Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level.
Sci Rep
; 5: 7729, 2015 Jan 12.
Article
en En
| MEDLINE
| ID: mdl-25578556
ABSTRACT
Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Análisis de la Célula Individual
/
Degradación de ARNm Mediada por Codón sin Sentido
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Sci Rep
Año:
2015
Tipo del documento:
Article
País de afiliación:
Rusia