RESUMEN
Angelman syndrome (AS) is a neurogenetic disorder characterized by severe mental retardation, ataxia, seizures, EEG abnormalities and bouts of inappropriate laughter. AS individuals fail to inherit a normal active maternal copy of ubiquitin protein ligase E3A (UBE3A). UBE3A is subject to genomic imprinting, with predominant transcription of the maternal allele in brain. The known genetic causes of AS are maternal deletion of chromosome 15q11-q13, paternal chromosome 15 uniparental disomy, UBE3A mutation and an abnormality of the imprinting process, termed imprinting defect. There remain major questions concerning the molecular pathogenesis of AS, including: 1) the mechanisms underlying the imprinting defect class of AS, 2) the identity of proteins targeted by UBE3A, 3) the role of a noncoding antisense transcript in regulating UBE3A imprinting and 4) the contribution of other genes such as methyl-binding CpG-binding protein 2 and gamma-aminobutyric acid A receptor, subunit beta3 to the AS phenotype.
Asunto(s)
Síndrome de Angelman/genética , Ubiquitina-Proteína Ligasas/genética , Síndrome de Angelman/diagnóstico , Animales , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Encéfalo/metabolismo , Epigénesis Genética , Impresión Genómica , Humanos , Síndrome de Prader-Willi/genética , ARN sin Sentido/metabolismo , Técnicas Reproductivas Asistidas/efectos adversosRESUMEN
The detection of specific RNA molecules in situ is routinely performed using haptenated probes, which are detected by either enzymatic amplification or direct fluorescence. A drawback of fluorescence labeling has been the reduced sensitivity relative to that of methods that use enzymes as signal generators. Reliable fluorescence detection methods often require the use of multiple oligonucleotide probes for each gene target. Here, we demonstrate that single haptenated DNA probes specific for actin mRNA may be detected in situ using antibody-coupled rolling circle amplification (immuno-RCA). This fluorescence-based detection method offers remarkable sensitivity due to the use of signal amplification and yet retains the ability to count hybridization signals as discrete objects. We demonstrate the detection of actin-specific immuno-RCA signals in the cytoplasm and use 3D image deconvolution of multiple z axis sections to show that there are hundreds of signals per cell. With some modifications, this method may be adaptable to the simultaneous detection of several RNA species, including low-copy-number mRNA.