RESUMEN
In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5'-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.
Asunto(s)
Proteínas Argonautas/metabolismo , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/genética , Femenino , Genómica , Masculino , Mesocricetus , Metafase , Fosforilación , ARN Interferente Pequeño/genética , Testículo/metabolismoRESUMEN
PIWI-interacting RNAs (piRNAs), a subclass of germ cell-specific noncoding small RNAs, are essential for de novo DNA methylation of retrotransposon genes in embryonic testes. PIWIL2/MILI, one of three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably via piRNA, and normal spermatogenesis. In vitro analysis using germline stem cells (GS cells) revealed that glycerol-3-phosphate acyltransferase 2 (GPAT2), which is a mitochondrial outer membrane protein involved in generation of lysophosphatidic acid (LPA) and highly expressed in testes, plays important roles in spermatogenesis. Namely, GPAT2 binds to PIWIL2 and is closely involved in the biogenesis of piRNAs; this process is independent of its enzymatic activity on LPA. However, GS cells recapitulate only a limited phase of spermatogenesis and the biological functions of GPAT2 remain largely unknown. In this study, we generated GPAT2-deficient mice and conducted comprehensive analyses. The deficient mice showed defective piRNA production and subsequent de-silencing of IAP and Line-1 retrotransposons in fetal testes. In addition, apoptosis of pachytene spermatocytes was observed. These abnormalities were all common to the phenotype of PIWIL2-deficient mice, in which piRNA production was impaired. GPAT2-deficient mice exhibited apoptosis in spermatogonia at the neonatal stage, which was not observed in PIWIL2-deficient mice. These data show that GPAT2 plays a critical role in preventing apoptosis in spermatogonia.
Asunto(s)
Silenciador del Gen/fisiología , Glicerol-3-Fosfato O-Aciltransferasa/fisiología , ARN Interferente Pequeño/biosíntesis , Retroelementos/genética , Espermatogonias/fisiología , Animales , Proliferación Celular/genética , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Glicerol-3-Fosfato O-Aciltransferasa/genética , Masculino , Ratones , Ratones Noqueados , ARN Interferente Pequeño/genética , Espermatogénesis/genética , Espermatogonias/citología , Testículo/citología , Testículo/metabolismoRESUMEN
Proper biogenesis of a sperm-specific organelle, the acrosome, is essential for gamete interaction. An acrosomal matrix protein, ACRBP, is known as a proacrosin-binding protein. In mice, two forms of ACRBP, wild-type ACRBP-W and variant ACRBP-V5, are generated by pre-mRNA alternative splicing of Acrbp Here, we demonstrate the functional roles of these two ACRBP proteins. ACRBP-null male mice lacking both proteins showed a severely reduced fertility, because of malformation of the acrosome. Notably, ACRBP-null spermatids failed to form a large acrosomal granule, leading to the fragmented structure of the acrosome. The acrosome malformation was rescued by transgenic expression of ACRBP-V5 in ACRBP-null spermatids. Moreover, exogenously expressed ACRBP-W blocked autoactivation of proacrosin in the acrosome. Thus, ACRBP-V5 functions in the formation and configuration of the acrosomal granule during early spermiogenesis. The major function of ACRBP-W is to retain the inactive status of proacrosin in the acrosome until acrosomal exocytosis.
Asunto(s)
Acrosoma/metabolismo , Empalme Alternativo , Proteínas Portadoras/genética , Precursores del ARN/genética , Espermatogénesis , Espermatozoides/metabolismo , Animales , Proteínas Portadoras/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores del ARN/metabolismo , Espermatozoides/crecimiento & desarrolloRESUMEN
Periostin is a matricellular protein involved in development, maintenance, and regulation of tissues and organs via by binding to cell surface integrin receptors. Pathologically, periostin plays an important role in the process of wound healing: as a deficiency of the Postn gene delays wound closure and periostin is consistently up-regulated in response to injury and skin diseases. However, the functional role of elevated periostin in the process of wound healing has not been tested. In this study, we generated Postn-transgenic mice under the control of the CAG promoter/enhancer to investigate the effects of constitutive overexpression of full length periostin during its pathophysiological roles. Transgenic mice showed significant overexpression of periostin in skin, lung, and heart, but no morphological changes were observed. However, when these transgenic mice were injured, periostin overexpression delayed the closure of excisional wounds. Expression of IL-1ß and TNFα, pro-inflammatory cytokines important for wound healing, was significantly decreased in the transgenic mice, prior to delayed healing. Infiltration of neutrophils and macrophages, the main sources of IL-1ß and TNFα, was also down-regulated in the transgenic wound sites. From these data, we conclude that enforced expression of periostin delays wound closure due to reduced infiltration of neutrophils and macrophages followed by down-regulation of IL-1ß and TNFα expression. This suggests that regulated spatiotemporal expression of periostin is important for efficient wound healing and that constitutive periostin overexpression interrupts the normal process of wound closure.
Asunto(s)
Moléculas de Adhesión Celular/genética , Regulación de la Expresión Génica , Cicatrización de Heridas/genética , Heridas y Lesiones/patología , Animales , Biomarcadores , Biopsia con Aguja , Citocinas/genética , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Distribución Aleatoria , Valores de Referencia , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genética , Regulación hacia Arriba , Cicatrización de Heridas/fisiologíaRESUMEN
In mammals, sperm migrate through the female reproductive tract to reach the egg; however, our understanding of this journey is highly limited. To shed light on this process, we focused on defining the functions of seminal vesicle secretion 2 (SVS2). SVS2(-/-) male mice produced sperm but were severely subfertile, and formation of a copulatory plug to cover the female genital opening did not occur. Surprisingly, even when artificial insemination was performed with silicon as a substitute for the plug, sperm fertility in the absence of SVS2 remained severely reduced because the sperm were already dead in the uterus. Thus, our results provide evidence that the uterus induces sperm cell death and that SVS2 protects sperm from uterine attack.
Asunto(s)
Proteínas de Secreción de la Vesícula Seminal/metabolismo , Vesículas Seminales/metabolismo , Espermatozoides/fisiología , Útero/química , Reacción Acrosómica/fisiología , Animales , Southern Blotting , Movimiento Celular/fisiología , Femenino , Fertilidad/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica , Reacción en Cadena de la Polimerasa , Colorantes de Rosanilina , Proteínas de Secreción de la Vesícula Seminal/genética , Espermatozoides/ultraestructura , Estadísticas no ParamétricasRESUMEN
Using transgenic mice with spermatozoa expressing enhanced green fluorescent protein in their acrosome and red fluorescent protein in their midpiece mitochondria, we followed the behavior of spermatozoa within the female genital tract after natural mating. When examined 15 min after coitus, many spermatozoa were around the opening of the uterotubal junction. Spermatozoa that entered the uterotubal junction were seemingly not moving, yet they steadily migrated toward the isthmus at a speed only time-lapse video recording could demonstrate. Many spermatozoa reaching the lower isthmus were motile. The site where spermatozoa attached and detached from the isthmus epithelium shifted from the lower to the upper segment of the isthmus with time. Virtually all the live spermatozoa within the lower isthmus were acrosome intact, whereas many of the actively motile spermatozoa in the upper isthmus were acrosome reacted. As far as we could observe, all the spermatozoa we found within the lumen of the ampulla and the cumulus oophorus were acrosome reacted. Even though we saw only a very few spermatozoa within the ampulla during fertilization, all were associated with, or were already within, oocytes, indicating that mouse fertilization in vivo is extremely efficient.
Asunto(s)
Fertilización , Espermatozoides/fisiología , Reacción Acrosómica , Animales , Femenino , Masculino , Ratones , Ratones Transgénicos , OviductosRESUMEN
Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) is a matricellular protein. Previously, we demonstrated that Tinagl1 expression was restricted to extraembryonic regions during the postimplantation period and detected marked expression in mouse Reichert's membranes. In uteri, Tinagl1 is markedly expressed in the decidual endometrium during the postimplantation period, suggesting that it plays a physical and physiological role in embryo development and/or decidualization of the uterine endometrium during pregnancy. In the present study, in order to determine the role of Tinagl1 during embryonic development and pregnancy, we generated Tinagl1-deficient mice. Although Tinagl1(-/-) embryos were not lethal during development to term, homologous matings of Tinagl1(-/-) females and Tinagl1(-/-) males showed impaired fertility during pregnancy, including failure to carry pregnancy to term and perinatal lethality. To examine ovarian function, ovulation was induced with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); the number of ovulated oocytes did not differ between Tinagl1(-/-) and Tinagl1(flox/flox). In vitro fertilization followed by embryo culture also demonstrated the normal developmental potential of Tinagl1-null embryos during the preimplantation period. Our results demonstrate that Tinagl1 deficiency affects female mice and results in subfertility phenotypes, and they suggest that although the potential of Tinagl1(-/-) oocytes is normal, Tinagl1 is related to fertility in adult females but is not essential for either fertilization or preimplantation development in vitro.
Asunto(s)
Fertilidad/genética , Lipocalinas/genética , Proteínas de Neoplasias/genética , Alelos , Animales , Gonadotropina Coriónica/metabolismo , Cruzamientos Genéticos , Técnicas de Cultivo de Embriones , Implantación del Embrión/efectos de los fármacos , Desarrollo Embrionario , Endometrio/metabolismo , Femenino , Fertilización In Vitro , Vectores Genéticos , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/citología , Ovulación , Fenotipo , Útero/metabolismoRESUMEN
Melanin-concentrating hormone (MCH) is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area. Recent studies have reported that MCH neurons are active during rapid eye movement (REM) sleep, but their physiological role in the regulation of sleep/wakefulness is not fully understood. To determine the physiological role of MCH neurons, newly developed transgenic mouse strains that enable manipulation of the activity and fate of MCH neurons in vivo were generated using the recently developed knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction system. The activity of these cells was controlled by optogenetics by expressing channelrhodopsin2 (E123T/T159C) or archaerhodopsin-T in MCH neurons. Acute optogenetic activation of MCH neurons at 10 Hz induced transitions from non-REM (NREM) to REM sleep and increased REM sleep time in conjunction with decreased NREM sleep. Activation of MCH neurons while mice were in NREM sleep induced REM sleep, but activation during wakefulness was ineffective. Acute optogenetic silencing of MCH neurons using archaerhodopsin-T had no effect on any vigilance states. Temporally controlled ablation of MCH neurons by cell-specific expression of diphtheria toxin A increased wakefulness and decreased NREM sleep duration without affecting REM sleep. Together, these results indicate that acute activation of MCH neurons is sufficient, but not necessary, to trigger the transition from NREM to REM sleep and that MCH neurons also play a role in the initiation and maintenance of NREM sleep.
Asunto(s)
Hormonas Hipotalámicas/fisiología , Melaninas/fisiología , Neuronas/metabolismo , Hormonas Hipofisarias/fisiología , Sueño/fisiología , Vigilia/fisiología , Animales , Ratones , Ratones Transgénicos , OptogenéticaRESUMEN
Preeclampsia is a relatively common pregnancy-related disorder. Both maternal and fetal lives will be endangered if it proceeds unabated. Recently, the placenta-derived anti-angiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFLT1) and soluble endoglin (sENG), have attracted attention in the progression of preeclampsia. Here, we established a unique experimental model to test the role of sFLT1 in preeclampsia using a lentiviral vector-mediated placenta-specific expression system. The model mice showed hypertension and proteinuria during pregnancy, and the symptoms regressed after parturition. Intrauterine growth restriction was also observed. We further showed that pravastatin induced the VEGF-like angiogenic factor placental growth factor (PGF) and ameliorated the symptoms. We conclude that our experimental preeclamptic murine model phenocopies the human case, and the model identifies low-dose statins and PGF as candidates for preeclampsia treatment.
Asunto(s)
Modelos Animales de Enfermedad , Pravastatina/farmacología , Preeclampsia/prevención & control , Proteínas Gestacionales/metabolismo , Animales , Animales Recién Nacidos , Línea Celular , Femenino , Células HEK293 , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inyecciones Intraperitoneales , Lentivirus/genética , Masculino , Ratones , Ratones Transgénicos , Placenta/metabolismo , Factor de Crecimiento Placentario , Pravastatina/administración & dosificación , Preeclampsia/genética , Preeclampsia/metabolismo , Embarazo , Proteínas Gestacionales/genética , Transducción Genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell-surface proteins, such as Thy-1 and CD48. GPI-anchored proteins (GPI-APs) play important roles in many biological processes, such as signal transduction and cell-cell interaction, through their association with lipid rafts. Fatty acid remodeling of GPI-APs in the Golgi apparatus is required for their efficient association with lipid rafts, i.e., the unsaturated fatty acid at the sn-2 position of the PI moiety is exchanged for the saturated fatty acid by PGAP2 and PGAP3. To investigate the immunological role of the fatty acid remodeling of GPI-APs, we generated a Pgap3 knockout mouse. In this mouse, GPI-APs are expressed on the cell surface without fatty acid remodeling, and fail to associate with lipid rafts. Male Pgap3 knockout mice were born alive at a ratio lower than expected from Mendel's law, whereas the number of female mice followed Mendel's law. All mice exhibited growth retardation and abnormal reflexes such as limb grasping. We focused T cell function in these mice and found that T cell development in the absence of Pgap3 was normal. However, the response of T cells was enhanced in Pgap3 knockout mice in both in vitro and in vivo studies, including alloreactive response, antigen-specific immune response, and experimental autoimmune encephalomyelitis. Cross-linking of Thy-1 in wild-type cells inhibited the signal transduced by the T cell receptor (TCR), whereas cross-linking of Thy-1 in Pgap3 knockout cells enhanced the TCR signal. These results suggest that GPI-APs localized in lipid rafts may modulate signaling through the TCR.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Hidrolasas de Éster Carboxílico/genética , Encefalomielitis Autoinmune Experimental/inmunología , Ácidos Grasos/inmunología , Glicosilfosfatidilinositoles/inmunología , Activación de Linfocitos/genética , Microdominios de Membrana/inmunología , Antígenos Thy-1/inmunología , Animales , Antígenos/inmunología , Femenino , Masculino , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Many animals have a conserved adaptive genome defence system known as the Piwi-interacting RNA (piRNA) pathway, which is essential for germ cell development and function. Disruption of individual mouse Piwi genes results in male but not female sterility, leading to the assumption that PIWI genes play little or no role in mammalian oocytes. Here, we report the generation of PIWI-defective golden hamsters, which have defects in the production of functional oocytes. The mechanisms involved vary among the hamster PIWI genes, whereby the lack of PIWIL1 has a major impact on gene expression, including hamster-specific young transposon de-silencing, whereas PIWIL3 deficiency has little impact on gene expression in oocytes, although DNA methylation was reduced to some extent in PIWIL3-deficient oocytes. Our findings serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes, including humans.
Asunto(s)
Mesocricetus/metabolismo , Oocitos/metabolismo , ARN Interferente Pequeño/genética , Testículo/metabolismo , Animales , Proteínas Argonautas/genética , Cricetinae , Metilación de ADN/fisiología , Expresión Génica/fisiología , Células Germinativas/metabolismo , MasculinoRESUMEN
Neural tube defects (NTDs) are the second most common structural birth defect. Senescence, a state of permanent cell cycle arrest, occurs only after neural tube closure. Maternal diabetes-induced NTDs are severe diabetic complications that lead to infant mortality or lifelong morbidity and may be linked to premature senescence. Here, we report that premature senescence occurs in the mouse neuroepithelium and disrupts neurulation, leading to NTDs in diabetic pregnancy. Premature senescence and NTDs were abolished by knockout of the transcription factor Foxo3a, the miR-200c gene, and the cell cycle inhibitors p21 and p27; transgenic expression of the dominant-negative FoxO3a mutant; or the senomorphic rapamycin. Double transgenic expression of p21 and p27 mimicked maternal diabetes in inducing premature neuroepithelium senescence and NTDs. These findings integrate transcription- and epigenome-regulated miRNAs and cell cycle regulators in premature neuroepithelium senescence and provide a mechanistic basis for targeting premature senescence and NTDs using senomorphics.
RESUMEN
Lentiviral vectors efficiently integrate into the host genome of both dividing and nondividing cells, and so they have been used for stable transgene expression in biological and biomedical studies. However, recent studies have highlighted the risk of insertional mutagenesis and subsequent oncogenesis. Here, we used an integrase-defective lentiviral (IDLV) vector to decrease the chance of random integration and examined the feasibility of lentiviral vector-mediated gene targeting into murine embryonic stem (ES) cells. After transduction with wild-type lentiviral vectors, none of the 512 G418 resistant clones were found to be homologous recombinant clones. Although the transduction efficiency was lower with the IDLV vectors (5.9% of wild-type), successful homologous recombination was observed in nine out of the 941 G418 resistant clones (0.83 +/- 1.32%). Pluripotency of the homologous recombinant ES cells was confirmed by the production of chimeric mice and subsequent germ line transmission. Because lentiviral vectors can efficiently transduce a variety of stem cell types, our strategy has potential relevance for secure gene-manipulation in therapeutic applications.
Asunto(s)
Células Madre Embrionarias/metabolismo , Marcación de Gen/métodos , Vectores Genéticos/genética , Integrasas/genética , Animales , Animales Recién Nacidos , Blastómeros/citología , Blastómeros/metabolismo , Southern Blotting , Proteínas de Unión al Calcio/genética , Virus Defectuosos/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/trasplante , Femenino , Lentivirus/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Modelos Genéticos , Chaperonas Moleculares/genética , Mutagénesis Insercional , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Reacción en Cadena de la Polimerasa , Embarazo , Trasplante de Células Madre , Transfección/métodosRESUMEN
CD52 is a glycosylphosphatidylinositol (GPI)-anchored antigen expressed on lymphocytes and in epididymal epithelial cells. CD52 is also known as "maturation-associated sperm antigen" but its function is unknown. We therefore generated Cd52 disrupted mice. The resulting Cd52 null mice were healthy, even though Cd52 is expressed on cells of the immune system. We then examined a possible role for CD52 in reproduction. Sperm from Cd52-deficient males were investigated and the viability, motility, morphology, and incidence of spontaneous acrosome reactions were found to be all similar to values for wild-type sperm. In in vitro fertilization system, the sperm showed normal fertilizing ability. As CD52 was found to be transferred onto sperm only after they had migrated into the vas deferens, we examined the behavior of sperm from Cd52-deficient mice in vivo. The mice mated naturally and we observed that a normal number of sperm passed through the uterotubal junction, known to the crucial hurdle for various gene knockouts resulting in infertile sperm. As a consequence, there was no difference in the litter size from the wild-type and Cd52-null males. Our results therefore indicate CD52 is not required for fertilization in the mouse either in vivo or in vitro.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Epidídimo/metabolismo , Fertilización , Glicoproteínas/metabolismo , Animales , Antígenos CD/genética , Antígenos de Neoplasias/genética , Antígeno CD52 , Expresión Génica , Glicoproteínas/genética , Masculino , Ratones , Especificidad de Órganos , Espermatozoides/metabolismoRESUMEN
In the original HTML version of this Article, the affiliation details for Hirosuke Shiura, Hidetoshi Hasuwa and Takashi Kohda were incorrect, as detailed in the associated Publisher Correction. These errors have been corrected in both the HTML version of the Article.
RESUMEN
X-chromosome inactivation (XCI) is an essential epigenetic process in female mammalian development. Although cell-based studies suggest the potential importance of the Ftx long non-protein-coding RNA (lncRNA) in XCI, its physiological roles in vivo remain unclear. Here we show that targeted deletion of X-linked mouse Ftx lncRNA causes eye abnormalities resembling human microphthalmia in a subset of females but rarely in males. This inheritance pattern cannot be explained by X-linked dominant or recessive inheritance, where males typically show a more severe phenotype than females. In Ftx-deficient mice, some X-linked genes remain active on the inactive X, suggesting that defects in random XCI in somatic cells cause a substantially female-specific phenotype. The expression level of Xist, a master regulator of XCI, is diminished in females homozygous or heterozygous for Ftx deficiency. We propose that loss-of-Ftx lncRNA abolishes gene silencing on the inactive X chromosome, leading to a female microphthalmia-like phenotype.
Asunto(s)
Microftalmía/genética , Microftalmía/patología , ARN Largo no Codificante/metabolismo , Inactivación del Cromosoma X/genética , Animales , Ojo/patología , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Femenino , Humanos , Patrón de Herencia/genética , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Linaje , Fenotipo , ARN Largo no Codificante/genética , Transcriptoma/genéticaRESUMEN
Laminin-integrin interactions regulate various adhesion-dependent cellular processes. γ1C-Glu, the Glu residue in the laminin γ1 chain C-terminal tail, is crucial for the binding of γ1-laminins to several integrin isoforms. Here, we investigated the impact of γ1C Glu to Gln mutation on γ1-laminin binding to all possible integrin partners in vitro, and found that the mutation specifically ablated binding to α3, α6, and α7 integrins. To examine the physiological significance of γ1C-Glu, we generated a knock-in allele, Lamc1 EQ , in which the γ1C Glu to Gln mutation was introduced. Although Lamc1 EQ/EQ homozygotes developed into blastocysts and deposited laminins in their basement membranes, they died just after implantation because of disordered extraembryonic development. Given the impact of the Lamc1 EQ allele on embryonic development, we developed a knock-in mouse strain enabling on-demand introduction of the γ1C Glu to Gln mutation by the Cre-loxP system. The present study has revealed a crucial role of γ1C-Glu-mediated integrin binding in postimplantation development and provides useful animal models for investigating the physiological roles of laminin-integrin interactions in vivo.
RESUMEN
Von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells (ECs) and has two main roles: to carry and protect coagulation factor VIII (FVIII) from degradation by forming VWF-FVIII complex; and to mediate platelet adhesion and aggregation at sites of vascular injury. Previous research using the HEK293 cell line revealed that the VWF K1362 mutation interacted directly with platelet glycoprotein Ib (GPIb). Vwf K1362A knock-in (KI) mice were therefore generated to verify the in vivo function of residue 1362 in binding to platelet GPIb. The Cre-loxP system was employed to introduce the Vwf K1362A mutation systemically in mice. In blood coagulation analysis, the VWF antigen (VWF:Ag) of Lys1362Ala KI homozygous (homo) mice was below the sensitivity of detection by enzyme-linked immunosorbent assay. FVIII activities (FVIII:C) were 47.9 ± 0.3 and 3.3 ± 0.3% (K1362A heterozygous (hetero) and K1362A KI homo mice, respectively) compared to wild-type mice. Immunohistochemical staining analysis revealed that VWF protein did not exist in ECs of K1362A KI homo mice. These results indicated that VWF protein synthesis of K1362A was impaired after transcription in mice. K1362 seems to represent a very important position not only for VWF function, but also for VWF synthesis in mice.
Asunto(s)
Biosíntesis de Proteínas/genética , Factor de von Willebrand , Animales , Células Endoteliales/metabolismo , Factor VIII/metabolismo , Megacariocitos/metabolismo , Ratones Endogámicos C57BL , Mutación , Adhesividad Plaquetaria/genética , Agregación Plaquetaria/genética , Factor de von Willebrand/biosíntesis , Factor de von Willebrand/genética , Factor de von Willebrand/fisiologíaRESUMEN
The mouse testis has served as a popular model system to study a wide range of biological processes, including germ cell development, meiosis, epigenetic changes of chromatin, transposon silencing, and small RNA-mediated epigenetic modifications. PIWI-interacting RNAs (piRNAs) are a class of small RNAs that are almost exclusively expressed in animal gonads. They repress transposons by forming effector complexes with PIWI proteins to maintain genome integrity of the germline. Here we describe detailed procedures of how to produce monoclonal antibodies against a mouse nuclear PIWI protein, MIWI2, which functions in de novo DNA methylation of target transposon loci. We then describe how to use the antibodies to isolate associated complexes and to detect MIWI2 immunohistochemically.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Proteínas Argonautas/inmunología , ARN Interferente Pequeño/genética , Animales , Epigénesis Genética , Redes Reguladoras de Genes , Inmunización , Masculino , Ratones , Células 3T3 NIH , Testículo/inmunología , Testículo/metabolismoRESUMEN
Ovarian cancer is the most frequent cause of cancer death among all gynecologic cancers. We demonstrate here that lysophosphatidic acid (LPA)-induced ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF) is a critical to tumor formation in ovarian cancer. We found that among the epidermal growth factor receptor (EGFR) family of growth factors, HB-EGF gene expression in cancerous tissues and HB-EGF protein levels in patients' ascites fluid were significantly elevated. The human ovarian cancer cell lines SKOV3 and RMG-1 form tumors in nude mice. Tumor formation of these cells was enhanced by exogenous expression of pro-HB-EGF and completely blocked by pro-HB-EGF gene RNA interference or by CRM197, a specific HB-EGF inhibitor. Transfection with mutant forms of HB-EGF indicated that the release of soluble HB-EGF is essential for tumor formation. LPA, which is constitutively produced by ovarian cancer cells, induced HB-EGF ectodomain shedding in SKOV3 and RMG-1 cells, resulting in the transactivation of EGFR and the downstream kinase extracellular signal-regulated kinase/mitogen-activated protein kinase. LPA-induced transactivation was abrogated by HB-EGF gene RNA interference or by CRM197. Introduction of lipid phosphate phosphohydrolase, which hydrolyzes LPA, decreased the constitutive shedding of HB-EGF, EGFR transactivation, and the tumorigenic potential of SKOV3 and RMG-1 cells. These results indicate that HB-EGF is the primary member of the EGFR family of growth factors expressed in ovarian cancer and that LPA-induced ectodomain shedding of this growth factor is a critical step in tumor formation, making HB-EGF a novel therapeutic target for ovarian cancer.