Construction of Drosophila melanogaster lines knockout for Ldh and Gpo1 using the CRISPR technique

• Grant number: 23/10746-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2023
• End date: February 28, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Marcos Túlio de Oliveira
• Grantee: Hosana de Sousa Petita
• Host Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil
• Associated research grant: 21/06711-2 - Modulation of tissue growth and biomass accumulation by the mitochondrial alternative oxidase, AP.JP2

Abstract

The use of the fruit fly, Drosophila melanogaster, as a model organism has been increasingly targeted by the scientific community. Drosophilae have a rapid life cycle, making it possible to obtain different generations in a short period of time. In our laboratory, we have been studying the alternative oxidase enzyme AOX and the way in which its expression in drosophilae alters their metabolism during development. Unpublished unpublished data from our laboratory suggest the influence of AOX expression on two very important redox systems for larval development: lactate dehydrogenase (Ldh) and the glycerol-3-phosphate shuttle. Through lactic fermentation, Ldh utilizes the product of glycolysis, pyruvate, to form lactate and NAD+. The glycerol-3-phosphate shuttle, in turn, has a cytosolic component, the cytosolic enzyme glycerol-3-phosphate dehydrogenase, which transforms dihydroxyacetone into glycerol-3-phosphate, also oxidizing NADH to NAD+. The mitochondrial component, the enzyme mitochondrial glycerol-3-phosphate dehydrogenase, transforms glycerol-3-phosphate into dihydroxyacetone, sending the electrons directly to the respiratory chain. Both systems therefore contribute to the redox balance of the organism, allowing glycolysis to continue functioning by regenerating NAD+. Glycolytic metabolism is important during larval biomass accumulation, and also during tumor growth. Studies have shown that both tumor cells and drosophila larvae that have an inactivation of Ldh show a compensation with an increased performance of the glycerol-3-phosphate shuttle. Thus, constructing drosophila strains genetically edited for the genes encoding Ldh and the glycerol-3-phosphate shuttle is an interesting way to further understand the genetic and metabolic interactions of these enzymes with AOX. In D.melanogaster, a major challenge in constructing transgenic lines to create null mutants is that these mutations do not necessarily have any easily identifiable phenotype, which makes it difficult to visualize the success of the technique employed in the individual's genome. Thus, the co-CRISPR technique allows more than one mutational event to be made in the organism - in the gene of interest and in an easily identifiable phenotypic marker gene. Thus, we will employ the method developed by Kane and collaborators (2017) to construct deleterious strains for genes encoding Ldh and glycerol-3-phosphate shuttle with the ebony marker gene, which generates a dominant change in adult body coloration. In this sense, the main objective of the work is the construction of drosophila strains that are knockout for the genes Ldh (which encodes Ldh) and Gpo1 (which encodes the main isoform of mitochondrial glycerol-3-phosphate dehydrogenase) by means of the CRISPR technique. Specifically, the gRNAs (RNA fragments that direct the Cas9 enzyme to the portion of the DNA of interest to be cleaved) will be cloned to the intended targets in the pCFD3:U6:3-gRNA vector - for the design of the Ldh and Gpo1 targets we will use the FlyCrispr platform. After construction of the plasmid vectors, embryos of the y[1] M{w[+mC]=nos-Cas9.P}ZH-2A strain (BDSC 54591 - constitutively expressing Cas9 protein in the germ cell lineage) will be microinjected. The progeny of this lineage, which should indeed present the deletion of the genes of interest in its genome, will be characterized genetically and functionally. Finally, to prove the success of the technique, the animals will be sequenced.