Sık Kullanılan Bazı Hücre Hatları için Kalite Kontrol: Mikoplazma Kontaminasyon Tespiti, Sitokrom B ve Sitokrom Oksidaz Alt Birim I Genlerinin DNA Dizi Analizlerinin Gerçekleştirilmesi

Year 2022, Issue 18, 770 - 786, 31.12.2022

Habibe KAHYA Tugce KARADUMAN

https://doi.org/10.38079/igusabder.1114239

Abstract

Amaç: Laboratuvarlarda sık kullanılan serviks epitelyal karsinom (HeLa), insan periferal kan promiyelösitik lösemi (HL-60), fare C3/bağ dokusu (L929), Madin Darby köpek böbrek (MDCK), fare nöroblastom (Neuro-2a) gibi bazı hücre hatlarının mikoplazma kontaminasyon kontrollerinin yapılması, kimlik doğrulamalarının gerçekleştirilmesi ve klonalitelerinin belirlenmesidir.

Yöntem: Bu çalışmada üç farklı türe ait beş hücre hattı kullanılmıştır. Çalışılan tüm hatların Bisbenzimid (Hoechst 33258) ile deoksiribonükleik asit (DNA) floresan işaretlemesi yapılarak mikoplazma kontaminasyonu kontrolleri gerçekleştirilmiştir. Hücre hatlarından DNA izolasyonları yapılmış, elde edilen DNA örneklerinden sitokrom B (CYTB) geninin bölgesel amplifikasyonu için L14816 ve H15173 primerleri; sitokrom oksidaz alt birim I (COI) geni için ise LCO 1490 and HCO 2198 primerleri kullanılmıştır. İlgili amplifikasyonların DNA dizi analiz sonuçları, biyoinformatik araçlar kullanılarak referans dizilerle karşılaştırmalı olarak değerlendirilmiştir.

Bulgular: Çalışmada ilgili hücrelerin, Bisbenzimid (Hoechst 33258) ile üretici firmanın protokollerine göre belirlenen konsantrasyon ve sürede yapılan boyama sonucunda mikoplazma kontaminasyonuna rastlanılmamıştır. Ayrıca CYTB gen bölgesi için veritabanında yer alan referans dizi ile yapılan karşılaştırma sonucu 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 “HeLa, HL-60, L929, MDCK ve Neuro-2a” hücre hatları için sırasıyla %95, %99, %96, %96 ve %98 olarak bulunmuştur.

Sonuç: Bu bağlamda, çalışmadan elde edilen Bisbenzimid (Hoechst 33258) işaretleme ve DNA dizi analiz sonuçları, pek çok araştırmada kullanılan bu hücre hatlarının kalitesi konusunda kabul edilebilir bir belirteç ve güven sağlamıştır.   

Keywords

Hücre kültürü, CYTB, COI, DNA dizi analizi, mikoplazma, Hoechst 33258

Quality Control for Some Commonly Used Cell Lines: Mycoplasma Contamination Detection, DNA Sequence Analysis of Cytochrome B and Cytochrome Oxidase Subunit I Genes

Abstract

Aim: Mycoplasma contamination controls, identity verification and clonality determination of some cell lines such as cervix epithelioid carcinoma (HeLa), human peripheral blood promyelocytic leukemia (HL-60), mouse C3 / a connective tissue (L929), madin darby canine kidney (MDCK), mouse neuroblastoma (Neuro-2a) that are frequently used in laboratories.

Method: Five cell lines from three different species were used in this study. Mycoplasma contamination controls of all studied lines were performed by deoxyribonucleic acid (DNA) fluorescent labeling with Bisbenzimid (Hoechst 33258). DNA isolation was performed on cell lines and DNA samples were obtained. Regional amplification of obtained DNA samples was performed as follows: L14816 and H15173 primers were used for cytochrome B (CYTB) gene; LCO 1490 and HCO 2198 primers were used for cytochrome oxidase subunit I (COI) gene. Results of DNA sequence analysis for respective amplifications were compared with reference sequences using bioinformatics tools.

Results: In the study, no mycoplasma contamination was observed as a result of staining relevant cells with Bisbenzimid (Hoechst 33258) at the concentration and time determined by the manufacturer's protocols. In addition, comparing the results for CYTB gene region with the reference sequence in the database resulted in 97% similarity for HL-60, and 98% similarity for “HeLa, L929, MDCK, Neuro-2a” cell lines. Similarity rates for COI gene region were 95%, 99%, 96%, 96%, and 98% for “HeLa, HL-60, L929, MDCK and Neuro-2a” cell lines respectively.

Conclusion: In this context, Bisbenzimid (Hoechst 33258) labeling and DNA sequence analysis results obtained from the study provided an acceptable indicator and confidence for the quality of these cell lines used in many studies.

Keywords

Cell culture, CYTB, COI, DNA sequence analysis, mycoplasma, Hoechst 33258

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