Designing and constructing genetic constructs for the production of Tg(pdx1:GFP) transgenic zebrafish as a model for drug screening in diabetes

Document Type : Research Paper

Authors

1 M.Sc. in Genetics, Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Karaj, Iran

2 Ph.D. in Fish Biotechnology, Department of Stem Cells and Developmental Biology, Stem Cell Biology and Technology Institute, Royan Institute, ACECR, Tehran, Iran

3 Associate Professor in Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Karaj, Iran

4 Associate Professor in Department of Stem Cells and Developmental Biology, Stem Cell Biology and Technology Institute, Royan Institute, ACECR, 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 Tg(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 2500 and 6600 base pairs 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) was 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 colony 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.

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Main Subjects

References

Ausubel F.M. 1992. Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology. Wiley, USA. 804P. doi: 10.1002/abio.370130118
Brown T.A. 2020. Gene cloning and DNA analysis: An introduction. John Wiley and Sons, UK. 432P.
Chawla P., Silva L.F.D. and Ninov N. 2020. Insights on β-cell regeneration from the zebrafish shoal: From generation of cells to functional integration. Current Opinion in Physiology, 14: 27–34. doi: 10.1016/j.cophys.2019.11.009
Choi T.Y., Choi T.I., Lee Y.R., Choe S.K. and Kim C.H. 2021. Zebrafish as an animal model for biomedical research. Experimental and Molecular Medicine, 53(3): 310–317. doi: 10.1038/s12276-021-0 0571-5
Devlin R.H., Biagi C.A., Yesaki T.Y., Smailus D.E. and Byatt J.C. 2001. Growth of domesticated transgenic fish. Nature, 409(6822): 781–782. doi: 10.1038/35057314
Djurkin Kusec I., Radisic Z., Komlenic M. and Kusec G. 2015. Comparison of commercial DNA kits and traditional DNA extraction procedure in PCR detection of pork in dry/fermented sausages. Poljoprivreda, 21(1): 199–202. doi: 10.18047/poljo.21.1.sup.47
Green M.R. and Sambrook J. 2020. Cloning in plasmid vectors: Directional cloning. Cold Spring Harbor Protocols, 2020(11): 485–488. doi: 10.1101/pdb.prot101238
Helker C.S.M., Mullapudi S.T., Mueller L.M., Preussner J., Tunaru S., Skog O., Kwon H.B., Kreuder F., Lancman J.J., Bonnavion R., Dong P.D.S., Looso M., Offermanns S., Korsgren O., Spagnoli F.M. and Stainier D.Y.R. 2019. A whole organism small molecule screen identifies novel regulators of pancreatic endocrine development. Development, 146(14): 1–12 (dev172569). doi: 10.1242/dev.1725 69
Huang H., Vogel S. S., Liu N., Melton D. A. and Lin S. 2001. Analysis of pancreatic development in living transgenic zebrafish embryos. Molecular and Cellular Endocrinology, 177(1-2): 117–124. doi: 10.1016/s0303-7207 (01)00408-7
Karami F., Asgari Abibeiglou B., Pahlavanneshan S., Farrokhi A., Tamadon A., Basiri M., Khalooghi K., Fallahi M. and Tahamtani Y. 2022. Enhanced characterization of beta cell mass in a Tg (Pdx1-GFP) mouse model. BioImpacts: 12(5): 463–470. doi: 10.34172/bi.2022.23840
Kaufman D. and Evans G. 1990. Restriction endonuclease cleavage at the termini of PCR products. BioTechniques, 9(3): 304–306.
Kawakami K., Shima A. and Kawakami N. 2000. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proceedings of the National Academy of Sciences, 97(21): 11403–11408. doi: 10.1073/ pnas.97.21.11403
Kawakami K., Takeda H., Kawakami N., Kobayashi M., Matsuda N. and Mishina M. 2004. A transposon-mediated gene trap approach identifies develop-mentally regulated genes in zebrafish. Developmental Cell, 7(1): 133–144. doi: 10.1016/j.devcel. 2004.06.005
Kimmel R.A., Dobler S., Schmitner N., Walsen T., Freudenblum J., and Meyer D. 2015. Diabetic pdx1-mutant zebrafish show conserved responses to nutrient overload and anti-glycemic treatment. Scientific Reports, 5(1): 1–14 (14241). doi: 10.1038/srep142 41
Kimmel R.A., Onder L., Wilfinger A., Ellertsdottir E. and Meyer D. 2011. Requirement for Pdx1 in specification of latent endocrine progenitors in zebrafish. BMC Biology, 9(1): 1–15. doi: 10.1186/17 41-7007-9-75
Lavergne A., Tarifeno-Saldivia E.J., Pirson J., Reuter A.S., Flasse L., Manfroid I., Voz M.L. and Peers B. 2020. Pancreatic and intestinal endocrine cells in zebrafish share common transcriptomic signatures and regulatory programmes. BMC Biology, 18: 1–19. doi: 10.1186/s12 915-020-00840-1
Lee Y., Choi H.Y., Kwon A., Park H., Park M.H., Kim J.W., Kim M.J., Kim Y.O., Kwak S. and Koo S.K. 2019. Generation of a PDX1-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-3, using the CRISPR/Cas9 system. Stem Cell Research, 41: 1–4 (101632). doi: 10.1016/j.scr.2019.101632
Liu C.J., Jiang H., Wu L., Zhu L.Y., Meng E. and Zhang D.Y. 2017. OEPR cloning: An efficient and seamless cloning strategy for large-and multi-fragments. Scientific Reports, 7(1): 1–7. doi: 10.1038/srep 44648
Liu K.C., Villasenor A., Bertuzzi M., Schmitner N., Radros N., Rautio L., Mattonet K., Matsuoka R.L., Reischauer S., Stainier D.Y. and Andersso O. 2021. Insulin-producing β-cells regenerate ectopically from a mesodermal origin under the perturbation of hemato-endothelial specification. eLife, 10: 1–23 (65758). doi: 10.7554/eLife.65758
Mi J., Liu K.C. and Andersson O. 2023. Decoding pancreatic endocrine cell differentiation and β cell regeneration in zebrafish. Science Advances, 9(33): 1–23 (eadf5142). doi: 10.1126/sciadv.adf5 142
Mohseni A., Majidzadeh K. and Soleimani M. 2012. A new screening method for selection of desired recombinant plasmids in molecular cloning. African Journal of Biotechnology, 11(64): 12777–12780. doi: 10.5897/AJB11.3185
Nora L.C., Westmann C.A., Martins‐Santana L., Alves L.D.F., Monteiro L.M.O., Guazzaroni M.E. and Silva‐Rocha R. 2019. The art of vector engineering: Towards the construction of next‐generation genetic tools. Microbial Biotechnology, 12(1): 125–147. doi: 10.1111/1751-7915.13318
Pourghadamyari H., Rezaei M., Basiri M., Tahamtani Y., Asgari B., Hassani S.N., Meshkani R., Golmohammadi T. and Baharvand H. 2019. Generation of a transgenic zebrafish model for pancreatic beta cell regeneration. Galen Medical Journal, 8: 1–8 (e1056). doi: 10.31661/gmj.v8i0.1056
Salehpour A., Rezaei M., Khoradmehr A., Tahamtani Y. and Tamadon A. 2021. Which hyperglycemic model of zebrafish (Danio rerio) suites my type 2 diabetes mellitus research? A scoring system for available methods. Frontiers in Cell and Developmental Biology, 9: 1–15 (652061). doi: 10.3389/fcell.2021.65 2061
Scharf S.J., Horn G.T., and Erlich H.A. 1986. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science, 233(4768): 1076–1078. doi: 10.1126/science.3461561
Subramanian S. 2021. Zebrafish as a model organism- Can a fish mimic human? Journal of Basic and Clinical Physiology and Pharmacology, 34(5): 559–575. doi: 10.1515/jbcpp-2021-0113
Wu R. 2012. Recombinant DNA Methodology II. Academic Press, USA. 904P. doi: 10.1016/C2009-0-0 2410-6
Zang L., Maddison L.A. and Chen W. 2018. Zebrafish as a model for obesity and diabetes. Frontiers in Cell and Developmental Biology, 6: 1–31 (91). doi: 10.3389/fcell.2018. 00091
Aquatic Physiology and Biotechnology
Volume 13, Issue 2
November 2025
Pages 71-94

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