Development of transgenic lines of transgenic fish for the growth hormone (GH) gene using the Zebrafish Danio rerio (Cypriniformes; Cyprinidae) as an experimental model

Author: Marcio de Azevedo Figueiredo (Currículo Lattes)
Supervisor: Dr Luis Fernando Fernandes Marins

Abstract

The objective of this work was to produce transgenic germ lines from São Paulo (Danio rerio) for the growth hormone (GH) gene through the co-injection technique using the fluorescent green protein marker (GFP) gene. To achieve this objective, in the first part of this work, the conditions to be used for the production of transgenic fish were determined. For this, two different concentrations of the genetic construct containing the GFP gene associated with the carp -actin promoter (Cyprinus carpio) were microinjected into freshly fertilized eggs. The results obtained showed that the concentration of 18.3 ng / L was more efficient in the production of transgenic individuals in relation to the concentration of 3.7 ng / L. In the second part of this study,the GH gene co-injection strategy with the marker gene was used to assess the degree of mosaicism in vivo and to identify transgenic paulistinhas with the potential to generate germ lines for the gene of interest. The two constructions, regulated by the same promoter, were co-injected linearized in an equimolar ratio (1: 1). The GH gene used was that of the sea kingfish Odonthestes argentinensis (msGH). After hatching, the larvae were observed under an epifluorescence microscope, and those that showed a strong expression for GFP were cultured until sexual maturity, when they were reproduced with non-transgenic individuals. G1 fish positive for GFP expression were analyzed by genomic DNA PCR for the presence of the msGH gene.This allowed the identification of two G0 individuals (M0104 and F0104) who transmitted the two transgenes to G1. G1 animals that carried both transgenes were bred with non-transgenics. The G2 analysis showed that in the M0104 line the transgenes integrated into different chromosomes, and that in the F0104 line the two transgenes integrated into the same chromosome. In the third part of this work, growth experiments were carried out between homozygous, hemizygous and non-transgenic F0104 strains. The results of these experiments demonstrated that the hemizygous animals showed a significantly higher growth than the others, which can be explained by the significant increase in the expression of the IGF-I (insulin-like growth factor I) gene observed in this class.Although homozygous transgenics showed, as expected, a greater expression of the msGH gene, the expression of the IGF-I gene was not detected. This fact may explain the results of the growth experiments. The methodology applied in the present study allowed inferences about the integration events of the transgenes used, enabling the identification of a lineage that was transmitting both transgenes linked on the same chromosome. In the experimental model developed here, hemizygous transgenic fish proved to be more viable for cultivation. These results may, in the future, be applied to the production of genetically modified lines of commercially important fish species with higher growth performance.

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