Sık kullanılan bazı hücre hatları için kalite kontrol: Sitokrom B ve sitokrom oksidaz alt birim I genlerinin DNA dizi analizlerinin gerçekleştirilmesi
Yükleniyor...
Dosyalar
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Aksaray Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu tez çalışmasında, HeLa, Cervix epithelloid carcinom (İnsan), HL-60, Human peripheral blood promyelocytic leukemia (İnsan), L929, Mouse C3 / An connective tissue (Fare), MDCK, Madin Darby canine kidney (Köpek), Neuro-2a, Mouse neuroblastoma (Fare) hücre hatlarının, kontaminasyon kontrolleri yapılmış, kimlik doğrulamaları gerçekleştirilmiş ve klonaliteleri belirlenmiştir. İlk olarak, kültüre edilen hücrelerin DNA'larından amplifiye edilen mitokondriyal sitokrom b (CYTB) ve sitokrom c oksidaz alt birim I (COI) genlerinin DNA dizi analizleri gerçekleştirilmiştir. Bu genler evrensel primerler tarafından tanınan stabil dizilere ve aynı zamanda kimliklendirmeyi sağlayacak birtakım dizi çeşitliliklerine sahiptir. Tezde CYTB gen bölgesi için veritabanında yer alan referans dizi ile yapılan karşılaştırma sonuçları "HL-60 için %97; "HeLa, L929, MDCK, Neuro-2a" hücre hatları için ise %98 oranında benzerlik bulunmuştur. COI gen bölgesi için ise bu benzerlik oranları sırasıyla %95, %99, %96, %96 ve %98 olarak bulunmuştur. Bu bağlamda, elde edilen DNA dizi analiz sonuçları, pek çok araştırmada kullanılan "HeLa, HL-60, L929, MDCK, Neuro-2a hücre hatlarının kalitesi konusunda kabul edilebilir bir belirteç ve güven sağlamıştır. Aynı zamanda mikoplazma kontaminasyonu açısından değerlendirme yapılmış ve mikoplazma kontaminasyonuna rastlanılmamıştır. Ulaşılmak istenen hedefler ve beklenen çıktıların bilimsel, teknolojik ve sosyo-ekonomik ne tür katkılarda bulunabileceği, deneysel çalışmalarda kullanılan hücre hatlarının karakterizasyonunun belirlenmesi, bu hücrelerle gerçekleştirilen araştırma sonuçlarının güvenilirliği açısından önem arz etmektedir. Tamamlanan tez ile çalışmaların ana kaynağını oluşturan hücre hatlarının kimliği ortaya konulmuş, bu kimliklendirme, yürütülen hücre kültürü çalışmalarının kesinlik ve güvenilirliğine katkı sağlayacak veriler ortaya koymuştur.
In this thesis, HeLa, Cervix epithelloid carcinom (Human), HL-60, Human peripheral blood promyelocytic leukemia (Human), L929, Mouse C3 / An connective tissue (Mouse), MDCK, Madin Darby canine kidney (Dog), Neuro-2a, Mouse neuroblastoma (Mouse) cell lines were checked for contamination, their identity was verified and their clonality was determined. Theoretical approach and the outline of the method, in this study, DNA sequence analysis of mitochondrial cytochrome b (CYTB) and cytochrome c oxidase subunit I (COI) genes amplified from the DNA of cultured cells was aimed. These genes have stable sequences recognized by universal primers and a number of sequence variants to provide identification at the same time. In the thesis, the results of the comparison with the reference sequence in the database for the CYTB gene region were found to be 97% similar for "HL-60" and 98% for "HeLa, L929, MDCK, Neuro-2a" cell lines. For the COI gene region, these similarity rates were found to be 95%, 99%, 96%, 96% and 98%, respectively. In this context, DNA sequence analysis results obtained provided an acceptable indicator and confidence in the quality of "HeLa, HL-60, L929, MDCK, NA" cell lines used in many studies. At the same time, an evaluation was made in terms of mycoplasma contamination and no mycoplasma contamination was found. Targets and scientific, technological and socio-economic contributions: The determination of the characterization of cell lines used in experimental studies is important in terms of the reliability of the results of the research carried out with these cells. With the completed thesis, the identity of the cell lines that constitute the main source of the studies has been revealed, and this identification has revealed data that will contribute to the precision and reliability of the cell culture studies.
In this thesis, HeLa, Cervix epithelloid carcinom (Human), HL-60, Human peripheral blood promyelocytic leukemia (Human), L929, Mouse C3 / An connective tissue (Mouse), MDCK, Madin Darby canine kidney (Dog), Neuro-2a, Mouse neuroblastoma (Mouse) cell lines were checked for contamination, their identity was verified and their clonality was determined. Theoretical approach and the outline of the method, in this study, DNA sequence analysis of mitochondrial cytochrome b (CYTB) and cytochrome c oxidase subunit I (COI) genes amplified from the DNA of cultured cells was aimed. These genes have stable sequences recognized by universal primers and a number of sequence variants to provide identification at the same time. In the thesis, the results of the comparison with the reference sequence in the database for the CYTB gene region were found to be 97% similar for "HL-60" and 98% for "HeLa, L929, MDCK, Neuro-2a" cell lines. For the COI gene region, these similarity rates were found to be 95%, 99%, 96%, 96% and 98%, respectively. In this context, DNA sequence analysis results obtained provided an acceptable indicator and confidence in the quality of "HeLa, HL-60, L929, MDCK, NA" cell lines used in many studies. At the same time, an evaluation was made in terms of mycoplasma contamination and no mycoplasma contamination was found. Targets and scientific, technological and socio-economic contributions: The determination of the characterization of cell lines used in experimental studies is important in terms of the reliability of the results of the research carried out with these cells. With the completed thesis, the identity of the cell lines that constitute the main source of the studies has been revealed, and this identification has revealed data that will contribute to the precision and reliability of the cell culture studies.
Açıklama
Anahtar Kelimeler
Hücre Kültürü, CYTB, COI, DNA Dizi Analizi, Mikoplazma, Hoechst 33258, Cell Culture, DNA Sequence Analysis, Mycoplasma
