Design and Fabrication of Gene Cons tructs to GenerateA Transgenic Zebrafish Model to S tudy PancreaticProgenitor Cells during Beta Cell Ablation

Objective: Zebrafish are widely used in beta-cell regenerativeresearch because of its high regeneration capacity, easy genomicmanipulation, and potential for high throughput drug screening.Pancreatic duodenal homeobox۱ (PDX۱) is essential for the developmentof pancreatic exocrine and endocrine cells includingglucose-responsive (insulin-producing) beta cells. Pdx۱ mutantzebrafish have the key diabetic features of reduced beta cells,decreased insulin, and elevated glucose. Therefore, the pdx۱transgenic zebrafish is an essential model for diabetes research.Materials and Methods: In this s tudy, Pdx۱:GFP cons tructwas designed using the TOL۲ transposase cons truct to generatetransgenic zebrafish (Tg(pdx: GFP)) model. Because theregulatory region of the pdx۱ gene isn’t defined we design twopairs of primers to amplify ۲ kb and ۶ kb ۵´-flanking sequenceups tream of the ATG s tart codon in zebrafish pdx۱ gene andres triction site of SalI enzyme added to ends of pieces. Highfidelity polymerase was used to reduce the mismatch in the amplificationof these polymerase chain reaction (PCR) products.Results: Amplified products inserted ups tream of GFP usingSalI enzyme and recombinant plasmids were confirmed bycolony PCR and diges tion, and proper fragment sizes were obtainedin both tes ts. This vector could be used for the generationof transgenic zebrafish Tg(pdx۱:GFP) by co-injection withtransposase mRNA in one-cell s tage zebrafish embryos.Conclusion: This transgenic line could be used for further drugscreening experiments and because of pdx۱ expression in endocrineprogenitor cells this model is useful for the s tudy ofneogenesis mechanism in beta cell regeneration research.

کلیدواژه ها:

Beta Cell ، Diabetes ، Tol۲ ، Transposase ، Zebrafish