Navegando por Palavras-chave "Reverse Genetics"

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    Avaliação da combinação de ferramentas de expressão gênica condicional em Plasmodium falciparum para estudos de genômica funcional e screening de potenciais antimaláricos
    (Universidade Federal de São Paulo (UNIFESP), 2020-09-04) Santos, Caroline Lima dos [UNIFESP]; Azevedo, Mauro Ferreira de [UNIFESP]; http://lattes.cnpq.br/1657599115711431; http://lattes.cnpq.br/6563769993198823; Universidade Federal de São Paulo
    Introduction: Despite the advances, malaria remains a major public health problem, leading to the death of thousands of people every year, mainly children under 5 years old. The study of essential genes for the parasite's life cycle is important for the development and validation of new antimalarials. Using reverse genetics, it has been possible to characterize important genes for the development of P. falciparum. It is common to use conditional gene expression to carry out these studies, but there’s still an expression leak that prevents a wider use. Methods for detecting the action of antimalarial drugs currently used take a long time to generate results, some of which are of low sensitivity and practicality, requiring the development of a fast method, of low cost and capable of being used on large scale. Objectives: To enhance conditional gene expression systems based on the combination of existing tools and to evaluate their application in assays for the detection of fast-acting antimalarials and in studies of essential genes. Methods: In order to evaluate the performance of conditional gene expression systems, plasmids were constructed with different combinations of these, being fused with Nluc-GFP, to assess regulation. Synchronized parasites in ring or trophozoite stages were grown in different combinations of the ligands used to regulate the systems and the results of gene expression determined by bioluminescence and fluorescence. For future functional analyzes of essential genes, strains were generated with some plasmids integrated in the genomic loci of interest. Results: The fusion of the conditional gene expression systems enhanced the induction capacity by up to 41x, with the 5D fusion being faster to induce in rings and the ribozyme glmS in trophozoites. Using the Nluc- 5D-glmS strain, which contains the 3 systems combined, as a method of detecting the action of antimalarials, it was possible to detect a 50% decay activity by the action of chloroquine in just 4 hours and to differentiate slow-acting antimalarials from fast-acting ones. In preliminary experiments, it was possible to observe a phenotypic effect in the parasites due to the modulation of the expression of the kinases CDPK1 and CDPK5 using some of the combinations of systems generated. Conclusion: Notably, when constructing a plasmid with different domains in fusion, its regulatory potential is multiplied and by using the parasites that express this construction it is possible to identify the action of drugs quickly, with high sensitivity and at low cost. You can also use them to conditionally knock out essential genes such as CDPK1 and CDPK5.