JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Immunology and Infection

Lambda seçin CII mutasyon tespit sistemi

Published: April 26, 2018
doi:
10.3791/57510
,
1Department of Preventive Medicine, USC Keck School of Medicine,University of Southern California

Summary

Biz Lambda seçin CII mutasyon tahlil transgenik kemirgenler kültürlü hücrelerdeki veya faiz kimyasal/fiziksel ajanı ile tedavi karşılık gelen hayvanlar için detaylı bir protokol tanımlamak. Bu yaklaşım yaygın kanserojen memeli hücreleri, mutagenisitesinin test etmek için kullanılmıştır.

Abstract

Transgenik hayvan modelleri ve mutasyon tespit sistemleri bir dizi memeli hücreleri kanserojen mutagenisitesinin test için geliştirilmiştir. Bunlar transgenik fareler ve Lambda (λ) Select CII mutasyon tespit sistemi mutagenisitesinin deneyler için birçok araştırma grupları tarafından dünya çapında istihdam edilmiştir. Burada, biz transgenik fareler/fareler kültürlü hücrelere uygulanan Lambda seçin CII mutasyon tahlil için detaylı bir protokol tanımlamak veya faiz kimyasal/fiziksel Aracısı ile ilgili hayvan tedavi. Protokol aşağıdaki adımlardan oluşur: (1) yalıtım hücrelerden genomik DNA’ın veya organ/Transjenik hayvanlar dokuların vitro veya vivo içindesırasıyla, bir test karışımla; tedavi (2) (Yani, CII transgene) mutational muhabir gen genomik DNA taşıyan lambda Servisi vektör kurtarılması; (3) bulaşıcı bacteriophages içine kurtarıldı vektörlerin ambalaj; (4) bir ana bilgisayar bakteri hastalık ve seçici koşullar altında indüklenen CII mutasyonlar yayılmasını sağlamak için kültür; ve (5) skorlama CII-mutantlar ve DNA dizi mutasyon spektrum ve CII mutant sıklığı sırasıyla belirlemek için analizi.

Introduction

Transgenik hayvan modelleri ve mutasyon tespit sistemleri geniş bir memeli hücreleri kanserojen mutagenisitesinin test için geliştirilmiştir. Bunlar transgenik Big Blue (bundan sonra BB adlandırılır) fareler ve λ Select CII mutasyon tespit sistemi mutagenisitesinin deneyler için bu grup ve birçok diğer araştırma grupları dünya çapında1,2tarafından istihdam edilmiştir, 3,4,5,6,7,8,9. Son 16 yıldır, biz bu Transjenik hayvanlar kullanarak çeşitli kimyasal ve/veya fiziksel ajanların mutajenik etkileri araştırdık veya onların karşılık gelen embriyonik fibroblast hücre kültürlerinde bir test karışımı ile tedavi ve daha sonra analiz λ Select CII tahlil ve DNA sıralama, sırasıyla10,11,12,13,14 CII transgene genotip ve fenotip , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24. bu Transjenik hayvanlar genom kromozom 4 Çoklu kopya kafa kuyruk concatemer1,2,25üzerinde entegre bir bakteriyofaj λ Servisi vektör (λLIZ) içerir. ΛLIZ Servisi vektör iki mutational muhabir gen taşır yani lacI ve CII transgenes1,2,25,26,27, 28,29,30,31,32,33,34,35,36, 37,38,39,40,41,42,43,44,45 , 46 , 47. λ Select CII tahlil λLIZ Servisi vektörel çizimler kurtarma organ/dokulardan Transjenik hayvanlar1,2,25 türetilmiş hücre genomik DNA dayanır . Kurtarılan λLIZ Servisi vektörel çizimler sonra bir göstergesi ana Escherichia colibulaşmasını yetenekli λ fajının zihinlerine paketlenmiştir. Daha sonra virüs bulaşan bakteriler puanlama ve CII transgene1,3mutasyon analizi için izin vermek için seçici koşullar altında yetiştirilmektedir.

Burada, biz hücreleri/Transjenik hayvanlar tedavi vitroorganlarının genomik DNA izolasyonu oluşur λ Select CII tahlil için detaylı bir protokol tarif /vivo λLIZ test bileşik, alımı ile olan yakındaki vektörel çizimler genomik DNA vektörler ambalaj içine bulaşıcı λ phages, bacteriophages, CIItanımlaması ile ana E. coli enfeksiyonu- CII mutant frekansını belirlemek için mutantlar seçici koşullar altında ve DNA dizi analizi CII mutasyon spektrum oluşturmak için. Protokol transgenik mouse/fare hücre kültürleri tedavi vitro ilgi bir kimyasal/fiziksel maddesi ile uygulanabilir veya doku/organ karşılık gelen hayvanların vivo içinde test kimyasal/ajan1ile tedavi, 2,4,48,49,50,51,52. λ Select CII tahlil şematik bir sunumunu şekil 1‘ de gösterilen.

Protocol

1. fare embriyonik fibroblastlar genomik DNA izolasyonu Not: Birincil fare embriyonik fibroblastlar göre yayımlanmış protokolü53C57BL/6 genetik arka plan ile BB transgenik fareler türetilen embriyo etkilenmezsiniz. Bu iletişim kuralı için başlangıç materyali 1 x 106 -bileşik karşı test denetimi ile tedavi 1 x 107 embriyonik fibroblast hücreleri oluşur. Hasat ve standart yöntemlerle bu hücrelerin sayma başvurular<sup class…

Representative Results

Veri dağıtımı bağlı olarak, parametrik ya da non-parametrik testler (spontan mutant Frekanslar karşı indüklenenYani) tedavi ve kontrol grupları arasında CII mutant frekans farkı önemini belirlemek için kullanılır . İndüklenen CII mutant frekansları farklı tedavi grupları arasında karşılaştırılması yapılmış çeşitli tarafından (ikili) uygulanabilir istatistiksel testler. χ2 test veya Varyans analizi (ANOVA), gibi diğe…

Discussion

λ Select CII tahlil organ/dokulardan BB kemirgenler3türetilmiş hücre genomik DNA kurtarıldı CII transgene mutasyonların tespiti için kullanılır. Bu Transjenik hayvanlar genom CII taşıdığı genetik entegre λLIZ Servisi vektör, birden çok tandem kopyalarını içerir (294 bp) ve lacI (1,080 bp) transgenes, mutational muhabir gen1, 2 , 25. λ Select CI…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Tüm meslektaşlarımızın katkıları ve ortak çalışanlar, sonuçlar için bu el yazması (görsel amacıyla) sevk edilmiştir orijinal çalışmalarımız için kabul etmek istiyoruz. Yazarların iş hibe Ulusal Enstitüsü diş ve ulusal sağlık Enstitüleri (1R01DE026043) AB için kraniyofasiyal araştırma ve California Tobacco-Related hastalığı araştırma programı için AB (Üniversitesi tarafından desteklenmektedir TRDRP-26IR-0015) ve ST (TRDRP-25IP-0001). Sponsorlar çalışmanın çalışma tasarım, veri toplama, veri analizi, veri yorumu, rapor yazma veya yayın için göndermek için karar herhangi bir rolü yoktu.

Materials

Agar MO Bio Laboratories, Inc. 12112-05 Bacteriological grade
BigDye Terminator v3.1 Cycle Sequencing Kit Thermo Fisher Scientific 4337455 None
Casein Peptone Alfa Aesar H26557 None
Gelatine J. T. Baker 2124-01 Powder
Glycerol Fisher Scientific BP 229-1 / M-13750 None
LB Agar Fisher Scientific BP 9724-500 None
QIAquick PCR purification kit Qiagen 8104 50 PCR purification reactions
Sodium Acetate Trihydrate Fisher Scientific M-15756 None
Taq5000 DNA Polymerase  Qiagen 201207 None
Thiamine Hydrochloride Macron Fine Chemicals 2722-57 None
Transpack Packaging Extract Stratagene Corp., Acquired by BioReliance | Sigma-Aldrich Corp. 200223 50 packaging reactions
Tris Base Fisher Scientific BP 152-1 / EC 201-064-4 None
Trypton Biosciences RC-110 None
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Lambda Select CIIMutation DetectionTransgenic RodentsMutagenicity TestingMammalian CellsReporter GenePackaging ReactionSM BufferChloroformPlating CultureTiteringScreeningE. ColiPhagesTB1 Top Agar

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Besaratinia, A., Tommasi, S. The Lambda Select cII Mutation Detection System. J. Vis. Exp. (134), e57510, doi:10.3791/57510 (2018).
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