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

ラムダを選択cII変異検出システム

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

Summary

トランスジェニックげっ歯類の培養細胞でラムダを選択cII突然変異試験または興味の化学/物理エージェントで治療対応する動物のための詳しいプロトコルについて述べる。このアプローチは、哺乳類細胞における発がん性物質の変異原性試験のため広く使用されています。

Abstract

トランスジェニック動物モデルおよび突然変異検出システムの数は、哺乳類細胞における発がん性物質の変異原性試験のために開発されています。これらのトランスジェニック マウスとラムダ (λ) の選択cII変異検出システム世界中の多くの研究グループによって変異原性実験のため採用されています。ここでは、ラムダを選択cII突然変異試験は、トランスジェニック マウス/ラットの培養細胞に適用することができるための詳しいプロトコルについて述べるまたは興味の化学/物理エージェントに対応する動物が扱われます。プロトコルは、次の手順で構成されます: (1) 分離細胞からゲノム dna やトランスジェニック動物の臓器・組織生体外でまたは生体内で、それぞれで治療テスト混合物;(ゲノム DNA からの (すなわち、 cII遺伝子) の変異レポーター遺伝子を運ぶラムダ シャトルベクターの回復 2)(伝染性のバクテリオファージに救助されたベクトルの包装 3)(4) ホストの細菌に感染して誘導cII突然変異の伝播を許可する選択的な条件下で培養(5) 得点cII-変異体と DNA シーケンスをそれぞれcII突然変異頻度および突然変異スペクトルを分析します。

Introduction

トランスジェニック動物モデルおよび突然変異検出システムの広い範囲は、哺乳類細胞における発がん性物質の変異原性試験のために開発されています。これらの大きなブルー (以下 bb) トランスジェニック マウスと λ 選択cII変異検出システムで採用されている変異原性実験のためこのグループは、多くの他研究グループ世界中1,の2、 3,4,5,6,7,8,9。過去 16 年間、これらのトランスジェニック動物を用いた化学的および/または物理的な剤の変異原性の効果を調べたか、対応する胚性線維芽細胞培養テスト混合物と扱われ、その後の分析、λ 選択cIIアッセイおよび DNA の配列、それぞれ10、11,12,の13,14によってcII遺伝子の遺伝子型と表現型,15,16,17,18,19,20,21,22,23,24. これらのトランスジェニック動物のゲノムに複数コピー頭-尾コンカテマー1,2,25, 染色体 4 統合バクテリオファージ λ シャトル ベクトル (λLIZ) が含まれています。ΛLIZ シャトルベクターを運ぶ 2 つの変異レポーター遺伝子、すなわち、ラッチとcII遺伝子1,2,25,26,27、 28,29,30,31,32,33,34,35,36、 37,38,39,40,41,42,43,44,45,46,47. λ 選択cIIアッセイに基づいて λLIZ シャトル ベクトルのトランスジェニック動物1,2,25 の臓器・組織由来細胞のゲノム DNA からの回復.回復 λLIZ シャトル ベクトル、インジケーター ホスト大腸菌に感染可能な λ ファージの頭にパッケージ化されます。その後、感染した細菌は、得点とcII遺伝子1,3における変異解析を可能にする選択的な条件のもとで栽培しています。

ここ、体外治療トランスジェニック動物の細胞・臓器からゲノム DNA の隔離から成っている λ 選択cIIの試金のための詳しいプロトコルについて述べる/シャトル生体内でλLIZ の化合物は、取得テスト ベクトルgenomic DNA から、バクテリオファージ、 cIIの同定とホストエシェリヒア属大腸菌の感染感染 λ ファージにベクトルの包装- cIIの突然変異頻度を決定する選択的な条件の下で突然変異体とCII変異スペクトルを確立する dna 塩基配列解析。トランスジェニック マウス ・ ラット細胞培養体外興味の化学/物理エージェントに適用できるプロトコルまたは対応する動物の組織/臓器治療体内テスト化学/エージェント1、 2,4,48,49,50,51,52。Λ 選択cIIアッセイの図式を図 1に示します。

Protocol

1 マウス胚性線維芽細胞からゲノム DNA の隔離 注: プライマリ マウス胚性線維芽細胞は、公開されたプロトコル53によると BB C57BL/6 遺伝的背景を持つトランスジェニック マウス由来の胚から分離されます。このプロトコルのための開始材料は、1 x 106 1 x 107胚性線維芽細胞細胞制御化合物とテストで構成されます。収穫と標準的な方?…

Representative Results

データ ディストリビューションによってパラメトリックまたはノンパラのテストを使用して、処置および制御グループ (すなわち、自発の突然変異体の周波数対誘導) のcIIの突然変異頻度の違いの重要性を判断します.誘導cII変異周波数別の治療グループ間での比較が行われます各種 (一対)、適用可能な統計的テスト。Χ2テストや分散分析 (…

Discussion

Λ 選択cIIアッセイは、3BB の齧歯動物の臓器・組織由来細胞のゲノム DNA から回復したcII遺伝子変異の検出に使用されます。これらの形質転換動物ゲノムには染色体統合 λLIZ シャトル ベクトルは、 cIIを運ぶ複数タンデム コピーが含まれます (294 bp) とラッチ(1,080 bp) 遺伝子変異レポーター遺伝子1,2,</su…

Disclosures

The authors have nothing to disclose.

Acknowledgements

すべての同僚の貢献と (説明のため) この原稿に呼ばれている、結果が私たちの元の研究に協力したいと思います。作家の作品は、国立歯科・頭蓋顔面研究、国立衛生研究所 (1R01DE026043) ab の AB (California Tobacco-Related 病研究プログラムの大学からの補助金によってサポートされてTRDRP-26IR-0015) および ST (TRDRP-25IP-0001)。研究のスポンサーには、研究デザイン、データ収集、データ分析、データ解釈、レポートの執筆、出版のために提出する決定の役割はなかった。

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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