Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB
Аутори
Parezanović, MarinaAnđelković, Marina
Stevanović, Nina
Klaassen, Kristel
Spasovski, Vesna
Ugrin, Milena
Komazec, Jovana
Stanković, Sara
Jocić, Nikola
Pavlović, Sonja
Stojiljković, Maja
Skakić, Anita
Конференцијски прилог (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
Introduction: Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder characterized by fasting hypoglycemia and the accumulation of glycogen in the liver, kidneys and intestinal mucosa. Recent studies revealed that chronic endoplasmic reticulum (ER) stress and increased apoptosis
play a role in the progression of disease manifestations. Although dietary control is commonly utilized
to manage hypoglycemia, there is still a lack of effective pharmacological therapy. Therefore, the establishment of proper model system is essential for testing novel treatment approaches.
Methods: To create GSD-Ib in vitro model system, CRISPR/Cas9-knockout (KO) method was used to introduce a deletion in SLC37A4 gene in the FlpInHEK293 cells. Characterization of CRISPR/Cas9-KO model
system was performed using Sanger sequencing, RT-qPCR and Western Blot. Additionally, the expression analysis of ER stress and apoptotic markers was performed.
Results: Sanger sequencing confirmed the p...resence of c.14_100del in SLC37A4 gene. The expression
level of SLC37A4 was decreased to 26.8% in the SLC37A4-/- cell line compared to the SLC37A4 wild-type
along with Western blot analysis, which confirmed reduced target protein level in SLC37A4-/- clones. Furthermore, ER stress (ATF4, DDIT3, HSPA5, XBP1s) and apoptotic (BCL2, BAX, CASP3, CASP7) markers expression levels were significantly altered in SLC37A4-/- clones compared to wild-type, which proved that
we created a suitable GSD-Ib in vitro model system.
Conclusion: Utilizing CRISPR/Cas9 technology, we established cellular GSD-Ib modelsystem that mirrors
increased ER stress and apoptosis. This model system could be used to facilitate a comprehensive understanding of disease mechanisms and enable testing of potential treatment effectiveness.
Кључне речи:
CRISPR/Cas9 knockout / Glycogen storage disease type Ib / in vitro disease modelling / endoplasmic reticulum stress / apoptosisИзвор:
CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia, 2023, 65-65Издавач:
- Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade
Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200042 (Универзитет у Београду, Институт за молекуларну генетику и генетичко инжењерство) (RS-MESTD-inst-2020-200042)
Институција/група
Institut za molekularnu genetiku i genetičko inženjerstvoTY - CONF AU - Parezanović, Marina AU - Anđelković, Marina AU - Stevanović, Nina AU - Klaassen, Kristel AU - Spasovski, Vesna AU - Ugrin, Milena AU - Komazec, Jovana AU - Stanković, Sara AU - Jocić, Nikola AU - Pavlović, Sonja AU - Stojiljković, Maja AU - Skakić, Anita PY - 2023 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/2121 AB - Introduction: Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder characterized by fasting hypoglycemia and the accumulation of glycogen in the liver, kidneys and intestinal mucosa. Recent studies revealed that chronic endoplasmic reticulum (ER) stress and increased apoptosis play a role in the progression of disease manifestations. Although dietary control is commonly utilized to manage hypoglycemia, there is still a lack of effective pharmacological therapy. Therefore, the establishment of proper model system is essential for testing novel treatment approaches. Methods: To create GSD-Ib in vitro model system, CRISPR/Cas9-knockout (KO) method was used to introduce a deletion in SLC37A4 gene in the FlpInHEK293 cells. Characterization of CRISPR/Cas9-KO model system was performed using Sanger sequencing, RT-qPCR and Western Blot. Additionally, the expression analysis of ER stress and apoptotic markers was performed. Results: Sanger sequencing confirmed the presence of c.14_100del in SLC37A4 gene. The expression level of SLC37A4 was decreased to 26.8% in the SLC37A4-/- cell line compared to the SLC37A4 wild-type along with Western blot analysis, which confirmed reduced target protein level in SLC37A4-/- clones. Furthermore, ER stress (ATF4, DDIT3, HSPA5, XBP1s) and apoptotic (BCL2, BAX, CASP3, CASP7) markers expression levels were significantly altered in SLC37A4-/- clones compared to wild-type, which proved that we created a suitable GSD-Ib in vitro model system. Conclusion: Utilizing CRISPR/Cas9 technology, we established cellular GSD-Ib modelsystem that mirrors increased ER stress and apoptosis. This model system could be used to facilitate a comprehensive understanding of disease mechanisms and enable testing of potential treatment effectiveness. PB - Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade C3 - CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia T1 - Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB EP - 65 SP - 65 UR - https://hdl.handle.net/21.15107/rcub_imagine_2121 ER -
@conference{ author = "Parezanović, Marina and Anđelković, Marina and Stevanović, Nina and Klaassen, Kristel and Spasovski, Vesna and Ugrin, Milena and Komazec, Jovana and Stanković, Sara and Jocić, Nikola and Pavlović, Sonja and Stojiljković, Maja and Skakić, Anita", year = "2023", abstract = "Introduction: Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder characterized by fasting hypoglycemia and the accumulation of glycogen in the liver, kidneys and intestinal mucosa. Recent studies revealed that chronic endoplasmic reticulum (ER) stress and increased apoptosis play a role in the progression of disease manifestations. Although dietary control is commonly utilized to manage hypoglycemia, there is still a lack of effective pharmacological therapy. Therefore, the establishment of proper model system is essential for testing novel treatment approaches. Methods: To create GSD-Ib in vitro model system, CRISPR/Cas9-knockout (KO) method was used to introduce a deletion in SLC37A4 gene in the FlpInHEK293 cells. Characterization of CRISPR/Cas9-KO model system was performed using Sanger sequencing, RT-qPCR and Western Blot. Additionally, the expression analysis of ER stress and apoptotic markers was performed. Results: Sanger sequencing confirmed the presence of c.14_100del in SLC37A4 gene. The expression level of SLC37A4 was decreased to 26.8% in the SLC37A4-/- cell line compared to the SLC37A4 wild-type along with Western blot analysis, which confirmed reduced target protein level in SLC37A4-/- clones. Furthermore, ER stress (ATF4, DDIT3, HSPA5, XBP1s) and apoptotic (BCL2, BAX, CASP3, CASP7) markers expression levels were significantly altered in SLC37A4-/- clones compared to wild-type, which proved that we created a suitable GSD-Ib in vitro model system. Conclusion: Utilizing CRISPR/Cas9 technology, we established cellular GSD-Ib modelsystem that mirrors increased ER stress and apoptosis. This model system could be used to facilitate a comprehensive understanding of disease mechanisms and enable testing of potential treatment effectiveness.", publisher = "Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade", journal = "CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia", title = "Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB", pages = "65-65", url = "https://hdl.handle.net/21.15107/rcub_imagine_2121" }
Parezanović, M., Anđelković, M., Stevanović, N., Klaassen, K., Spasovski, V., Ugrin, M., Komazec, J., Stanković, S., Jocić, N., Pavlović, S., Stojiljković, M.,& Skakić, A.. (2023). Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade., 65-65. https://hdl.handle.net/21.15107/rcub_imagine_2121
Parezanović M, Anđelković M, Stevanović N, Klaassen K, Spasovski V, Ugrin M, Komazec J, Stanković S, Jocić N, Pavlović S, Stojiljković M, Skakić A. Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia. 2023;:65-65. https://hdl.handle.net/21.15107/rcub_imagine_2121 .
Parezanović, Marina, Anđelković, Marina, Stevanović, Nina, Klaassen, Kristel, Spasovski, Vesna, Ugrin, Milena, Komazec, Jovana, Stanković, Sara, Jocić, Nikola, Pavlović, Sonja, Stojiljković, Maja, Skakić, Anita, "Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB" in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia (2023):65-65, https://hdl.handle.net/21.15107/rcub_imagine_2121 .