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Development of an in vitro model of dysferlinopathy via crispr/cas-mediated transcriptional activation of the dysf gene
- Authors: Yakovlev I.A.1,2, Slesarenko Y.S.2, Starostina I.G.3, Shaimardanova A.A.3, Solovyova V.V.3, Bobrovsky P.A.4, Grafskaia E.N.4, Belikova L.D.4, Bardakov S.N.1, Rizvanov A.A.3,5, Isaev A.A.1, Deev R.V.1,2,6
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Affiliations:
- Artgene Biotech
- OOO Genotarget, Skolkovo Innovation Center
- Kazan (Volga Region) Federal University
- Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine
- Division of Medical and Biological Sciences, Tatarstan Academy of Sciences
- Avtsyn Research Institute of Human Morphology, Petrovsky National Research Centre of Surgery
- Issue: Vol 66, No 4 (2024)
- Pages: 380-392
- Section: Articles
- URL: https://ruspoj.com/0041-3771/article/view/669502
- DOI: https://doi.org/10.31857/S0041377124040064
- EDN: https://elibrary.ru/QCPXOW
- ID: 669502
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Fig. 1. CRISPR/Cas9-SAM transcriptional activation system.
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Fig. 2. Distribution of fibroblasts isolated from the gums of a patient with dysferlinopathy by surface markers (immunophenotyping). Vertical axis – cell number; horizontal axis – glow intensity of the probe bound to the corresponding antibody. a – Cells negative for markers CD4 5, CD34, CD 1 1b, CD 19 and HLA-DR; b – CD90+ cells (94.4%), b – CD 10 5+ cells (90.5%), d – CD73+ cells (91.5%); d, e – histogram of cell population distribution by GFP fluorescent signal intensity before and after sorting, respectively. Flow cytometry.
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Fig. 3. Detection of p53 knockout in fibroblasts. a – Fibroblasts from a patient with dysferlinopathy transduced with LVUHshp53-gfp.3 lentivirus. b – Western blot: KO – immortalized skin fibroblasts from a patient with dysferlinopathy obtained by knockout of the p53 tumor suppressor gene (53 kDa), WT – skin fibroblasts from a patient with dysferlinopathy. M – molecular marker.
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Fig. 4. Detection of DYSF mRNA and protein in fibroblasts and HEK 293T cells before (wo TA) and after (TA) transcriptional activation of DYSF using CRISPR/dCas9 SAM. a – Relative expression of dysferlin mRNA (ΔΔCT, RT-PCR) in human myoblasts without a mutation in the DYSF gene used as control cells (HMb), patient fibroblasts after transcriptional activation (TA) of DYSF (HF-mut26-p53_TA) and patient fibroblasts lacking a component of the activation system (HF-mut26-p53 wo TA). b – Relative (ΔΔCT) expression of dysferlin mRNA in HEK 293T cells before and after TA DYSF and C 2C 1 2 cells, which served as a positive control. c, d – Western blots with antibodies to the dysferlin protein and their densitograms (D – optical density), respectively. a, b: Data are presented as mean ± standard error of the mean.
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