RESUMO
Modern experimental techniques, such as whole-genome sequencing and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endogenous genome editing, are enabling researchers to identify and further characterize the roles of proteins that were previously thought of as well defined. In the December 2016 issue of GENETICS, an article by Jaramillo-Lambert et al. identified a new role for the enzyme topoisomerase II in Caenorhabditis elegans male meiosis. This Primer article is designed to provide essential background information on C. elegans spermatogenesis and the relevant scientific techniques that will assist students and instructors in their understanding and discussion of the related article.Related article in GENETICS: Jaramillo-Lambert, A., A. S. Fabritius A. S., T. J. Hansen T. J., H. E. Smith H. E., and A. Golden A., 2016 The identification of a novel mutant allele of topoisomerase II in Caenorhabditis elegans reveals a unique role in chromosome segregation during spermatogenesis. Genetics204: 1407-1422.
Assuntos
Alelos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Mutação , Espermatogênese/genética , Animais , Sistemas CRISPR-Cas , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , DNA Topoisomerases/genética , DNA Topoisomerases/metabolismo , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas , Miose/genética , Oogênese/genética , Interferência de RNA , Sequenciamento Completo do GenomaAssuntos
Terapia Genética/métodos , Leishmaniose/terapia , Proteínas de Membrana/antagonistas & inibidores , Interferência de RNA/fisiologia , RNA Interferente Pequeno/antagonistas & inibidores , Regulação para Baixo/genética , Humanos , Leishmaniose/genética , Leishmaniose/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Peroxinas , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: The early secreted antigenic target 6-kDa protein (ESAT-6) of Mycobacterium tuberculosis (Mtb) not only acts as a key player for virulence but also exhibits a strong immunotherapeutic potential against Mtb. However, little is known about the molecular basis for its potential in immunotherapy. The present study was designed to unravel the role of miRNA-155 in ESAT-6-mediated enhancement of host immunity and apoptosis in macrophages. METHODS: Lentivirus-mediated miR-155 sponge and miR-155 and SOCS1 overexpression vectors were developed in macrophages. TLR2- or p65-specific siRNA knockdown was employed to silence TLR2 or p65. Quantitative polymerase chain reaction and western blotting analyses were performed to determine mRNA and protein expression levels, respectively. Macrophage apoptosis was analyzed by flow cytometry. RESULTS: ESAT-6 significantly increased miR-155 expression, which was dependent on TLR2/NF-κB activation in macrophages. Induced expression of miRNA-155 was required for the ESAT-6-mediated protective immune response and macrophage apoptosis. ESAT-6 promoted macrophage apoptosis by targeting the miR-155-SOCS1 pathway. The differential expression levels of TLR2, BIC, and SOCS1 were involved in regulating the immune response in human peripheral blood mononuclear cells of patients with active tuberculosis (TB) and latent TB (LTB). CONCLUSION: ESAT-6 promotes apoptosis of macrophages via targeting the miRNA155-SOCS1 interaction.
Assuntos
Antígenos de Bactérias/farmacologia , Apoptose/efeitos dos fármacos , MicroRNAs/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Tuberculose/patologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Linhagem Celular , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Interferência de RNA , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Supressoras da Sinalização de Citocina/genética , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Tuberculose/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis. The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.