Desenvolvimento de um protocolo para microinjeção em insetos utilizando drosophila melanogaster como organismo modelo

Abstract

Drosophila melanogaster is one of the most used species in genetic experiments, being of extreme importance for biology. Over the last few years there has been an increase in the use of molecular tools that aid the development of biological studies. Several revolutionary studies with different model organisms have been carried out, from complete genome sequencing to the production of transgenic organisms using, for example, transposable elements. Transposable elements, also known as mobile DNA, are DNA sequences that move from one place to another in the genome. One of the most widely used systems in recent years is the transposon piggyBac, which features a wide range of hosts, ranging from insects to mammalian use. The technique most used to produce genetically modified insects is microinjection, which consists in the delivery of exogenous DNA directly into the nucleus of the target cell. Taking this in account, the objective of this work was to establish a microinjection protocol for the creation and development of transgenic insects using D. melanogaster as a model organism. The plasmid used to establish the microinjection protocol was the pBac[3xp3-EGFPattB]. The needles used in microinjection were made using four types of PC-10 configurations. To obtain eggs of D. melanogaster, we transferred the flies to oviposition media and two recipes were tested. In order to perform the dechorionation, two methods were tested: The first method was performed by using, 1% CaCl2 solution in distilled water was used. The second method, the eggs were bathed in distilled water and ethyl alcohol 92,8°. After these processes, 10 eggs were fixed on each microscope slide with transparent double-sided tape and covered with mineral oil. In each egg was injected 1 μL of pBac [3xp3-EGFPattB] plasmid (181 ηg/μL). 40 eggs were injected, 2 were developed and reached adulthood. The best results were obtained using needles with a diameter ranging from 45 μm. The two oviposition media were efficient. In the dechorionated, distilled water plus 92,8° alcohol presented better removal of the chorion, and naturol mineral oil was effective against desiccation. It was not possible to analyze the expression of the plasmid because the construct did not possess the transposase gene, which able the stability of exogenous DNA in the genome. Thus, our main objectives were achieved, establishing a low cost of consumables microinjection protocol. However, further testing will be required to aiming the expression of exogenous DNA into the model organism.