Engineering a DYRK1B R102C mutation: insights into metabolic syndrome pathogenesis through lentiviral gene delivery

Abstract

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.

Keywords

• dyrk1b protein
• human
• metabolic syndrome
• mutagenesis
• cloning
• organism
• lentivirus
• genetic vectors