RESUMEN
To quantify the transcription level of a gene, we have conceived a novel concept, transcription level of messenger RNA (mRNA) per gene copy, which was determined with a dual-spike-in strategy. In this strategy, an exogenous DNA was added as the spike reference for target DNA in addition to the exogenous RNA as the reference for target RNA. After the mRNA-to-DNA ratio of a target gene was estimated by real-time polymerase chain reaction (PCR), it was first normalized with the mRNA-to-DNA ratio of the exogenous reference. The normalized ratio was multiplied by the ratio of exogenous RNA to exogenous DNA to obtain the transcription level of mRNA per gene copy. This quantified transcription value allows one to compare the expression of a target gene in different tissues or the expression in a specified tissue under different conditions.
Asunto(s)
Dosificación de Gen , ARN Mensajero/genética , Transcripción Genética , Animales , Bombyx , ADN/genética , Cartilla de ADN/química , Larva/genética , Reacción en Cadena de la Polimerasa , ARN/análisis , ARN/genética , ARN Mensajero/análisis , ARN Ribosómico 28S/análisis , ARN Ribosómico 28S/metabolismo , Estándares de ReferenciaRESUMEN
Cassettes harboring luciferase reporter driven by Bombyx mori cytoplasmic actin gene promoter (A3) (671 bp) and B. mori nuclear polyhedrosis virus immediate-early promoter (IE-1) (580 bp) were transferred to the bacmid AcDeltaEGT to generate the recombinant Autographa californica nuclear polyhedrosis viruses, AcNPVA3Luc and AcNPVIELuc, respectively. Recombinant baculoviruses were injected into the hemocoele of newly ecdysed 5th instar larvae. The activities of the A3 and IE-1 promoters in various tissues were measured by luciferase activity assay and normalized by the copy number of recombinant virus. Results showed that the activity of the A3 promoter was approximately 10-fold higher than the IE-1 promoter. The promoter activities of A3 and IE-1 were highest in the silk gland, followed by fat body, middle gut, malpighian tubule, and hemocyte. In silk gland, activity of the two promoters was highest in posterior silk gland, followed by middle and anterior silk glands. The difference in promoter activities reflects the growth speed of tissue in silkworm larvae. The activity of the A3 promoter remained unchanged and was not inhibited significantly by viral factors at least 3-4 d post injection of rAcNPV.
Asunto(s)
Actinas/metabolismo , Bombyx/fisiología , Vectores Genéticos/genética , Nucleopoliedrovirus/genética , Regiones Promotoras Genéticas/genética , Ingeniería de Proteínas/métodos , Transfección/métodos , Actinas/genética , Animales , Citoplasma/genética , Citoplasma/metabolismo , Mejoramiento Genético/métodos , Proteínas Recombinantes/genéticaRESUMEN
A cassette harboring luciferase reporter driven by Bombyx mori A3 promoter was transferred to the bacmid AcDeltaEGT to generate the recombinant virus AcNPVA3Luc (where Ac represents Autographa californica, NPV represents nucleopolyhedrovirus, and A3Luc represents the firefly luciferase reporter cassette driven by the A3 promoter). Recombinant baculovirus was injected into the hemocoele of newly ecdysed fifth instar larvae of the silkworm. The infection of virus in various silkworm tissues was determined by real-time PCR. The profile of viral infection showed that the copy number of recombinant AcNPV (rAcNPV) increased the fastest in the hemocyte, followed by the fat body, Malpighian tubule, middle gut, and silk gland. Detecting in nonpermissive strain silkworm showed that there was no significant difference in the entry of rAcNPV into all tested tissues. The difference in viral infection reflected mainly the big difference in replication of rAcNPV in various tissues of silkworm larvae. Real-time quantitative RT-PCR showed that it was due to the different expression of genes involved in viral DNA replication.
