Transdiferencijacija alfa ćelija pankreasa miša u ćelije koje proizvode insulin ciljanom metilacijom DNK primenom Epi-CRISPR sistema
Transdifferentiation of mouse pancreatic alpha to insulin-producing cells using Epi-CRISPRs directed DNA methylation
Abstract:
Aristaless-related homeobox (Arx) gene expression level is regulated by DNA methylation, plaing an important role in the maintenance of pancreatic alpha cell identity. Diabetes is characterized by a disturbed source of insulin, representing a good candidate for cell reprogramming strategy in diabetes therapy by Arx targeting. The aim of this doctoral dissertation was to examine the transdifferentiation ability of murine pancreatic alpha cells into insulin-producing cells induced by the targeted DNA methylation in the Arx promoter. The expression of beta specific marker was analiysed in transiently transfected αTC1-6 cells with a synthetic epigenetic tool for gene repression. The optimization of αTC1-6 cells nucleofection was established conditions by which was achieved an efficiency of 71.1% with an 80% of cell viability. The high efficiency of methylation induction by the dCas9-Dnmt3a3L-KRAB fusion (EpiCRISPR) construct was confirmed by targeted bisulfite sequencing. The Arx silencing followed by induction of Ins2 expression on 5 and 7 days after transfection was detected by RT-qPCR and transcriptome analysis. The insulin protein level was detected immunocytochemicaly until the 12th post-transfection day, and released insulin was detected by the enzyme immunoassay on the 7th post-transfection day. The initiation of the transdifferentiation process of αTC1-6 cells was examined by analyzing the presence of beta cell specific markers. The results showed that a single transient transfection initiate the transdifferentiation of ~1% of alpha cells into cells that produce 35% more insulin compared to mock-transfected cells. Acting on the epigenome plastic nature, the direct reprogramming of pancreatic alpha cells into insulin-producing cells was successfully initiated.
Keywords:
Arx; CRISPR/Cas9; pancreatic alpha cells; diabetes; epigenetic editing; targeted DNA methylation; transfectionSource:
University of Belgrade, Faculty of Biology, 2023, 1-92Funding / projects:
- Signaling molecules in diabetes: search for potential targets in intrinsic pathways for prediction and intervention in diabetes (RS-MESTD-Basic Research (BR or ON)-173020)
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200007 (University of Belgrade, Institute for Biological Research 'Siniša Stanković') (RS-MESTD-inst-2020-200007)
- Evropska Fondacije za istraživanja u dijabetesu (EFSD) - grant kompanije AstraZeneca