, no significant fluctuations in litter size and sex ratios have been observed. Extensive genetic analyses of chromosome C-banding pattern, biochemical markers and microsatellite DNA (MIT) markers of this strain have demonstrated the characteristics of MUS. A phylogenetic tree constructed from MIT markers has confirmed the MUS nature of MSM strain. Taken together with its genetic remoteness from CIS, MSM appears to maintain many valuable alleles for investigation of biological functions and diseases. Some of these alleles have avoided selection during breeding as either fancy mice or laboratory mice. The MSM-specific genetic traits discovered to date are discussed.
Genetic Differences Among C57BL/6 Substrains
The C57BL/6 mouse is the most well-known inbred mouse strain, and has been widely used as a genetic background for congenic and mutant mice. A number of C57BL/6 substrains have been derived from the C57BL/6 founder line and are reported to differ in several phenotypes. There are several major sources of C57BL/6 substrains for the biomedical research community. The importance of their genetic and phenotypic differences among substrains, however, has not yet been well recognized by biomedical researchers. Here, we report the result of screening of the functional deletion of the nicotinamide nucleotide transhydrogenase (Nnt) gene and 1,446 SNPs genotyping among seven C57BL/6 substrains from different sources, such as C57BL/6J, C57BL/6JJcl, C57BL/6JJmsSlc, C57BL/6NJcl, C57BL/6NCrlCrlj, C57BL/6NTac, and C57BL/6CrSlc. The deletion of exon 7-11 in the Nnt gene that was previously reported in C57BL/6J was also observed in other C57BL/6J substrains, indicating that this functional deletion probably occurred at an early stage in the establishment of C57BL/6J substrains. The genotyping of SNP loci clearly demonstrate genetic differences between C57BL/6J and C57BL/6N substrains at 11 loci. Besides, we found another SNP differing between C57BL/6J and other C57BL/6J substrains available from commercial breeders. No genetic difference was detected among C57BL/6N substrains. The C57BL/6CrSlc mouse, originally derived from the National Cancer Institute of the NIH was found to be the same as the C57BL/6N substrains by the SNP pattern. These data will be useful for accurate genetic monitoring of genetically engineered mice with the C57BL/6 background.
Sex-reversed Somatic Cell Cloning in the Mouse
Somatic cell nuclear transfer has many potential applications in the fields of basic and applied sciences. However, it has a disadvantage that can never be overcome technically-the inflexibility of the sex of the offspring. Here, we report an accidental birth of a female mouse following nuclear transfer using an immature Sertoli cell. We produced a batch of 27 clones in a nuclear transfer experiment using Sertoli cells collected from neonatal male mice. Among them, one pup was female. This "male-derived female" clone grew into a normal adult and produced offspring by natural mating with a littermate. Chromosomal analysis revealed that the female clone had a 39,X karyotype, indicating that the Y chromosome had been deleted in the donor cell or at some early step during nuclear transfer. This finding suggests the possibility of resuming sexual reproduction after a single male is cloned, which should be especially useful for reviving extinct or endangered species.
Simultaneous Detection of Multiple Transgenes for Genetically-modified Mouse Strains
Recent advances in the genetic manipulation of mice have enabled us to generate transgenic and knockout mice. However, it is not easy to maintain these genetically-modified mice with the high-quality necessary to meet both scientific and legal requirements. RIKEN BRC has collected various transgenic, knockout, and conditional knockout mice. To examine the genetic modifications in these strains quickly and thoroughly, we established a simultaneous PCR test to detect multiple transgenes. We have called this, the "KO-survey". The PCR condition was optimized to detect neo, puro, pgk-neo, lacZ, and HSVtk-neo in set I, and hyg, IRES, cre, flp, and Gfp in set II. This "KO-survey" is useful for providing users with mouse strains of the highest genetic quality and accurate information on their genetic modifications.
NBRP Databases: Databases of Biological Resources in Japan
The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.
Genetic Variation of Melatonin Productivity in Laboratory Mice Under Domestication
Melatonin is a pineal hormone produced at night; however, many strains of laboratory mice are deficient in melatonin. Strangely enough, the gene encoding HIOMT enzyme (also known as ASMT) that catalyzes the last step of melatonin synthesis is still unidentified in the house mouse (Mus musculus) despite the completion of the genome sequence. Here we report the identification of the mouse Hiomt gene, which was mapped to the pseudoautosomal region (PAR) of sex chromosomes. The gene was highly polymorphic, and nonsynonymous SNPs were found in melatonin-deficient strains. In C57BL/6 strain, there are two mutations, both of which markedly reduce protein expression. Mutability of the Hiomt likely due to a high recombination rate in the PAR could be the genomic basis for the high prevalence of melatonin deficiency. To understand the physiologic basis, we examined a wild-derived strain, MSM/Ms, which produced melatonin more under a short-day condition than a long-day condition, accompanied by increased Hiomt expression. We generated F2 intercrosses between MSM/Ms and C57BL/6 strains and N2 backcrosses to investigate the role of melatonin productivity on the physiology of mice. Although there was no apparent effect of melatonin productivity on the circadian behaviors, testis development was significantly promoted in melatonin-deficient mice. Exogenous melatonin also had the antigonadal action in mice of a melatonin-deficient strain. These findings suggest a favorable impact of melatonin deficiency due to Hiomt mutations on domestic mice in breeding colonies.
The RIKEN Integrated Database of Mammals
The RIKEN integrated database of mammals (http://scinets.org/db/mammal) is the official undertaking to integrate its mammalian databases produced from multiple large-scale programs that have been promoted by the institute. The database integrates not only RIKEN's original databases, such as FANTOM, the ENU mutagenesis program, the RIKEN Cerebellar Development Transcriptome Database and the Bioresource Database, but also imported data from public databases, such as Ensembl, MGI and biomedical ontologies. Our integrated database has been implemented on the infrastructure of publication medium for databases, termed SciNetS/SciNeS, or the Scientists' Networking System, where the data and metadata are structured as a semantic web and are downloadable in various standardized formats. The top-level ontology-based implementation of mammal-related data directly integrates the representative knowledge and individual data records in existing databases to ensure advanced cross-database searches and reduced unevenness of the data management operations. Through the development of this database, we propose a novel methodology for the development of standardized comprehensive management of heterogeneous data sets in multiple databases to improve the sustainability, accessibility, utility and publicity of the data of biomedical information.
Kbus/Idr, a Mutant Mouse Strain with Skeletal Abnormalities and Hypophosphatemia: Identification As an Allele of 'Hyp'
The endopeptidase encoded by Phex (phosphate-regulating gene with homologies to endopeptidases linked to the X chromosome) is critical for regulation of bone matrix mineralization and phosphate homeostasis. PHEX has been identified from analyses of human X-linked hypophosphatemic rickets and Hyp mutant mouse models. We here demonstrated a newly established dwarfism-like Kbus/Idr mouse line to be a novel Hyp model.
