RESUMEN
The immunogenicity testing of oligonucleotide drugs using an antibody bridging assay has been scarcely investigated. We developed a highly sensitive antibody bridging assay model and assessed it using probe alteration link self-assembly reactions (PALSAR) technology as a signal amplifier. Methods: The concentration of each probe was optimized, and the bridging assay model was compared with and without signal amplification. Cut-point and analytical sensitivity were determined, and accuracy, precision and drug tolerance were evaluated. Results: The PALSAR bridging assay achieved a net signal 21-36 times higher than that obtained with the conventional method. The analytical sensitivity achieved was 48.8 ng/ml, with adequate accuracy, precision and drug tolerance. Conclusion: PALSAR technology is feasible for developing an antibody bridging assay using oligonucleotides as capture and detection probes.
Asunto(s)
Anticuerpos , Tecnología , Estudios de Factibilidad , Tolerancia a MedicamentosRESUMEN
A subset of genes in mammals are subject to genomic imprinting. The mouse H19 gene, for example, is active only when maternally inherited and the neighboring Igf2 gene is paternally expressed. This imprinted expression pattern is regulated by the imprinting control region (ICR) upstream of the H19 gene. A maternally inherited H19 ICR inhibits Igf2 gene activation by the downstream enhancer due to its insulator function while it suppresses H19 gene transcription by promoter DNA methylation when paternally inherited. These parent-of-origin specific functions depend on the allele-specific methylation of the ICR DNA, which is established during gametogenesis. Therefore, the ICR may also function as a landmark for epigenetic modifications. To examine whether the ICR confers these activities autonomously, we introduced a 2.9-kbp ICR-containing DNA fragment into a human beta-globin yeast artificial chromosome at the 3' end of the locus control region and established transgenic mouse lines. Expression of all of the beta-like globin genes was higher when the transgene was paternally inherited. In accord with this result, transgenic ICR DNA from nucleated erythrocytes was more heavily methylated when paternally transmitted. Chromatin immunoprecipitation assays confirmed that CCCTC binding factor is preferentially recruited to the maternal transgenic ICR in vivo. Surprisingly however, the parent-of-origin specific methylation pattern was not observed in germ cell DNA in testis, demonstrating that methylation was established after fertilization. Thus, the ICR autonomously recapitulated imprinting within the normally nonimprinted transgenic beta-globin gene locus, but the temporal establishment of imprinting methylation differs from that at the endogenous Igf2/H19 locus.
Asunto(s)
Metilación de ADN , Regulación de la Expresión Génica/genética , Impresión Genómica/genética , Globinas/genética , Animales , Inmunoprecipitación de Cromatina , Cromosomas Artificiales de Levadura/genética , Clonación Molecular , Eritrocitos/metabolismo , Femenino , Humanos , Patrón de Herencia/genética , Factor II del Crecimiento Similar a la Insulina , Masculino , Ratones , Ratones Transgénicos , Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/metabolismo , Activación TranscripcionalRESUMEN
The human beta-like globin genes (5'-epsilon-Ggamma-Agamma-delta-beta-3') are temporally expressed in sequential order from the 5' to 3' end of the locus, but the nonadult epsilon- and gamma-globin genes are autonomously silenced in adult erythroid cells. Two cis elements have been proposed to regulate definitive erythroid gamma-globin repression: the DR (direct repeat) and CCTTG elements. Since these two elements partially overlap, and since a well-characterized HPFH point mutation maps to an overlapping nucleotide, it is not clear if both or only one of the two participate in gamma-globin silencing. To evaluate the contribution of these hypothetical silencers to gamma-globin regulation, we generated point mutations that individually disrupted either the single DR or all four CCTTG elements. These two were separately incorporated into human beta-globin yeast artificial chromosomes, which were then used to generate gamma-globin mutant transgenic mice. While DR element mutation led to a dramatic increase in Agamma-globin expression only during definitive erythropoiesis, the CCTTG mutation did not affect adult stage transcription. These results demonstrate that the DR sequence element autonomously mediates definitive stage-specific gamma-globin gene silencing.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Globinas/genética , Regiones Promotoras Genéticas/fisiología , Secuencias Repetitivas de Ácidos Nucleicos/fisiología , Elementos de Respuesta/fisiología , Animales , Secuencia de Bases , Cromosomas Artificiales de Levadura/genética , Células Eritroides/metabolismo , Eritropoyesis/genética , Eritropoyesis/fisiología , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación Puntual/genética , Regiones Promotoras Genéticas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Elementos de Respuesta/genética , Transcripción Genética/genética , Transcripción Genética/fisiologíaRESUMEN
The function of putative regulatory sequences identified in cell transfection experiments can be elucidated only through in vivo experimentation. However, studies of gene regulation in transgenic mice (TgM) are often compromised by the position effects, in which independent transgene insertions differ in expression depending on their location in the genome. In order to overcome such a dilemma, a method called transgene coplacement has been developed in Drosophila melanogaster. In this method, any two sequences can be positioned at exactly the same genomic site by making use of Cre/loxP recombination. Here we applied this method to mouse genetics to characterize the function of direct repeat (DR) sequences in the promoter of the human angiotensinogen (hAGT) gene, the precursor of the vasoactive octapeptide angiotensin II. We modified a hAGT bacterial artificial chromosome to use Cre/loxP recombination in utero to generate TgM lines bearing a wild-type or a mutant promoter-driven hAGT locus integrated at a single chromosomal position. The expression analyses revealed that DR sequences contribute 50 or >95% to hAGT transcription in the liver and kidneys, respectively, whereas same sequences are not required in the heart and brain. This is the first in vivo dissection of DNA cis elements that are demonstrably indispensable for regulating both the level and cell type specificity of hAGT gene transcription.
Asunto(s)
Angiotensinógeno/genética , Marcación de Gen/métodos , Regiones Promotoras Genéticas , Recombinación Genética/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Angiotensinógeno/metabolismo , Animales , Secuencia de Bases , Línea Celular , Cromosomas Artificiales Bacterianos , Regulación hacia Abajo , Humanos , Integrasas/genética , Ratones , Datos de Secuencia Molecular , Mutación/genética , Distribución Tisular , Transfección , Transgenes , Proteínas Virales/genéticaRESUMEN
The human beta-globin locus contains five developmentally regulated beta-type globin genes. All five genes depend on the locus control region (LCR), located at the 5' end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I-hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human beta-globin yeast artificial chromosome transgenic mice and observed reduced expression of all the beta-type globin genes regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. Here, we describe genetic evidence demonstrating that human HS5 includes enhancer-blocking (insulator) activity that is both CTCF and developmental stage dependent. Curiously, we also observed an attenuating activity in HS5 that was specific to the epsilon-globin gene at the primitive stage and was independent of the HS5 CTCF binding site. These observations demonstrate that the phenotype observed in the LCR-inverted locus was in part attributable to placing the HS5 insulator between the LCR HS enhancers (HS1 to HS4) and the promoter of the beta-globin gene.