Document Type : Research Paper
Authors
1
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
2
Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
10.22124/japb.2024.28064.1549
Abstract
The pdx1 gene, which is also known as insulin factor 1, is the main key to the growth and development of the pancreas, as well as the maturation of beta cells and their survival. By targeting this gene, various transgenic models have been produced in stem cells as well as animal models that are used in the field of diabetes research. In this research, the aim is to make gene plasmids based on Tol2 using conventional cloning method, which can be used to create Pdx1:GFP zebrafish transgenic model. In order to label this gene, a part of its promoter sequence needs to be placed upstream of the reporter gene. Since the regulatory region of the pdx1 gene has not been defined, two pairs of primers were designed to amplify the 5' sequence of 2 kilobase and 6 kilobase upstream of the ATG start codon in the zebrafish pdx1 gene, and to the 5' and 3' ends of each of these primers, the sequence of restriction enzymes (SalI, Sph1) is placed. For this purpose, after designing suitable primers for two different lengths of the promoter sequence of the pdx1 gene expressed in beta cells, these fragments were amplified and inserted in the Tol2 plasmid containing the green fluorescent reporter gene using the conventional cloning method including enzymatic digestion and the ligation reaction. Confirmation of the correctness of the cloning process was done using PCR, enzymatic digestion and sequencing. The results showed that the conventional cloning process, in addition to the low cost, has good efficiency for cloning of fragments, with different sizes. The construction of these gene plasmids can be used in the next step to produce pdx1:GFP transgenic zebrafish, which is a suitable model for drug discovery studies for diabetes. By producing this transgenic model, the significant effect of this factor in the regeneration of beta cells after the induction of type 1 diabetes can be investigated, and this model can be used for extensive studies such as drug screening.
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