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

La Lambda selezionare cII , sistema di rilevazione di mutazione

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

Summary

Descriviamo un protocollo dettagliato per l’analisi di mutazione di Lambda selezionare cII in cellule coltivate di roditori transgenici o corrispondenti animali trattati con un agente chimico/fisiche di interesse. Questo approccio è stato ampiamente utilizzato per il test di mutagenicità degli agenti cancerogeni in cellule di mammifero.

Abstract

Una serie di modelli animali transgenici e sistemi di rilevazione di mutazione è stata sviluppata per il test di mutagenicità degli agenti cancerogeni in cellule di mammifero. Di questi, topi transgenici e la Lambda (λ) Select cII sistema di rilevazione di mutazione sono stati impiegati per esperimenti di mutagenesi da molti gruppi di ricerca in tutto il mondo. Qui, descriviamo un protocollo dettagliato per il Lambda selezionare cII test di mutazione, che può essere applicato alle cellule coltivate di topi/ratti transgenici o gli animali corrispondenti trattati con un agente chimico/fisiche di interesse. Il protocollo prevede i passaggi seguenti: (1) isolamento di DNA genomic dalle cellule o tessuti o organi di animali transgenici trattati in vitro o in vivo, rispettivamente, con un composto in esame; (2) il recupero del vettore navetta lambda portatrice di un gene reporter mutazionale (cioè, cII transgene) dal DNA genomico; (3) imballaggio dei vettori hanno salvati in infettivi batteriofagi; (4) che infetta un batterio ospite e coltura in condizioni selettive per consentire la propagazione delle mutazioni indotte cII ; e (5) segnando il cII-analisi per determinare la frequenza dei mutanti cII e spettro di mutazione, rispettivamente di sequenza del DNA e mutanti.

Introduction

Una vasta gamma di modelli animali transgenici e sistemi di rilevazione di mutazione sono stati sviluppati per il test di mutagenicità degli agenti cancerogeni in cellule di mammifero. Di questi, topi transgenici Big Blue (di seguito chiamato BB) e λ Select cII sistema di rilevazione di mutazione sono stati impiegati per esperimenti di mutagenesi da questo gruppo e molti altri ricerca gruppi in tutto il mondo1,2, 3,4,5,6,7,8,9. Per i 16 anni, abbiamo studiato gli effetti mutageni di vari agenti chimici e/o fisici, utilizzando questi animali transgenici o loro colture di cellule embrionali del fibroblasto corrispondente trattato con un composto in esame e successivamente analizzato il fenotipo e genotipo del transgene cII dal saggio Select cII λ e sequenziamento del DNA, rispettivamente10,11,12,13,14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24. il genoma di questi animali transgenici contiene un vettore navetta λ di batteriofago (λLIZ) integrato sul cromosoma 4 come multi-copia testa-coda concatemer1,2,25. Il vettore λLIZ navetta trasporta due geni reporter mutazionale, vale a dire i lacI e cII transgeni1,2,25,26,27, 28,29,30,31,32,33,34,35,36, 37,38,39,40,41,42,43,44,45 , 46 , 47. il saggio di Select cII λ si basa sul recupero dei vettori navetta λLIZ dal DNA genomico delle cellule derivate da tessuti o organi di animali transgenici1,2,25 . I vettori di navetta λLIZ recuperati vengono quindi assemblati in teste di fago λ in grado di infettare un ospite indicatore Escherichia coli. Successivamente, i batteri infetti sono coltivati in condizioni selettive per consentire per il punteggio e l’analisi delle mutazioni in cII transgene1,3.

Qui, descriviamo un protocollo dettagliato per il dosaggio di Select cII λ, che consiste di isolamento del DNA genomico da cellule/organi di animali transgenici trattati in vitro/in vivo con un test composto, recupero della λLIZ navetta vettori da DNA genomic, imballaggio dei vettori in fagi λ infettiva, infezione dell’ospite e. coli con batteriofagi, identificazione di cII-mutanti sotto condizioni selettive per determinare la frequenza mutante cII , e Analisi di sequenza del DNA per stabilire lo spettro di mutazione di cII . Il protocollo può essere applicato a transgenici topo/ratto cell culture trattate in vitro con un agente chimico/fisiche di interesse, o tessuti/organi degli animali corrispondenti trattati in vivo con il test chimico/agente1, 2,4,48,49,50,51,52. Una presentazione schematica del dosaggio Select cII λ è illustrata nella Figura 1.

Protocol

1. isolamento genomic del DNA da fibroblasti embrionali del Mouse Nota: Fibroblasti embrionali del mouse primario sono isolate da embrioni ottenuti da topi transgenici BB con background genetico C57BL/6, secondo il protocollo pubblicato53. Il materiale di partenza per questo protocollo è costituito da 1 x 106 a 1 x 107 embrionali del fibroblasto cellule trattate con un test composto rispetto al controllo. La raccolta e il conteggio di quest…

Representative Results

A seconda della distribuzione dei dati, parametrici o non parametrici test vengono utilizzati per determinare il significato di differenza nella frequenza mutante cII tra gruppi di controllo e trattamento (cioè, indotta contro frequenze mutante spontanee) . Confronto delle frequenze mutante indotto cII attraverso diversi gruppi di trattamento è fatto da varie (pairwise) test statistici, come applicabile. La prova di ipergeometrica di Adams e Skopek comunemente…

Discussion

Il dosaggio di Select cII λ viene utilizzato per il rilevamento delle mutazioni nel transgene cII recuperato dal DNA genomico delle cellule derivate da organi/tessuti di BB roditori3. Il genoma di questi animali transgenici contiene più tandem copie del vettore navetta cromosomicamente integrato λLIZ, che trasporta il cII (294 bp) e lacI (1.080 bp) transgeni, come il reporter mutazionale di geni1, 2 <…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vorremmo riconoscere i contributi di tutti i colleghi e collaboratori per i nostri studi originali, i cui risultati sono stati denominati in questo manoscritto (a scopo esemplificativo). Lavoro degli autori è sostenuto da borse di studio dal National Institute of Dental e Craniofacial Research del National Institutes of Health (1R01DE026043) di AB e dall’Università di programma di ricerca di malattia di California Tobacco-Related a (AB TRDRP-26IR-0015) e ST (TRDRP-25IP-0001). Gli sponsor dello studio non avevano alcun ruolo nel disegno dello studio, raccolta dati, analisi dei dati, interpretazione dei dati, scrittura della relazione, o nella decisione di presentare per la pubblicazione.

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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