Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders.

Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications.

A novel imprinted transgene located near a repetitive element that exhibits allelic imbalance in DNA methylation during early development.

A mouse line carrying a lacZ transgene driven by the human EEF1A1/EF1 alpha promoter was established. Although the promoter is known to show ubiquitous activity, only paternal transgene alleles were expressed, resulting in a transgene imprinting. At mid-gestation, the promoter sequence was differentially methylated, hypomethylated for paternal and hypermethylated for maternal alleles. In germline, the promoter was a typical differentially methylated region. After fertilization, however, both alleles were hypermethylated. Thus, the differential methylation of the promoter required for transgene imprinting was re-established during later embryonic development independently of the germline differential methylation. Furthermore, also a retroelement promoter closely-flanking imprinted transgene and its wild type counterpart displayed similar differential methylation during early development. The retroelement promoter was methylated differentially also in germline, but in an opposite pattern to the embryonic differential methylation. These results suggest that there might be an unknown epigenetic regulation inducing transgene imprinting independently of DNA methylation in the transgene insertion site. Then, besides CpG dinucleotides, non-CpG cytosines of the retroelement promoter were highly methylated especially in the transgene-active mid-gestational embryos, suggesting that an unusual epigenetic regulation might protect the active transgene against de novo methylation occurring generally in mid-gestational embryo.

Structural abnormalities of corpus callosum and cortical axonal tracts accompanied by decreased anxiety-like behavior and lowered sociability in spock3- mutant mice.

Spock3/Testican-3 is a nervous system-expressed heparan sulfate proteoglycan belonging to a subgroup of the BM-40/SPARC/osteonectin family, the role of which in brain development is unclear. Because Spock1, a member of the Spock family, inhibits their attachment to substrates and the neurite outgrowth of cultured neuronal cells, Spock3 is also thought to be similarly involved in the neuronal development. In the present study, we established a Spock3-mutant mouse harboring a deletion extending from the presumptive upstream regulatory region to exon 4 of the Spock3 locus and performed histological and behavioral studies on these mutant mice. In wild-type (WT) mice, all Spock members were clearly expressed during brain development. In adults, intense Spock1 and Spock2 expressions were observed throughout the entire brain; whereas, Spock3 expression was no longer visible except in the thalamic nuclei. Thus, Spock3 expression is mostly confined to the developmental stage of the brain. In adult mutant mice, the cells of all cortical layers were swollen. The corpus callosum was narrowed around the central region along the rostral-caudal axis and many small spaces were observed without myelin sheaths throughout the entire corpus callosum. In addition, the cortical input and output fibers did not form into thick bundled fibers as well as the WT counterparts did. Moreover, a subpopulation of corticospinal axonal fibers penetrated into the dorsal striatum with moderately altered orientations. Consistent with these modifications of brain structures, the mutant mice exhibited decreased anxiety-like behavior and lowered sociability. Together, these results demonstrate that Spock3 plays an important role in the formation or maintenance of major neuronal structures in the brain.

Ataxia telangiectasia derived iPS cells show preserved x-ray sensitivity and decreased chromosomal instability.

Ataxia telangiectasia is a neurodegenerative inherited disease with chromosomal instability and hypersensitivity to ionizing radiation. iPS cells lacking ATM (AT-iPS cells) exhibited hypersensitivity to X-ray irradiation, one of the characteristics of the disease. While parental ataxia telangiectasia cells exhibited significant chromosomal abnormalities, AT-iPS cells did not show any chromosomal instability in vitro for at least 80 passages (560 days). Whole exome analysis also showed a comparable nucleotide substitution rate in AT-iPS cells. Taken together, these data show that ATM is involved in protection from irradiation-induced cell death.

Multiple renal cyst development but not situs abnormalities in transgenic RNAi mice against Inv::GFP rescue gene.

In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments.

Subcellular localization of dystrophin isoforms in cardiomyocytes and phenotypic analysis of dystrophin-deficient mice reveal cardiac myopathy is predominantly caused by a deficiency in full-length dystrophin.

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive muscle degenerative disorder that causes dilated cardiomyopathy in the second decade of life in affected males. Dystrophin, the gene responsible for DMD, encodes full-length dystrophin and various short dystrophin isoforms. In the mouse heart, full-length dystrophin Dp427 and a short dystrophin isoform, Dp71, are expressed. In this study, we intended to clarify the functions of these dystrophin isoforms in DMD-related cardiomyopathy. We used two strains of mice: mdx mice, in which Dp427 was absent but Dp71 was present, and DMD-null mice, in which both were absent. By immunohistochemical staining and density-gradient centrifugation, we found that Dp427 was located in the cardiac sarcolemma and also at the T-tubules, whereas Dp71 was specifically located at the T-tubules. In order to determine whether T tubule-associated Dp71 was involved in DMD-related cardiac disruption, we compared the cardiac phenotypes between DMD-null mice and mdx mice. Both DMD-null mice and mdx mice exhibited severe necrosis, which was followed by fibrosis in cardiac muscle. However, we could not detect a significant difference in myocardial fibrosis between mdx mice and DMD-null mice. Based on the present results, we have shown that cardiac myopathy is caused predominantly by a deficiency of full-length dystrophin Dp427.

Fetal skeletal muscle progenitors have regenerative capacity after intramuscular engraftment in dystrophin deficient mice.

Muscle satellite cells (SCs) are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs) isolated from Pax3(GFP/+) embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3(GFP/+) mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease.

Efficient and reproducible myogenic differentiation from human iPS cells: prospects for modeling Miyoshi Myopathy in vitro.

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70-90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.

In vitro modeling of paraxial mesodermal progenitors derived from induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells are generated from adult somatic cells by transduction of defined factors. Given their unlimited proliferation and differentiation potential, iPS cells represent promising sources for cell therapy and tools for research and drug discovery. However, systems for the directional differentiation of iPS cells toward paraxial mesodermal lineages have not been reported. In the present study, we established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture. The protocol was dependent on Activin signaling in addition to BMP and Wnt signaling which were previously shown to be effective for mouse ES cell differentiation. Independently of the cell origin, the number of transgenes, or the type of vectors used to generate iPS cells, the use of serum-free monolayer culture stimulated with a combination of BMP4, Activin A, and LiCl enabled preferential promotion of mouse iPS cells to a PDGFR-α(+)/Flk-1(-) population, which represents a paraxial mesodermal lineage. The mouse iPS cell-derived paraxial mesodermal cells exhibited differentiation potential into osteogenic, chondrogenic, and myogenic cells both in vitro and in vivo and contributed to muscle regeneration. Moreover, purification of the PDGFR-α(+)/KDR(-) population after differentiation allowed enrichment of human iPS cell populations with paraxial mesodermal characteristics. The resultant PDGFR-α(+)/KDR(-) population derived from human iPS cells specifically exhibited osteogenic, chondrogenic, and myogenic differentiation potential in vitro, implying generation of paraxial mesodermal progenitors similar to mouse iPS cell-derived progenitors. These findings highlight the potential of protocols based on the serum-free, stepwise induction and purification of paraxial mesodermal cell lineages for use in stem cell therapies to treat diseased bone, cartilage, and muscle.

Generation of a conditional null allele of Lbx1.

The homeobox gene Lbx1 not only plays critical roles in myogenesis and neurogenesis during embryonic development but is also expressed in activated satellite cells of adult mice. To address the potential postnatal functions of Lbx1, we generated conditional Lbx1-null mice using the Cre-loxP system. We generated a mouse in which Exon 2 of Lbx1 was floxed (Lbx1flox/flox), followed by cross-breeding between the Lbx1flox/flox mouse and either a transgenic mouse where a tamoxifen-inducible Cre-recombinase (Cre) was ubiquitously expressed, or a Myf5Cre mouse where Cre was inserted into the Myf5 locus. In both Lbx1-null mouse lines generated, Pax3-expressing limb muscle precursor cells were seriously reduced during embryonic development and eventually the limb extensor muscles were lost after birth. Since the conditional Lbx1-null mice generated were viable for a prolonged time, they will be useful in the investigation of Lbx1 function throughout the lifespan of the mouse.

Notch3 null mutation in mice causes muscle hyperplasia by repetitive muscle regeneration.

Satellite cells are skeletal muscle stem cells responsible for growth, maintenance, and repair of postnatal skeletal muscle. Although several studies have demonstrated that Notch signaling plays a critical role in muscle regeneration through promoting proliferation and self-renewal of satellite cells, the function of Notch3 is yet to be elucidated. We analyzed muscle regeneration in Notch3-deficient mutant mice. We found a remarkable overgrowth of muscle mass in the Notch3-deficient mice but only when they suffered repetitive muscle injuries. Immunochemical analysis found that Notch3 was expressed in Pax7(+)/MyoD(-) quiescent satellite cells and also in Pax7(+)/MyoD(+)-activated satellite cells, but the expression was restricted to around half the population of each cell type. In Notch3-deficient mice, the number of sublaminar quiescent satellite cells was significantly increased compared with those in control mice. We also found that primary cultured myoblasts isolated from the Notch3-deficient mice proliferated faster than those from control mice. Analysis of cultured myofibers revealed that the number of self-renewing Pax7-positive satellite cells attached to the myofiber was increased in the Notch3-deficient mice when compared with control mice. The data obtained in this study suggested that Notch3 pathway might be distinct from Notch1 in muscle regeneration. Because overexpression of Notch3 activated the expression of Nrarp, a negative feedback regulator of Notch signaling, Notch3 might act as a Notch1 repressor by activating Nrarp.

Spatially and temporally regulated expression of specific heparan sulfate epitopes in the developing mouse olfactory system.

Heparan sulfate (HS) comprises a structurally diverse group of glycosaminoglycans present ubiquitously on cell surfaces and in the extracellular matrix. The spatially and temporally regulated expression of specific HS structures is essential for various developmental processes in the nervous system but their distributions in the mouse olfactory system have not been explored. Here, we examined the spatiotemporal distribution of particular HS species in the developing mouse olfactory system using three structure-specific monoclonal antibodies (HepSS-1, JM403 and NAH46). The major findings were as follows. (i) During olfactory bulb morphogenesis, the HepSS-1 epitope was strongly expressed in anterior telencephalic cells and coexpressed with fibroblast growth factor receptor 1. (ii) In early postnatal glomeruli, the JM403 epitope was expressed at different levels among individual glomeruli. The expression pattern and levels of the JM403 epitope were both associated with those of ephrin-A3. (iii) In the vomeronasal system, the JM403 epitope was expressed in all vomeronasal axons but became increasingly restricted to vomeronasal axons terminating in the anterior region of the accessory olfactory bulb by 3 weeks of age. Our results demonstrate that each HS epitope exhibits a unique expression pattern during the development of the mouse olfactory system. Thus, each HS epitope is closely associated with particular developmental processes of the olfactory system and might have a particular role in developmental events.

Targeted deletion of the tybe IIb Na(+)-dependent Pi-co-transporter, NaPi-IIb, results in early embryonic lethality.

NaPi-IIb encodes a Na(+)-dependent Pi co-transporter, which is expressed in various adult tissues and mediates transport of extracellular Pi ions coupling with Na(+) ion. To define the role of NaPi-IIbin vivo, NaPi-IIb gene deficient mice were generated utilizing targeted mutagenesis, yielding viable, heterozygous NaPi-IIb mice. In contrast, homozygous NaPi-IIb mice died in utero soon after implantation, indicating that NaPi-IIb was essential for early embryonic development. In situ hybridization revealed NaPi-IIb mRNA expression in the parietal endoderm, followed by the visceral endoderm, at a time point prior to establishment of a functioning chorio-allantoic placenta. At the time point of functional placenta development, the main site of NaPi-IIb production resided in the labyrinthine zone, where embryonic and maternal circulations were in closest contact. Expression patterns of NaPi-IIb suggest that NaPi-IIb plays an important role in Pi absorption from maternal circulation.

Skeletogenesis in Xenopus tropicalis: characteristic bone development in an anuran amphibian.

In mammals and birds, most of the skeletal bones develop via endochondral ossification. Chondrocytes in the cartilaginous anlagen undergo processes of maturation such as hypertrophy, calcification and apoptosis. Concomitantly, osteoblasts are recruited to replace the cartilage scaffold gradually with bone matrix and become osteocytes in the trabecular bones. Throughout the successive development of bones, several gene products have been identified as being the components of the molecular mechanism regulating bone development. Transcription factor SOX9 plays essential roles during developmental steps from undifferentiated mesenchymal cells to proliferating chondrocytes, meanwhile, it inhibits transition of proliferating chondrocytes to hypertrophy. Other transcription factors RUNX2 and OSTERIX are critical in osteoblast differentiation, and RUNX2 is also essential for chondrocyte maturation such as hypertrophy and matrix mineralization. GDF5, a protein belonging to the transforming growth factor beta superfamily, is involved in joint formation and chondrogenesis. The limb skeleton of one of the ancestral tetrapod, anuran amphibians also develops through cartilaginous anlagen to bones, but their skeletogenesis has some unique characteristics compared with that of mammals and birds. Anuran amphibians develop and grow with less bone trabeculae and poor epiphyseal growth plates, and its endochondral ossification was found to be a delayed process. In order to address the characteristic skeletal development of anuran amphibians, we cloned Xenopus tropicalis RUNX2 (Xt-runx2), OSTERIX (Xt-osterix) and GDF5 (Xt-gdf5) homologue, and observed expression patterns together with Xt-sox9. In X. tropicalis limbs, histological observation and section in situ hybridization analysis suggest that Xt-SOX9 is involved in chondrogenesis, Xt-RUNX2 and Xt-OSTERIX are involved in osteogenesis, and Xt-GDF5 is involved in joint formation. In the cartilaginous anlagen, Xt-runx2 expression was found in perichondrium and immature chondrocytes as seen in other vertebrates. However, Xt-runx2 expression in enlarged chondrocytes was weak and dissimilar to common hypertrophic chondrocytes. These observations suggest that weak Xt-runx2 expression in maturing chondrocytes affects characteristic bone development in X. tropicalis long bones.

Loss of short dystrophin isoform Dp71 in olfactory ensheathing cells causes vomeronasal nerve defasciculation in mouse olfactory system.

The Duchenne muscular dystrophy (DMD) gene encodes dystrophin, which is a protein defective in DMD patients, as well as a number of shorter isoforms, which have been shown to be expressed in various non-muscle, primarily neural, tissues. As of yet, the physiological function of the various dystrophin isoforms is not fully understood. In the present study, we investigated the neurological phenotype that arises in the DMD-null mice, where expression of all dystrophin isoforms had been disrupted. We demonstrate that vomeronasal axons in the DMD-null mice are defasciculated, and some of the defasciculated vomeronasal axons aberrantly entered into the main olfactory bulb, which indicates that the product(s) of the DMD gene plays an important role in vomeronasal nerve organization. Through western blot and immunofluorescence analyses, we determined that the dystrophin isoform Dp71 was exclusively expressed in the mouse olfactory system: mainly in the olfactory ensheathing cells (OECs), an olfactory system-specific glia cell that ensheaths fascicles of the olfactory nerve. In the OECs, Dp71 was co-localized with beta-dystroglycan, utrophin, laminin, and perlecan. Since beta-dystroglycan and perlecan expression was decreased in the OECs of DMD-null mice, we hypothesize that Dp71 expressed in the OECs participates in fasciculation of the vomeronasal nerve, most likely through interactions with extracellular matrix.

Functional analysis of homeodomain-containing transcription factor Lbx1 in satellite cells of mouse skeletal muscle.

Satellite cells are usually mitotically quiescent muscle stem cells, located between the sarcolemma and the basement membrane of muscle fibers. When muscles are damaged, satellite cells become activated, proliferate and differentiate to form multinucleate myofibers. The molecular mechanisms underlying these processes are poorly understood. In the present study, we found that, following treatment with cardiotoxin, homeodomain-containing transcription factor Lbx1 was strongly expressed in the satellite cells of regenerating adult skeletal muscle. Our immunohistochemical and northern blot analyses indicate that Lbx1 is expressed in activated but not quiescent satellite cells. In vitro, this Lbx1 expression was gradually downregulated when satellite cells differentiate into mature myofibers. Transfection and forced expression of Lbx1 in satellite-cell-derived C2C12 myoblast cells resulted in severe depression of myogenic differentiation and incomplete myotube formation, concomitantly with the activation of the paired-box transcription factor Pax7 and depression of the myogenic regulatory factor MyoD. Moreover, knockdown of Lbx1 in in-vitro-cultured satellite cells resulted in downregulation of Pax7. These results suggest that Lbx1 plays important roles in differentiation and maintenance of satellite cells of mature myofibers, probably through the regulation of Pax7.

Epimorphin acts extracellularly to promote cell sorting and aggregation during the condensation of vertebral cartilage.

Formation of vertebrae occurs via endochondral ossification, a process involving condensation of precartilaginous cells. Here, we provide the first molecular evidence of mechanism that underlies initiation of this process by showing that the extracellular factor, Epimorphin, plays a role during early steps in vertebral cartilage condensation. Epimorphin mRNA is predominantly localized in the vertebral primordium. When provided exogenously in ovo, it causes precocious differentiation of chondrocytes, resulting in the formation of supernumerary vertebral cartilage in chicken embryos. To further analyze its mode of action, we used an in vitro co-culture system in which labeled 10T1/2 or sclerotomal prechondrogenic cells were co-cultured with unlabeled Epimorphin-producing cells. In the presence of Epimorphin, the labeled cells formed tightly packed aggregates, and sclerotomal cells displayed augmented accumulation of NCAM and other early markers of chondrocyte differentiation. Finally, we found that the Epimorphin expression is initiated during vertebrogenesis by Sonic hedgehog from the notochord mediated by Sox 9. We present a model in which successive action of Epimorphin in recruiting and stacking sclerotomal cells leads to a sequential elongation of a vertebral primordium.

Functional redundancy of the Notch gene family during mouse embryogenesis: analysis of Notch gene expression in Notch3-deficient mice.

The Notch3 gene, a member of the Notch gene family, is expressed in a wide variety of tissues during development. We generated and analyzed Notch3-deficient mice to assess the in vivo role of the Notch3 gene. Consistent with previous observation of Krebs et al. [Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation, Genesis 37 (3) (2003) 139-143], the Notch3-/- mice were viable, fertile, and developed normally despite abundant expression of Notch3 in various embryonic tissues. We examined the details of Notch1, 2, and 4 expressions in the Notch3-/- embryos compared with those in wild-type embryos. As a result, we found that a deficiency in Notch3 did not affect the expression of Notch1, 2, and 4, and that either Notch1 or Notch2, or sometimes both, was always expressed in all Notch3-expressing tissues examined. These results support the idea that other Notch genes functionally compensate for Notch3 during embryonic development. We also surveyed the adult tissues of Notch3-/- mice and found significantly fewer thymocytes in 10-week-old mice. Therefore, the thymus might be a target tissue affected by Notch3 deficiency.

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins.

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) kappa locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igkappa region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.

A new model mouse for Duchenne muscular dystrophy produced by 2.4 Mb deletion of dystrophin gene using Cre-loxP recombination system.

Duchenne muscular dystrophy (DMD) is caused by mutation in the 2.4-Mb dystrophin (DMD) gene . This gene encodes a number of tissue-specific isoforms of dystrophin generated by transcription from at least seven promoters and also by alternative splicing. We deleted entire genomic region of the DMD gene on mouse chromosome X using a Cre-loxP recombination system. Introduction of a loxP site in dystrophin's first and last exon by homologous recombination in mouse embryonic stem (ES) cells generated "DMD-floxed" (flanked by loxP sites) ES cells, which we subjected to Cre-mediated excision leading to establishment of "DMD-null" ES cell lines. The DMD-null mice produced from the DMD-null ES cells were viable but displayed severe muscular hypertrophy and dystrophy. In addition to the muscular impairment, the DMD-null mouse exhibited some behavioral abnormality and male sterility. The DMD-floxed mice produced from the DMD-floxed ES cells were viable, phenotypically normal, and were born with the expected Mendelian frequency, despite the absence of brain (cortical)-type dystrophin (Dp427c) expression. Since production of multiple dystrophin isoforms due to alternative splicing or exon skipping is totally prevented in the DMD-null mouse, these new mutants will provide an improved model system for functional studies of dystrophin and its isoforms.