Expression of GFP gene in embryos and larvaе Dania rerio fishes after microinjection of genetic vectors with different promoters

The paper deals with the problems associated with obtaining Danio rerio transgenic embryos and larvae with the expression of the green fluorescent gene (GFP) after the introduction of genetic constructs with different promoters. Despite the fact that the optimal stage for obtaining transgenic organisms is a zygote, in the pronuclei of which DNA synthesis is actively proceeding, in fish, embryos contain a yolk that masks these pronuclei. The vectors initially fall into the yolk part of the embryo, which does not allow foreign genetic material to enter the period of the first round of DNA replication, therefore, in most cases, these species show a mosaic type of transgene integration and expression. As a rule, fully transgenic fish are obtained by crossing the offspring F1 or F2, in the germ cells of which the transgene was integrated. In our experiments, two types of promoters were used: in one vector, the fluorescent green protein was under the control of the human elongation factor promoter (plasmid pCEEGFP), in the other, under the control of the chicken beta actin promoter (plasmid pCX-eGFP).To obtain transgenic embryos and Danio rerio larvae, in the blastodisc area, 15-20 minutes after fertilization, a microinjector was used to microinject genetic constructs containing the GFP gene. The developmental stages and analysis of the expression of the fluorescent gene were performed using a Lumar- 12 stereomicroscope from Zeiss. 372 embryos were analyzed. After the introduction of two different genetic constructs in most embryos (from 80 to 88%), expression of the fluorescent gene is observed on the fourth day of development, and only about 30% of embryos have a mosaic pattern of expression on the second day of development only after microinjection of the recombinant plasmid pCEEEGFP. It is noteworthy that in more than 70% of embryos, expression of the fluorescent gene was detected in the area of the digestive system. The initial stages of GFP gene expression differed in fish embryos obtained after the introduction of different vectors: the earliest expression was detected at the epibolia stage (5 hours of development) using the pCEEGFP construct. After maintaining the pCX-EGFP vector, expression first appeared on the third to fourth day of development. Most often, the localization of the fluorescence core was located in the region of the digestive system, as well as in separate clusters of myocytes and epithelial cells. Thus, the initial stages of expression of the GFP gene differed in fish embryos obtained after the introduction of different vectors. Transgenic fish are widely used. The models of transgenic fish allow in vivo to identify regulatory DNA sequences, as well as to determine the functions of genes, and serve as indicators for identifying pollutants in the environment, they can be used for screening drugs. Danio rerio transgenic fish are used as model organisms in the development of a number of human diseases, because most genes and their regulation to control the development of tissues and organs are conservative.

GFP, Danio rerio, transgenic animals, plasmids, reporter genes, gene expression, GFP

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