RESUMEN
Familial amyloidotic polyneuropathy (FAP) is caused by a mutation in the transthyretin (TTR) gene. In addition, deposition of wild-type TTR can cause senile systemic amyloidosis (SSA). To date, we have produced several transgenic mouse models for FAP and SSA by introducing TTR genes with different promoters or mutations. However, mouse TTR can associate with human TTR to produce hybrid tetramers in transgenic mice. Thus, these transgenic mice cannot be used to test the efficacy of a new therapy. In this study, we attempted to construct an optimized mouse model to verify a new therapy. The TTR gene consists of 4 exons and 3 introns. We prepared two gRNAs, one for the exon 1 and the other for exon 4, and a single donor vector carrying the whole TTR gene in which mouse exons were replaced with human exons. Using these vectors, we produced a TTR exon-humanized mouse with human exons and mouse introns using genome editing technology. These TTR exon-humanized mice showed normal TTR expression patterns in terms of serum TTR level and spatial specificity. These TTR exon-humanized mice will be useful for devising new treatment methods for FAP, including gene therapy.
Asunto(s)
Polineuropatías/etiología , Prealbúmina/genética , Animales , Modelos Animales de Enfermedad , Exones , Regulación de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Polineuropatías/terapia , Prealbúmina/análisis , ARN Guía de Kinetoplastida/genéticaRESUMEN
To produce a humanized mouse, it is critical to obtain a correct expression of a human gene/cDNA after insertion into a mouse locus. We previously generated a targeted allele in which the PGK-neo cassette, flanked by lox71 and loxP, was inserted into the first exon of the mouse endogenous transthyretin (Ttr) gene in ES cells. Using these ES cells, we showed that a human transthyretin (TTR) cDNA with the PGK-puro cassette can be efficiently inserted into this locus by Cre-mediated recombination, and that the human TTR cDNA was expressed in a tissue-specific manner under the control of the mouse endogenous Ttr promoter. To examine whether the PGK-puro cassette or IRES could affect the expression of human TTR cDNA, we generated four mouse lines using Cre and Flp-mediated recombination. The mouse line containing the PGK-puro cassette, but not IRES, exhibited quantitatively and temporally similar expression of human TTR cDNA. Removal of the PGK-puro cassette significantly downregulated the expression of the cDNA. The insertion of IRES sequence upstream of the human TTR cDNA resulted in decreased expression, even in the presence of the PGK-puro cassette. The mouse line containing IRES, but not PGK-puro, showed the lowest level of expression. These results suggest that the PGK-puro cassette is necessary to obtain the enhanced expression of a co-existing human cDNA in the mouse Ttr locus, even though the expression of co-existing cDNA was under the control of the mouse endogenous promoter.
Asunto(s)
ADN Complementario/metabolismo , Regulación de la Expresión Génica , Fosfoglicerato Quinasa/genética , Prealbúmina/metabolismo , Regiones Promotoras Genéticas/genética , Puromicina/farmacología , Animales , Células Cultivadas , ADN Complementario/genética , Ingeniería Genética/métodos , Humanos , Integrasas/metabolismo , Hígado/metabolismo , Ratones , Fosfoglicerato Quinasa/metabolismo , Prealbúmina/genética , Recombinación Genética , Ribosomas/metabolismoRESUMEN
Human transthyretin (TTR) has about 110 variants, more than 90 of which are associated with human amyloidosis. Several groups have generated transgenic mice that carry various mutant TTR genes. However, formation of mouse/human TTR heterotetramers has been shown to be inhibitory to dissociation and subsequent amyloid formation. To avoid the effect of mouse Ttr and produce humanized mice carrying different TTR variants at high efficiency, we first produced a null allele in the mouse transthyretin locus using targeting vector that contained a neomycin resistance gene flanked by lox71 and loxP. Then, through Cre-mediated recombination, we created a replacement allele that carried either a human normal (Val30) or mutant (Met30) TTR cDNA. This replacement resulted in a humanized TTR mouse with similar tissue-specific profile of human TTR as that of the endogenous mouse Ttr gene. The expression levels of human TTR mRNA and protein in the liver of homozygous human TTR (Val30/Val30) mice were about twice those of heterozygous mouse/human TTR (+/Val30) mice. However, the serum human TTR levels in the Val30/Val30 mice were much less than those in the +/Val30 mice. This contradictory expression was due to unstable Val30 tetramers caused by low binding affinity to mouse retinol binding protein.
Asunto(s)
Regulación de la Expresión Génica , Hígado/metabolismo , Prealbúmina/genética , Prealbúmina/metabolismo , Alelos , Animales , Línea Celular , ADN Complementario/genética , Femenino , Humanos , Concentración de Iones de Hidrógeno , Hígado/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Especificidad de Órganos , Unión Proteica , Multimerización de ProteínaRESUMEN
Ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) is an important initial step for the production of ribosomes. The RNA polymerase 1-2 (Rpo1-2) gene is comprised of 15 exons and encodes 1135 amino acids (aa) of the second largest subunit in Pol I. In a gene trap screen, we have identified an insertional mutation (Rpo1-2(Gt)) in the 14th exon of Rpo1-2, resulting in a truncation of 312aa from the C-terminal. In Rpo1-2(Gt/Gt) embryos, the synthesis of rRNA was severely impaired. Rpo1-2(Gt/Gt) embryos could develop to the morula stage, and thereafter displayed nucleolus disruption and apoptotic cell death. These results indicate that the loss of rDNA transcription induced nucleolar structure disorganization and apoptosis in preimplantation embryos.
Asunto(s)
Blastocisto/fisiología , Predisposición Genética a la Enfermedad/embriología , Predisposición Genética a la Enfermedad/genética , ARN Polimerasa II/genética , ARN Polimerasa I/genética , Animales , Ratones , Mutación , Análisis de Supervivencia , Tasa de SupervivenciaRESUMEN
Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.
Asunto(s)
Células Germinativas/crecimiento & desarrollo , Ratones/embriología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Expresión Génica , Genotipo , Células Germinativas/citología , Células Germinativas/metabolismo , Gónadas/metabolismo , Inmunoquímica , Masculino , Ratones/metabolismo , Mutación/genética , Placenta/anatomía & histología , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Estrógenos/genéticaRESUMEN
Importin beta1 (Impbeta)/karyopherin beta1 (Kpnb1) mediates the nuclear import of a large variety of substrates. This study aimed to investigate the requirement for the Kpnb1 gene in mouse development, using a gene trap line, B6-CB-Ayu8108(GtgeoIMEG) (Ayu8108(geo)), in which the trap vector was inserted into the promoter region of the Kpnb1 gene, but in reverse orientation of the Kpnb1 gene. Ayu8108(geo/geo) homozygous embryos could develop to the blastocyst stage, but died before embryonic day 5.5, and expression of the Kpnb1 gene in homozygous blastocysts was undetectable. We also replaced the betageo gene with Impbeta cDNA through Cre-mediated recombination to rescue Impbeta expression. Homozygous mice for the rescued allele Ayu8108(Impbeta/Impbeta) were born and developed normally. These results demonstrated that the cause of post-implantation lethality of Ayu8108(geo/geo) homozygous embryos was impaired expression of the Kpnb1 gene, indicating indispensable roles of Impbeta1 in early development of mice.
Asunto(s)
Implantación Tardía del Embrión/fisiología , Pérdida del Embrión , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Nucleares/deficiencia , Animales , Línea Celular , Supervivencia Celular , Células Cultivadas , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Tasa de Supervivencia , beta CarioferinasRESUMEN
BACKGROUND & AIMS: Serine protease inhibitor Kazal type 1 (SPINK1), which is structurally similar to epidermal growth factor, is thought to inhibit trypsin activity and to prevent pancreatitis. Point mutations in the SPINK1 gene seem to predispose humans to pancreatitis; however, the clinical significance of SPINK1 mutations remains controversial. This study aimed to elucidate the role of SPINK1. METHODS: We generated Spink3-deficient (Spink3(-/-)) mice by gene targeting in mouse embryonic stem cells. Embryonic and neonatal pancreases were analyzed morphologically and molecularly. Specific probes were used to show the typical autophagy that occurs during acinar cell death. RESULTS: In Spink3(-/-) mice, the pancreas developed normally up to 15.5 days after coitus. However, autophagic degeneration of acinar cells, but not ductal or islet cells, started from day 16.5 after coitus. Rapid onset of cell death occurred in the pancreas and duodenum within a few days after birth and resulted in death by 14.5 days after birth. There was limited inflammatory cell infiltration and no sign of apoptosis. At 7.5 days after birth, residual ductlike cells in the tubular complexes strongly expressed pancreatic duodenal homeodomain-containing protein 1, a marker of pancreatic stem cells, without any sign of acinar cell regeneration. CONCLUSIONS: The progressive disappearance of acinar cells in Spink3(-/-) mice was due to autophagic cell death and impaired regeneration. Thus, Spink3 has essential roles in the maintenance of integrity and regeneration of acinar cells.
Asunto(s)
Muerte Celular/fisiología , Páncreas/citología , Pancreatitis/patología , Inhibidor de Tripsina Pancreática de Kazal/metabolismo , Animales , Biopsia con Aguja , Western Blotting , Células Cultivadas , Ceruletida , ADN/análisis , Modelos Animales de Enfermedad , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pruebas de Función Pancreática , Pancreatitis/metabolismo , Fagocitosis/fisiología , ARN/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Inhibidores de Serina Proteinasa/metabolismoRESUMEN
We have developed a new exchangeable gene trap vector, pU-17, carrying the intron-lox71-splicing acceptor (SA)-beta geo-loxP-pA-lox2272-pSP73-lox511. The SA contains three stop codons in-frame with the ATG of beta galactosidase/neomycin-resistance fusion gene (beta geo) that can function in promoter trapping. We found that the trap vector was highly selective for integrations in the introns adjacent to the exon containing the start codon. Furthermore, by using the Cre-mutant lox system, we successfully replaced the beta geo gene with the enhanced green fluorescent protein (EGFP) gene, established mouse lines with the replaced clones, removed the selection marker gene by mating with Flp-deleter mice, and confirmed that the replaced EGFP gene was expressed in the same pattern as the beta geo gene. Thus, using this pU-17 trap vector, we can initially carry out random mutagenesis, and then convert it to a gain-of-function mutation by replacing the beta geo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre-mediated recombination.