Design and Fabrication of Gene Cons tructs to GenerateA Double Transgenic Zebrafish Model for S tudyingPancreatic Beta Cell Regeneration

Objective: There are scientific evidences that show pancreaticalpha cells (pAC) are able to contribute to beta cell (pBC)compensation through diabetic pathophysiologic condition.Zebrafish is a well-suited transgenic animal model for regeneratives tudies due to easy accessibility to the embryos as wellas cell signaling and developmental similarities with mammals.Generating transgenic zebrafish models was done by tol۲ transposasesys tem in zebrafish labs.Materials and Methods: Here, we designed and cons tructedins:RFP-NTR and Gcga:eGFP plasmids to generate a doubletransgenic zebrafish model which demons trates endocrine cellplas ticity through pBC regeneration. Accordingly, ins-RFPNTRplasmid diges ted by Age۱ and BsrG۱ res triction enzymes(RE) and RFP was inserted downs tream of insulin (ins) promoter.Second cloning was performed to insert Glucagon (Gcga)promoter into tol۲-eGFP plasmid.Results: Designed primer were used for amplification of ۲kbof Gcga promoter to be inserted ups tream of eGFP by SalI RE.Recombinant plasmids were confirmed by colony polymerasechain reaction (PCR), diges tion, and sequencing; in colonyPCR and diges tion proper fragment sizes were obtained andno errors were detected through sequencing data. These plasmids,then, will be injected into one-cell embryos to generateTg(ins:RFP-NTR, Gcga:eGFP) zebrafish. Specific Beta-Cellablation could be occurred by a fusion protein in ins producingcells called NTR.Conclusion: Overlap of green and red color was observedwhich indicates the s tate transition of Gcga+ to ins+- cells usingconfocal fluorescent microscopy. This line could be used forfurther drug screening experiments and the discovery of newbeta regeneration mechanisms

کلیدواژه ها:

Diabetes ، Metronidazole ، Primer ، Tol۲