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

Lambda Vælg cII Mutation Detection System

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

Summary

Vi beskriver en detaljeret protokol for Lambda Vælg cII mutation assay i kulturperler celler af transgene gnavere eller de tilsvarende dyr behandlet med et kemisk/fysisk agent af interesse. Denne tilgang har været meget anvendt for mutagenicitet afprøvning af kræftfremkaldende stoffer i pattedyrsceller.

Abstract

En række af transgene dyremodeller og mutation detektionssystemer er blevet udviklet for mutagenicitet afprøvning af kræftfremkaldende stoffer i pattedyrsceller. Af disse, er Transgene mus og Lambda (λ) Vælg cII Mutation Detection System blevet ansat for mutagenicitet eksperimenter af mange forskergrupper verden over. Her, vi beskriver en detaljeret protokol for Lambda Vælg cII mutation analysen, som kan anvendes til kulturperler celler af Transgene mus/rotter eller de tilsvarende dyr behandlet med et kemisk/fysisk agent af interesse. Protokollen består af følgende trin: (1) isolering af genomisk DNA fra celler eller organer/væv af transgene dyr behandles i in vitro eller i vivo, henholdsvis med en test sammensatte; (2) inddrivelse af lambda shuttle vektor bærer mutationsmønstre reporter gen (dvs., cII transgen) fra genomisk DNA; (3) emballage af de reddede vektorer i smitsomme Bakteriofager; (4) inficere en vært bakterier og dyrkning på selektiv betingelser at tillade formering af de inducerede cII mutationer; og (5) scoring cII-mutanter og DNA sekvens analyse for at bestemme cII mutant frekvens og mutation spektrum, henholdsvis.

Introduction

En bred vifte af transgene dyremodeller og mutation detektionssystemer er blevet udviklet for mutagenicitet afprøvning af kræftfremkaldende stoffer i pattedyrsceller. Af disse, har transgene Big Blue (kaldet herefter BB) mus og λ Vælg cII Mutation Detection System været beskæftiget for mutagenicitet eksperimenter i denne gruppe og mange andre forskning grupper verden over1,2, 3,4,5,6,7,8,9. For de sidste 16 år, har vi undersøgt de mutagene virkninger af forskellige kemiske og/eller fysiske agenser ved hjælp af disse transgene dyr eller deres tilsvarende embryonale fibroblast cellekulturer behandles med en test sammensatte, og efterfølgende analyseres de fænotype og genotype af cII transgenet ved λ Vælg cII analyse og DNA-sekventering, henholdsvis10,11,12,13,14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24. genomet af disse transgene dyr indeholder en bacteriophage λ shuttle vektor (λLIZ) integreret på kromosom 4 som en multi kopi hoved-til-hale concatemer1,2,25. ΛLIZ shuttle vektor bærer to mutationsmønstre reporter gener, nemlig Lund og cII transgener1,2,25,26,27, 28,29,30,31,32,33,34,35,36, 37,38,39,40,41,42,43,44,45 , 46 , 47. λ Vælg cII assay er baseret på genvinding af λLIZ shuttle vektorer fra genomisk DNA af celler, der stammer fra organer/væv af transgene dyr1,2,25 . Gendannede λLIZ shuttle vektorer er derefter pakket ind i λ phage hoveder i stand til at inficere en indikator vært Escherichia coli. Efterfølgende, er de inficerede bakterier vokset selektiv betingelser at tillade scoring og analyse af mutationer i cII transgen1,3.

Her, vi beskriver en detaljeret protokol for λ Vælg cII assay, som består af isolation af genomisk DNA fra celler/organer fra transgene dyr behandlet in vitro-/i vivo med en test sammensatte, hentning af λLIZ shuttle vektorer fra genomisk DNA emballage af vektorerne i smitsomme λ phages, infektion af værten E. coli med Bakteriofager, identifikation af cII-mutanter selektiv betingelser at fastlægge cII mutant frekvens, og DNA Sekvensanalyse at etablere cII mutation spektrum. Protokollen kan anvendes til Transgene mus/rotte celle kulturer behandlet in vitro- med en kemisk/fysisk agent af interesse, eller væv/organer af de tilsvarende dyr behandlet i vivo test kemiske/agent1, 2,4,48,49,50,51,52. En skematisk præsentation af λ Vælg cII assay er vist i figur 1.

Protocol

1. genomisk DNA isoleret fra mus embryonale fibroblaster Bemærk: Primære mus embryonale fibroblaster er isoleret fra embryoner stammer fra BB Transgene mus med C57BL/6 genetiske baggrund, ifølge offentliggjorte protokol53. Udgangsmateriale for denne protokol består af 1 x 106 til 1 x 107 embryonale fibroblastceller behandles med en test sammensatte versus kontrol. Høst og optælling af disse celler ved hjælp af standard metoder er besk…

Representative Results

Afhængigt af datadistribution bruges parametrisk eller ikke-parametrisk test til at bestemme betydningen af forskellen i cII mutant frekvens mellem behandling og kontrol grupper (dvs., induceret versus spontane mutant frekvenser) . Sammenligning af de inducerede cII mutant frekvenser på tværs af forskellige behandlingsgrupper er lavet af forskellige (parvis) statistiske test, hvor det er relevant. Hypergeometrisk test af Adams og Skopek er almindeligt anvendt…

Discussion

Λ Vælg cII analysen bruges til påvisning af mutationer i cII transgenet inddrives fra genomisk DNA af celler, der stammer fra organer/væv af BB gnavere3. Genomet af disse transgene dyr indeholder flere tandem kopier af chromosomally integreret λLIZ shuttle vektor, som bærer cII (294 bp) og Lund (1.080 bp) transgener, som mutationsmønstre reporter gener1, 2 , 25. …

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi vil gerne anerkende bidragene fra alle kolleger og samarbejdspartnere til vores oprindelige undersøgelser, hvis resultater er blevet henvist til i dette manuskript (til orientering). Forfatternes arbejde støttes af tilskud fra National Institute for Dental og Craniofacial forskning af National Institutes of Health (1R01DE026043) til AB og fra University of California Tobacco-Related sygdom Research Program til AB ( TRDRP-26IR-0015) og ST (TRDRP-25IP-0001). Sponsorer af undersøgelsen havde ingen rolle i undersøgelse design, dataindsamling, analyse af data, data fortolkning, skrivning af rapporten eller i beslutningen om at indsende til offentliggørelse.

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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