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Volume 18, Issue 5 (Sep-Oct 2024)
mljgoums 2024, 18(5): 34-38 |
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Daneshparvar A, Jamhiri I, Razban V, Fallahi J, Hamidizadeh N, Moghtaderi B et al . Engineering a DYRK1B R102C mutation: insights into metabolic syndrome pathogenesis through lentiviral gene delivery. mljgoums 2024; 18 (5) :34-38
URL: http://mlj.goums.ac.ir/article-1-1763-en.html
URL: http://mlj.goums.ac.ir/article-1-1763-en.html
Afrooz Daneshparvar1 
, Iman Jamhiri2 , Vahid Razban3 , Jafar Fallahi3 , Nasrin Hamidizadeh2 , Behnam Moghtaderi4 , Mehdi Dianatpour5
, Iman Jamhiri2 , Vahid Razban3 , Jafar Fallahi3 , Nasrin Hamidizadeh2 , Behnam Moghtaderi4 , Mehdi Dianatpour5
1- Stem Cell Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran ; Molecular Dermatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
2- Molecular Dermatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
3- Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
4- Department of Pathobiology, Shiraz Veterinary Medicine, Shiraz University, Shiraz, Iran
5- Stem Cell Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; Department of Human Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran ,mdianatpur@gmail.com
2- Molecular Dermatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
3- Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
4- Department of Pathobiology, Shiraz Veterinary Medicine, Shiraz University, Shiraz, Iran
5- Stem Cell Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; Department of Human Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran ,
Abstract: (1002 Views)
Background: A rare heterozygous DYRK1B mutation (R102C) recently linked to a familial form of metabolic syndrome prompted this study to introduce the R102C mutation into the mouse DYRK1B gene, utilizing recombinant lentiviruses for long-term gene expression.
Methods: In the present fundamental study, the DYRK1B R102C mutation was generated via Overlap Extension-PCR (OE-PCR) and inserted into the LeGO-iG2 transfer vector with a GFP marker. Recombinant lentiviruses were produced by co-transfection of the transfer vector carrying DYRK1B R102C, psPAX2 (Packaging vector), and pMD2 (Envelope vector) into HEK-293T cells.
Results: The accuracy of the intended mutation was confirmed through OE-PCR and sequencing. Expression of DYRK1B and successful gene transfer were visualized using a fluorescence microscope to detect the GFP marker. Lentiviral titer was quantified using flow cytometry, with an infection efficiency of 108 TU/ml in HEK-293T cells.
Conclusion: DYRK1B plays a crucial role in the pathogenesis of metabolic syndrome, central obesity, early-onset coronary artery disease, hypertension, type 2 diabetes, and adipogenesis, suggesting its potential as a target for therapeutic interventions. Lentiviruses carrying the DYRK1B R102C mutation offer significant advantages for both in vitro and in vivo research on metabolic syndrome. This study showcases the successful application of recombinant lentiviral vectors for gene transfer into eukaryotic cells.
Methods: In the present fundamental study, the DYRK1B R102C mutation was generated via Overlap Extension-PCR (OE-PCR) and inserted into the LeGO-iG2 transfer vector with a GFP marker. Recombinant lentiviruses were produced by co-transfection of the transfer vector carrying DYRK1B R102C, psPAX2 (Packaging vector), and pMD2 (Envelope vector) into HEK-293T cells.
Results: The accuracy of the intended mutation was confirmed through OE-PCR and sequencing. Expression of DYRK1B and successful gene transfer were visualized using a fluorescence microscope to detect the GFP marker. Lentiviral titer was quantified using flow cytometry, with an infection efficiency of 108 TU/ml in HEK-293T cells.
Conclusion: DYRK1B plays a crucial role in the pathogenesis of metabolic syndrome, central obesity, early-onset coronary artery disease, hypertension, type 2 diabetes, and adipogenesis, suggesting its potential as a target for therapeutic interventions. Lentiviruses carrying the DYRK1B R102C mutation offer significant advantages for both in vitro and in vivo research on metabolic syndrome. This study showcases the successful application of recombinant lentiviral vectors for gene transfer into eukaryotic cells.
Keywords: DYRK1B protein, human, Metabolic syndrome, Mutagenesis, Cloning, Organism, Lentivirus, Genetic vectors
Research Article: Research Article |
Subject:
Molecular Medicine
Received: 2023/12/24 | Accepted: 2024/07/30 | Published: 2024/10/27 | ePublished: 2024/10/27
Received: 2023/12/24 | Accepted: 2024/07/30 | Published: 2024/10/27 | ePublished: 2024/10/27
References
1. Goh KK, Chen CY-A, Wu T-H, Chen C-H, Lu M-L. Crosstalk between schizophrenia and metabolic syndrome: The role of oxytocinergic dysfunction. International journal of molecular sciences. 2022; 23(13): 7092. [View at Publisher] [DOI] [PMID] [Google Scholar]
2. Bazmandegan G, Abbasifard M, Nadimi AE, Alinejad H, Kamiab Z. Cardiovascular risk factors in diabetic patients with and without metabolic syndrome: a study based on the Rafsanjan cohort study. Scientific Reports. 2023; 13(1): 559. [View at Publisher] [DOI] [PMID] [Google Scholar]
3. Farooqui AA, Farooqui T, Panza F, Frisardi V. Metabolic syndrome as a risk factor for neurological disorders. Cellular and Molecular Life Sciences. 2012; 69: 741-62. [View at Publisher] [DOI] [PMID] [Google Scholar]
4. Swarup S, Goyal A, Grigorova Y, Zeltser R. Metabolic syndrome. StatPearls [internet]: StatPearls Publishing; 2022. [View at Publisher] [PMID] [Google Scholar]
5. Alberti KGMM, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus. Provisional report of a WHO consultation. Diabetic medicine. 1998; 15(7): 539-53.
https://doi.org/10.1002/(SICI)1096-9136(199807)15:7<539::AID-DIA668>3.0.CO;2-S [View at Publisher] [DOI] [Google Scholar]
6. Alberti KGM, Zimmet P, Shaw J. The metabolic syndrome-a new worldwide definition. The Lancet. 2005;366(9491):1059-62. [View at Publisher] [DOI] [PMID] [Google Scholar]
7. Goossens GH. The metabolic phenotype in obesity: fat mass, body fat distribution, and adipose tissue function. Obesity facts. 2017; 10(3): 207-15. [View at Publisher] [DOI] [PMID] [Google Scholar]
8. Naghipour M, Joukar F, Nikbakht H-A, Hassanipour S, Asgharnezhad M, Arab-Zozani M, et al. High prevalence of metabolic syndrome and its related demographic factors in North of Iran: results from the Persian Guilan cohort study. International Journal of Endocrinology. 2021;2021. [View at Publisher] [DOI] [PMID] [Google Scholar]
9. De Silva ST, Niriella MA, Ediriweera DS, Kottahachchi D, Kasturiratne A, de Silva AP, et al. Incidence and risk factors for metabolic syndrome among urban, adult Sri Lankans: a prospective, 7-year community cohort, follow-up study. Diabetology & Metabolic Syndrome. 2019;11(1):1-7. [View at Publisher] [DOI] [PMID] [Google Scholar]
10. Gunawan S, Aulia A, Soetikno V. Development of rat metabolic syndrome models: A review. Veterinary World. 2021;14(7):1774. [View at Publisher] [DOI] [PMID] [Google Scholar]
11. Pucci G, Alcidi R, Tap L, Battista F, Mattace-Raso F, Schillaci G. Sex-and gender-related prevalence, cardiovascular risk and therapeutic approach in metabolic syndrome: A review of the literature. Pharmacological research. 2017;120:34-42. [View at Publisher] [DOI] [PMID] [Google Scholar]
12. Dubey P, Singh V, Venishetty N, Trivedi M, Reddy SY, Lakshmanaswamy R, et al. Associations of sex hormone ratios with metabolic syndrome and inflammation in US adult men and women. Front Endocrinol (Lausanne). 2024;15:1384603. [View at Publisher] [DOI] [PMID] [Google Scholar]
13. Bhat N, Narayanan A, Fathzadeh M, Shah K, Dianatpour M, Abou Ziki MD, et al. Dyrk1b promotes autophagy during skeletal muscle differentiation by upregulating 4e-bp1. Cellular Signalling. 2022; 90: 110186. [View at Publisher] [DOI] [PMID] [Google Scholar]
14. Charkoftaki G, Thompson DC, Golla JP, Garcia-Milian R, Lam TT, Engel J, et al. Integrated multi-omics approach reveals a role of ALDH1A1 in lipid metabolism in human colon cancer cells. Chemico-biological interactions. 2019; 304: 88-96. [View at Publisher] [DOI] [PMID] [Google Scholar]
15. Keramati AR, Fathzadeh M, Singh R, Choi M, Faramarzi S, Mane S, et al. Identification of a novel disease gene for early onset atherosclerosis, diabetes and metabolic syndrome by whole exome sequencing and linkage analysis. Am Heart Assoc. 2013; 128(22). [View at Publisher] [DOI] [Google Scholar]
16. Abu Jhaisha S, Widowati EW, Kii I, Sonamoto R, Knapp S, Papadopoulos C, et al. DYRK1B mutations associated with metabolic syndrome impair the chaperone-dependent maturation of the kinase domain. 2017;7(1):6420. [View at Publisher] [DOI] [PMID] [Google Scholar]
17. Mendoza-Caamal EC, Barajas-Olmos F, Mirzaeicheshmeh E, Ilizaliturri-Flores I, Aguilar-Salinas CA, Gómez-Velasco DV, et al. Two novel variants in DYRK1B causative of AOMS3: expanding the clinical spectrum. Orphanet journal of rare diseases. 2021; 16(1): 291. [View at Publisher] [DOI] [PMID] [Google Scholar]
18. Zhuang L, Jia K, Chen C, Li Z, Zhao J, Hu J, et al. DYRK1B-STAT3 drives cardiac hypertrophy and heart failure by impairing mitochondrial bioenergetics. Circulation. 2022;145(11):829-46. [View at Publisher] [DOI] [PMID] [Google Scholar]
19. Keramati AR, Fathzadeh M, Go GW, Singh R, Choi M, Faramarzi S, et al. A form of the metabolic syndrome associated with mutations in DYRK1B. The New England journal of medicine. 2014;370(20):1909-19. [View at Publisher] [DOI] [PMID] [Google Scholar]
20. Armanmehr A, Jafari Khamirani H, Zoghi S. Analysis of DYRK1B, PPARG, and CEBPB Expression Patterns in Adipose-Derived Stem Cells from Patients Carrying DYRK1B R102C and Healthy Individuals During Adipogenesis. 2022. [View at Publisher] [DOI] [PMID] [Google Scholar]
21. Pinhas-Hamiel O. Type 2 Diabetes, Metabolic Syndrome and Lipid Metabolism. Yearbook of Pediatric Endocrinology 2014: Endorsed by the European Society for Paediatric Endocrinology (ESPE). 2014; 383: 171. [View at Publisher] [DOI] [Google Scholar]
22. Martin KA, Mani MV, Mani A. New targets to treat obesity and the metabolic syndrome. European journal of pharmacology. 2015; 763: 64-74. [View at Publisher] [DOI] [PMID] [Google Scholar]
23. Yang W, Jiang LH. Site-directed mutagenesis to study the structure-function relationships of ion channels. Methods in molecular biology (Clifton, NJ). 2013; 998: 257-66. [View at Publisher] [DOI] [PMID] [Google Scholar]
24. Zhang K, Yin X, Shi K, Zhang S, Wang J, Zhao S, et al. A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation. Scientific Reports. 2021;11(1):10454. [View at Publisher] [DOI] [PMID] [Google Scholar]
25. Usman S, Bushaala A, Teh MT, Waseem A. Site-Directed Mutagenesis to Mutate Multiple Residues in a Single Reaction. Methods Mol Biol. 2024. [View at Publisher] [DOI] [PMID] [Google Scholar]
26. Liu H, Naismith JH. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC biotechnology. 2008; 8(1): 1-10. [View at Publisher] [DOI] [PMID] [Google Scholar]
27. Aiyar A, Xiang Y, Leis J. Site-directed mutagenesis using overlap extension PCR. In vitro mutagenesis protocols. 1996; 177-91. [View at Publisher] [DOI] [PMID] [Google Scholar]
28. Forloni M, Liu AY, Wajapeyee N. Creating insertions or deletions using overlap extension polymerase chain reaction (PCR) mutagenesis. Cold Spring Harbor Protocols. 2018;2018(8):pdb. prot097758. [View at Publisher] [DOI] [PMID] [Google Scholar]
29. Nelson MD, Fitch DH. Overlap extension PCR: an efficient method for transgene construction. Molecular methods for evolutionary genetics. 2011:459-70. [View at Publisher] [DOI] [PMID] [Google Scholar]
30. Alfranca A, Campanero MR, Redondo JM. New Methods for Disease Modeling Using Lentiviral Vectors. Trends in molecular medicine. 2018; 24(10): 825-37. [View at Publisher] [DOI] [PMID] [Google Scholar]
31. Tseng W-C, Lin J-W, Wei T-Y, Fang T-Y. A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Anal Biochem. 2008; 375(2): 376-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
32. Ku M-W, Charneau P, Majlessi L. Use of lentiviral vectors in vaccination. Expert Review of Vaccines. 2021;20(12):1571-86. [View at Publisher] [DOI] [PMID] [Google Scholar]
33. Sakuma T, Barry MA, Ikeda Y. Lentiviral vectors: basic to translational. Biochemical Journal. 2012; 443(3): 603-18. [View at Publisher] [DOI] [PMID] [Google Scholar]
34. McGinley L, McMahon J, Strappe P, Barry F, Murphy M, O'Toole D, et al. Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia. Stem cell research & therapy. 2011;2:1-18. [View at Publisher] [DOI] [PMID] [Google Scholar]
35. Magnani A, Semeraro M, Adam F, Booth C, Dupré L, Morris E, et al. Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome. Nature Medicine. 2022; 28(1): 71-80. [View at Publisher] [DOI] [PMID] [Google Scholar]
36. Milone MC, O'Doherty U. Clinical use of lentiviral vectors. Leukemia. 2018; 32(7): 1529-41. [View at Publisher] [DOI] [PMID] [Google Scholar]
37. Zheng CX, Wang SM, Bai YH, Luo TT, Wang JQ. Lentiviral Vectors and Adeno-Associated Virus Vectors: Useful Tools for Gene Transfer in Pain Research. 2018;301(5):825-36. [View at Publisher] [DOI] [PMID] [Google Scholar]
38. Jakobsson J, Lundberg C. Lentiviral vectors for use in the central nervous system. Molecular Therapy. 2006; 13(3): 484-93. [View at Publisher] [DOI] [PMID] [Google Scholar]
39. Azzouz M, Kingsman SM, Mazarakis ND. Lentiviral vectors for treating and modeling human CNS disorders. The Journal of Gene Medicine: A cross‐disciplinary journal for research on the science of gene transfer and its clinical applications. 2004; 6(9): 951-62. [View at Publisher] [DOI] [PMID] [Google Scholar]
40. Sano S, Wang Y, Evans MA, Yura Y, Sano M, Ogawa H, et al. Lentiviral CRISPR/Cas9-mediated genome editing for the study of hematopoietic cells in disease models. Journal of visualized experiments: JoVE. 2019; 152: 10.3791/59977. [View at Publisher] [DOI] [PMID] [Google Scholar]
41. Mátrai J, Chuah MK, VandenDriessche T. Recent advances in lentiviral vector development and applications. Molecular therapy. 2010;18(3):477-90. [View at Publisher] [DOI] [PMID] [Google Scholar]
42. Sinn P, Sauter S, McCray P. Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors-design, biosafety, and production. Gene therapy. 2005;12(14):1089-98. [View at Publisher] [DOI] [PMID] [Google Scholar]
43. Yu YY, Xu MS, Liang H, Wang HY, Yu CQ, Liu Q. Review and future perspectives on the integration characteristics for equine lentivirus in the host genome. Pol J Vet Sci. 2023; 26(1): 163-72. [View at Publisher] [DOI] [PMID] [Google Scholar]
44. Bokhoven M, Stephen SL, Knight S, Gevers EF, Robinson IC, Takeuchi Y, et al. Insertional gene activation by lentiviral and gammaretroviral vectors. Journal of virology. 2009;83(1):283-94. [View at Publisher] [DOI] [PMID] [Google Scholar]
45. Ghosh S, Brown AM, Jenkins C, Campbell K. Viral vector systems for gene therapy: a comprehensive literature review of progress and biosafety challenges. Applied Biosafety. 2020; 25(1):7-18. [View at Publisher] [DOI] [PMID] [Google Scholar]
46. Narayan O, Clements JE. Biology and pathogenesis of lentiviruses. Journal of General Virology. 1989;70(7):1617-39. [View at Publisher] [DOI] [PMID] [Google Scholar]
47. Kokkorakis N, Zouridakis M, Gaitanou M. Mirk/Dyrk1B Kinase Inhibitors in Targeted Cancer Therapy. Pharmaceutics. 2024;16(4): 528. [View at Publisher] [DOI] [PMID] [Google Scholar]
48. Becker W. A wake-up call to quiescent cancer cells - potential use of DYRK1B inhibitors in cancer therapy. Febs j. 2018;285(7):1203-11. [View at Publisher] [DOI] [PMID] [Google Scholar]
49. Gao J, Zhao Y, Lv Y, Chen Y, Wei B, Tian J, et al. Mirk/Dyrk1B mediates G0/G1 to S phase cell cycle progression and cell survival involving MAPK/ERK signaling in human cancer cells. Cancer Cell Int. 2013;13(1):2. [View at Publisher] [DOI] [PMID] [Google Scholar]
50. Peng S, Li W, Hou N, Huang N. A review of FoxO1-regulated metabolic diseases and related drug discoveries. Cells. 2020;9(1):184. [View at Publisher] [DOI] [PMID] [Google Scholar]
51. Mirshahi T, Murray MF, Carey DJ. The metabolic syndrome and DYRK1B. The New England journal of medicine. 2014;371(8):784-5. [View at Publisher] [DOI] [PMID] [Google Scholar]
52. Mohamed YA, Hassaneen H, El-Dessouky MA, Safwat G, Hassan NA-M, Amr K. Study of DYRK1B gene expression and its association with metabolic syndrome in a small cohort of Egyptians. Molecular Biology Reports. 2021;48:5497-502. [View at Publisher] [DOI] [PMID] [Google Scholar]
53. Haldar SM. Metabolic syndrome: A family affair. Science Translational Medicine. 2014;6(239):239ec96-ec96. [View at Publisher] [DOI] [Google Scholar]
54. Singh R, Dhanyamraju PK, Lauth M. DYRK1B blocks canonical and promotes non-canonical Hedgehog signaling through activation of the mTOR/AKT pathway. Oncotarget. 2017;8(1):833. [View at Publisher] [DOI] [PMID] [Google Scholar]
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