Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA
2017
Authors:
Vidaković, MelitaĐorđević, Marija
Arambašić Jovanović, Jelena
Tolić, Anja
Đorđević, Miloš
Mihailović, Mirjana
Grdović, Nevena
Uskoković, Aleksandra
Rajić, Jovana
Poznanović, Goran
Dinić, Svetlana
Jurkowski, Tomasz
Contributors
Brajušković, GoranĐorđević, Ana
Document Type:
Conference object (Published version)
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© 2017 University of Belgrade, Faculty of Biology
Metadata
Show full item recordAbstract:
Introduction and aim: Diabetes is the perfect candidate for cell replacement therapy since it is caused by either an absolute (type 1 diabetes) or relative (type 2 diabetes) defect of insulin-producing pancreatic beta cells (b-cells}. We focused on applying a novel synthetic epigenetic tool (Epi-CRISPRs) for a straightforward, one-step transdifferentiation of mouse pancreatic alpha (a-cells) to b-cell by targeted DNA methylation and suppression of genes essential for maintaining pancreatic cell identity (homeobox Arx gene (Arx)).
Methods: The a-cells were transiently transfected with four different Epi-CRISPR constructs and co-transfected with a single guided RNA (gRNA} or with a mix of different gRNAs all targeting different promoter regions of Arx. After 5, 8 and 12 days post-transfection, DNA and RNA were isolated and the cells were immunostained. The transdifferentiated cells were analysed for key features of bona fide cells, using qPCR to assess Arx expression, and immunostaining of insulin/glucagon and ELISA for measuring secreted insulin.
Results: We succeeded to transiently transfect a-cells with Epi-CRISPR constructs and 275 gRNA/mix gRNA. The suppression of Arx in a-cells was confirmed on days 5 and 8 post-transfection. The reduction of glucagon synthesis and beginning of insulin production in transfected a-cell was confirmed and visualised by immunostaining. Whether DNA methylation-mediated suppression of Arx in a-cells lead to their transdifferentiation to insulin-producing cells, will be confirmed by bisulfite sequencing.
Conclusion: We are on the right course of developing a clear-cut technology capable of providing a perfect delivery system for increasing the number of insulinproducing cells in vitro.
Funding / projects:
- European Cooperation in Science and Technology (COST) CM1406
- 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)
- European Foundation for the Study of Diabetes (EFSD): European Diabetes Research Programme in Cellular Plasticity Underlying the Pathophysiology of Type 2 Diabetes
In:
- Brajušković G, Đorđević A, editors. CoMBoS. Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia. Belgrade: University of Belgrade, Faculty of Biology; 2017. p. 10.