Tez özetleri Astronomi ve Uzay Bilimleri Anabilim Dalı

A Study On The Gene (Gfp) Transfer İn Fertilized Zebrafish (Danio Rerio Hamilton-Buchanan, 1822) Eggs

This study was carried out at the TUBITAK-MAM Gene Engineering and Biotechnology Institute, Transgene and Experimental Animals Laboratory. The aim of this study is to set up transgenic fish produce technology in Turkey. In this study enhancer green flourescent protein (EGFP) gene used as a marker gene. To produce transgenic fish circular plasmid (pEGFP-N1) and linear forms of EGFP gene construct, which was containing cyto megalo virus (CMV) promoter has been transferred in to fertilized eggs of zebrafish by microinjection technique.

In microinjection, the EGFP gene constructs, 2,5-4,5-35-40-65 ng/μl consantration of linear form and 65-100 ng/μl consantration of circular form, were transferred into cytoplasm of zebrafish embryos at single-cell stage. At the third days of gene transferred larvae, gene expression was observed at the flourescent microscope and green larvae were detected. Also, flourescence and non-flourescence transgenic candidate larvae were investigated according to gene integration and expression with the same molecular methods. We obtained 54 F₀ zebrafish after linear EGFP gene microinjection and 25 F₀ zebrafish after circular form microinjection. We observed that the gene expression efficiency of circular form in 65 ng/µl consantration was higher than at the same consantration of the linearized form in F₀ zebrafish larvae. The expression of the circular EGFP gene construct was observed ubiquites distributed at different parts of body (yolk sack, head, dorsal muscle of the belly and eyes) in transgenic zebrafish larvae. The expression of the linearized EGFP gene was observed at the dorsal area, yolk sack and eyes. After the microinjection of linear form we obtained 6 F₀ transgenic fish, after the mating of F₀ transgenic fish with wild-type fish. We obtained 520 F₁ larvae. It is shown flourescent light at 102 larvae (20%). The mating of 19 number F₀ female fish with wild-type fish, male and female F₁ transgenic fish obtained. These F₁ transgenic fish was mating each other to produce F₂ transgenic fish which flourescent light observed at 20%. In our study, to produce stable transgenic fish lines, using linear gene constructs were effective on gene expression. However, it is brought up to produce only F₀ transgenic fish used circular gene construct was effective.

In the conclusion, to produce transgenic fish technology was established at the first time in Turkey and transgenic zebrafish was obtained by using this technology. The result of the study will give an opportunity to produce transgenic zebrafish or other fish. However, investigation of gene functions will be study in vivo in transgenic fish systems.