RESUMEN
Reversely transfected cell microarrays (RTCM) have been introduced as a method for parallel high throughput analysis of gene functions in mammalian cells. Hundreds to thousands of different recombinant DNA or RNA molecules can be transfected into different cell clusters at the same time on a single glass slide with this method. This allows either the simultaneous overexpression or--by using the recently developed RNA interference (RNAi) techniques--knockdown of a huge number of target genes. A growing number of sophisticated detection systems have been established to determine quantitatively the effects of the transfected molecules on the cell phenotype. Several different cell types have been successfully used for this procedure. This review summarizes the presently available knowledge on this technique and provides a laboratory protocol.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Genes/genética , Genes/fisiología , Transcripción Reversa/genética , Análisis de Matrices Tisulares/métodos , Transfección/métodos , Animales , Células Cultivadas , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Silenciador del Gen , HumanosRESUMEN
Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma. We present a localization map of 85 HHV-8-encoded proteins in mammalian cells. Viral open reading frames were cloned with a Myc tag in expression plasmids, confirmed by full-length sequencing, and expressed in HeLa cells. Protein localizations were analyzed by immunofluorescence microscopy. Fifty-one percent of all proteins were localized in the cytoplasm, 22% were in the nucleus, and 27% were found in both compartments. Surprisingly, we detected viral FLIP (v-FLIP) in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally localized exclusively in the cytoplasm. This suggested that v-FLIP may exert additional or alternative functions compared to cellular FLIPs. In addition, it has been shown recently that the K10 protein can bind to at least 15 different HHV-8 proteins. We noticed that K10 and only five of its 15 putative binding factors were localized in the nucleus when the proteins were expressed in HeLa cells individually. Interestingly, in coexpression experiments K10 colocalized with 87% (13 of 15) of its putative binding partners. Colocalization was induced by translocation of either K10 alone or both proteins. These results indicate active intracellular translocation processes in virus-infected cells. Specifically in this framework, the localization map may provide a useful reference to further elucidate the function of HHV-8-encoded genes in human diseases.
