RESUMEN
The introduction of germ line modifications by gene targeting in mouse embryonic stem (ES) cells has proven a fundamental technology to relate genes to mammalian biology. Critical aspects required for successful gene targeting have traditionally been experimental enhancements that increase the frequency or detection of homologous recombination within ES cells; however, the utilization of such methods may still result in the failed isolation of a positively targeted ES cell clone. In this study, we discuss the current enhancement methods and describe an ES cell pooling strategy that maximizes the ability to detect properly targeted ES cells regardless of an inherent low targeting efficiency. The sensitivity required to detect correctly targeted events out of a pool of ES cell clones is provided by polymerase chain reaction (PCR), and only those pools containing positives need to be expanded and screened to find individually targeted clones. This method made it possible to identify targeted clones from a screen of approximately 2,300 ES cell colonies by performing only 123 PCR reactions. This technically streamlined approach bypasses the need to troubleshoot and re-engineer an existing targeting construct that is functionally suitable despite its low targeting frequency.
RESUMEN
Periodontal disease is one of the most prevalent chronic inflammatory diseases. There is a genetic component to susceptibility and resistance to this disease. Using a mouse model, we investigated the progression of alveolar bone loss by gene expression profiling of susceptible and resistant mouse strains (BALB/cByJ and A/J, respectively). We employed a novel and sensitive quantitative real-time PCR method to compare basal RNA transcription of a 48-gene set in the gingiva and the spleen and the subsequent changes in gene expression due to Porphyromonas gingivalis oral infection. Basal expression of interleukin-1 beta (Il1b) and tumor necrosis factor alpha (Tnf) mRNA was higher in the gingiva of the susceptible BALB/cByJ mice than in the gingiva of resistant A/J mice. Gingival Il1b gene expression increased further and Stat6 gene expression was turned on after P. gingivalis infection in BALB/cByJ mice but not in A/J mice. The basal expression of interleukin-15 (Il15) in the gingiva and the basal expression of p-selectin (Selp) in the spleen were higher in the resistant A/J mice than in the susceptible BALB/cByJ mice. In the resistant A/J mice the expression of no genes detectably changed in the gingiva after infection. These results suggest a molecular phenotype in which discrete sets of differentially expressed genes are associated with genetically determined susceptibility (Il1b, Tnf, and Stat6) or resistance (Il15 and Selp) to alveolar bone loss, providing insight into the genetic etiology of this complex disease.
Asunto(s)
Pérdida de Hueso Alveolar/inmunología , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Encía/metabolismo , Porphyromonas gingivalis/patogenicidad , Proteínas/metabolismo , Pérdida de Hueso Alveolar/genética , Pérdida de Hueso Alveolar/microbiología , Animales , Infecciones por Bacteroidaceae/genética , Infecciones por Bacteroidaceae/inmunología , Infecciones por Bacteroidaceae/microbiología , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Enfermedades de la Boca/genética , Enfermedades de la Boca/microbiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas/genética , Bazo/metabolismoRESUMEN
Two-dimensional electrophoresis (2DE) of DNA fragments, in which separation occurs first by size and then by sequence variation, is a method enabling large-scale comparison of complex genomes. Combining 2DE with probing for various classes of repetitive genomic elements allows rapid and efficient comparison of thousands of fragments and millions of base-pairs of DNA distributed across most genomic regions. This approach is demonstrated here by analyzing the extent of genomic relatedness of different inbred strains of mice. Such strains are shown to differ from each other by approximately 0.2-1% of their nucleotides, above which level reproductive speciation occurs. The 2DE method of assessing the overall relationship between two genomes represents an appropriate tool for analyzing members of a single species, but is too sensitive for use in interspecies comparisons.
Asunto(s)
ADN/análisis , Electroforesis en Gel Bidimensional , Animales , ADN/genética , Marcadores Genéticos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Repeticiones de Minisatélite , Muridae/genética , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Transgenic mice bearing a c-myc oncogene under control of the immunoglobulin heavy chain (Igh) enhancer (Eu-myc mice) (1, reviewed in 2) undergo a reproducible series of developmental stages and die from malignancies of the B lymphocyte lineage. To investigate the cellular events underlying tumorigenesis in this model, we quantified B lymphoid subpopulations and turnover at various stages of this process. An early stage was characterized by the presence in the blood of many large proliferating B lineage cells marked by surface antigen phenotype IgM+l-, B220low, CD5-, Mac-1low. During a prolonged intermediate 'remission' phase of different duration in each mouse, B lymphocytes in the periphery were non-proliferative, few, and of conventional phenotype (IgM+, B220+, CD5-, Mac-1-), while subsets of precursor B cells were both numerous and highly proliferative in the bone marrow. In the final stage of tumorigenesis, large proliferating cells similar in phenotype to those of the early period reappeared and increased rapidly in numbers. This B cell tumorigenic progression occurred independently of interactions with T lymphocytes. Evidence of massive cell death in the bone marrow during the intermediate phase, plus molecular characterization of the final tumors, suggested that the end of the peripheral 'remission' period and entry into the terminal stage of tumorigenesis may be due to a clone of cells acquiring the ability to circumvent normal processes of cell death and elimination that usually regulate the egress of B cells from the bone marrow to the periphery.
Asunto(s)
Genes myc , Leucemia de Células B/patología , Linfoma de Células B/patología , Factores de Edad , Animales , Linfocitos B/patología , Secuencia de Bases , Médula Ósea/patología , Transformación Celular Neoplásica , Elementos de Facilitación Genéticos , Femenino , Reordenamiento Génico de Cadena Pesada de Linfocito B/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/genética , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Microscopía Electrónica , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Linfocitos T/patología , Factores de TiempoRESUMEN
Denaturing gradient gel electrophoresis (DGGE) is based upon the different melting behaviors of DNA molecules in a chemical denaturant gradient according to their sequences. This technique has recently become a widespread tool to detect mutations. The introduction of a GC-clamp enables the detection of most single base differences between two DNA molecules. As a test system we have applied the polymerase chain reaction (PCR) in combination with DGGE to detect a number of mutations in the mouse H2Kb DNA sequence. A wide variety of spontaneous in vivo mutations of this haplotype have been reported in the C57BL/6J mouse strain and are clustered in the alpha 1 and alpha 2 domains. The combination of PCR and DGGE revealed almost all base changes present in the H2Kb mutants used. However, most of the PCR products of these mutants showed melting behavior which is not easily predicted. We suggest that in addition to current simple theory, which considers that the migration of a DNA molecule in a denaturing gradient depends primarily on its initial melting behavior, additional factors such as secondary structure in partially melted molecules may play a role and can be used to detect mutations.
Asunto(s)
ADN/genética , Electroforesis en Gel de Poliacrilamida/métodos , Mutación , Animales , Secuencia de Bases , ADN/aislamiento & purificación , Haplotipos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
We analyzed embryonal carcinoma cell lines infected with a recombinant Moloney murine leukemia virus. Lines that carried but did not express the neo gene retained a provirus of LTR-gag-pol-neo-LTR, where LTR is a long terminal repeat, whereas all G418-resistant lines deleted regions that included the primer binding site and the splicing donor site. This suggested the presence of multiple inhibitory elements.