CRISPR/CAS9 Genom Düzenleme Aracı Kullanarak TGFBI Geninde R124H Mutasyonu Oluşturmaya Yönelik gRNA ve Donör DNA Tasarlama ve DH5ALFA Vektörüne Klonlama
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2022
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Selçuk Üniversitesi Sağlık Bilimleri Enstitüsü
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info:eu-repo/semantics/openAccess
Abstract
TGFBI (tranforme edici büyüme faktörü beta ile indüklenen) proteini, kornea dahil pek
çok dokuda ifade olan matriks proteinidir. Özellikle korneada hücre dıĢı matriksin major bileĢeni
olarak bulunan TGFBI, farklı tip kollajenler ve integrinlerle etkileĢime girerek yapısal
bütünlüğün ve kornea Ģeffaflığının korunmasında önemli rol oynamaktadır. TGFBI proteinini
kodlayan TGFBI geninde meydana gelen mutasyonlar, korneada anormal TGFBI katlanması ve
kümelenmesi ile seyreden, ciddi görme bozukluğu ve kaybına neden olan farklı tipte granüler
kornea distrofi (GCD)’lerine yol açmaktadır. Korneal stromada birden fazla ayrı ve düzensiz
Ģekilli granüler opasitelerin biriktiği otozomal dominant veya resesif kalıtılan GCD formları
bilinmektedir. Primer kornea hücrelerinin eldesinde ve kültürüzasyonundaki güçlükler nedeniyle
kornea epitel hücrelerinin kimliklendirilmesi, biyolojisi, hücresel mekanizmaları ve bunların
hastalık patogenezindeki rolleri anlaĢılamamakta ve etkin ilaç deneme çalıĢmaları
yapılamamaktadır. Bu nedenle, en yaygın görülen GCD formlarından biri olan GCD Tip 2
(GCD2) kornea distrofisinin, CRISPR/Cas9 (düzenli aralıklarla bölünmüĢ kısa palindromik
tekrar kümeleri/CRISPR-iliĢkili nükleaz-9) genom düzenleme teknolojisi kullanılarak, hücresel
düzeyde modellenebilmesi amaçlandı. Bu çalıĢmada hücresel düzeyde GCD2 hastalığının
modellenebilmesi için gerekli klonlama ve doğrulama adımlarının yapılması hedeflendi. Bunun
için GCD2 kornea distrofisine neden olduğu bilinen TGFBI genindeki Arg124His (R124H)
mutasyonunu oluĢturmaya yönelik single guide RNA (sgRNA) ve donör DNA tasarlandı.
https://www.benchling.com/ online software programı kullanılarak tasarlanan ve ticari olarak
temin edilen gRNA’lar plazmit vektörüne (79145, Addgene) klonlandı. Klonlama aĢamasının
ardından bir E.coli varyantı olan DH5alpha bakterisine transformasyon yapıldı. Transformasyon
sonrası, koloni PZR ve dizi analizi gerçekleĢtirildi. Dizi analizi sonuçlarına göre gRNA’ların
baĢarıyla klonlandığı gösterildi. Böylece ökaryotik hücreye transfeksiyon için hazır
CRISPR/Cas9 vektörler elde edildi. Hücresel ve moleküler mekanizmaları hala tam olarak
bilinmeyen ve kesin tedavisi olmayan bu hastalığın modellenmesi hastalığın temel
mekanizmasının daha detaylı anlaĢılması ve hedefe yönelik tedavi yaklaĢımlarının
geliĢtirilebilmesine önemli katkılar sağlayacaktır.
TGFBI (transforming growth factor beta-induced) protein is a matrix protein that is expressed in many tissues, including the cornea. TGFBIp, which is a major component of the extracellular matrix especially in the cornea, plays an important role in maintaining the structural integrity and transparency of the cornea by interacting with different types of collagens and integrins. Mutations in the TGFBI gene which encodes the TGFBI protein, lead to different types of granular corneal dystrophy (GCD), characterized by abnormal folding and accumulation of TGFBIp in the cornea, causing severe visual impairment and loss. Autosomal dominant or recessive forms of GCD in which more than one discrete and irregularly shaped granular opacities accumulate in the corneal stroma, are known. Due to the difficulties of obtaining and culturing primary corneal cells, identification of corneal epithelial cells, their biology, cellular mechanisms and roles in the pathogenesis of the disease cannot be understood, and effective drug trials cannot be performed. Therefore, in our study, it was aimed to model GCD Type 2 (GCD2) corneal dystrophy, one of the most common forms of GCD, at cellular level using CRISPR/Cas9 genome editing technology targeting to perform the necessary cloning and validation steps for modeling. Guide RNA (gRNA) and donor DNA were designed to generate the Arg124His (R124H) mutation in the TGFBI gene, which is known to cause GCD2 corneal dystrophy. gRNAs, designed using the https://www.benchling.com/ online software program and obtained commercially, were cloned into the plasmid vector (79145, Addgene). Following the cloning step, vectors were transformed into DH5alpha bacteria, an E.coli variant. After transformation, colony PCR and sequence analysis were performed. Successfully cloned gRNAs were confirmed according to the results of the sequence analysis. By this way, CRISPR/CAS9 construct vectors ready for transfection into eukaryotic cell have been created. Modeling of this disease, which cellular and molecular mechanisms are still unknown and has not a definite treatment, will make important contributions to a more detailed understanding of the basic mechanism of the disease and the development of targeted treatment approaches.
TGFBI (transforming growth factor beta-induced) protein is a matrix protein that is expressed in many tissues, including the cornea. TGFBIp, which is a major component of the extracellular matrix especially in the cornea, plays an important role in maintaining the structural integrity and transparency of the cornea by interacting with different types of collagens and integrins. Mutations in the TGFBI gene which encodes the TGFBI protein, lead to different types of granular corneal dystrophy (GCD), characterized by abnormal folding and accumulation of TGFBIp in the cornea, causing severe visual impairment and loss. Autosomal dominant or recessive forms of GCD in which more than one discrete and irregularly shaped granular opacities accumulate in the corneal stroma, are known. Due to the difficulties of obtaining and culturing primary corneal cells, identification of corneal epithelial cells, their biology, cellular mechanisms and roles in the pathogenesis of the disease cannot be understood, and effective drug trials cannot be performed. Therefore, in our study, it was aimed to model GCD Type 2 (GCD2) corneal dystrophy, one of the most common forms of GCD, at cellular level using CRISPR/Cas9 genome editing technology targeting to perform the necessary cloning and validation steps for modeling. Guide RNA (gRNA) and donor DNA were designed to generate the Arg124His (R124H) mutation in the TGFBI gene, which is known to cause GCD2 corneal dystrophy. gRNAs, designed using the https://www.benchling.com/ online software program and obtained commercially, were cloned into the plasmid vector (79145, Addgene). Following the cloning step, vectors were transformed into DH5alpha bacteria, an E.coli variant. After transformation, colony PCR and sequence analysis were performed. Successfully cloned gRNAs were confirmed according to the results of the sequence analysis. By this way, CRISPR/CAS9 construct vectors ready for transfection into eukaryotic cell have been created. Modeling of this disease, which cellular and molecular mechanisms are still unknown and has not a definite treatment, will make important contributions to a more detailed understanding of the basic mechanism of the disease and the development of targeted treatment approaches.
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Keywords
CRISPR/Cas9, GCD2 Kornea distrofisi, Klonlama, R124H mutasyonu, TGFBI, Cloning, GCD2 Corneal Dystrophy, R124H mutation
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Kaldırım, B., (2022). CRISPR/CAS9 Genom Düzenleme Aracı Kullanarak TGFBI Geninde R124H Mutasyonu Oluşturmaya Yönelik gRNA ve Donör DNA Tasarlama ve DH5ALFA Vektörüne Klonlama. (Yüksek Lisans Tezi). Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, Konya.
