Name: in vivo alkaline comet assay and enzyme-modified alkaline comet assay for measuring dna strand breaks and oxidative dna damage in rat liver
Uploaded: 2016-05-04T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver at JoVE.com

This work was supported by the US Food and Drug Administration. We acknowledge the original publication of the CPA study by Elsevier B.V.: Ding W, Bishop ME, Peace MG, Davis KJ, White GA, Lyn-Cook LE, Manjanatha MG. Sex-specific dose-response analysis of genotoxicity in cyproterone acetate-treated F344 rats. Mutation Research 774: 1-7, 2014 (PMID: 25440904)

윤리 문 : 동물과 관련된 절차 독성 연구를위한 미국 FDA / 국립 센터의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었습니다. 참고 : 여기에 설명 된 연구 설계가 생체 내 설치류 알칼리 혜성 분석 (18)의 타당성에 대한 대체 방법의 유효성 검사를위한 일본 센터 (JaCVAM)에 의해 개발 된 프로토콜을 기반으로하고, 또한 OECD 가이드 라인의 권장 사항에 따라 ?...

<ol>
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이 프로토콜은 단일 세포 수준에서 쥐의 간에서 모두 직접 산화 DNA 손상의 동시 측정을 설명합니다. 일반적인 프로토콜 (즉, DNA 손상되지 테스트 에이전트하지만 동물 조직의 취급 및 가공에 의해 유도 된) 단일 세포 또는 핵 최소 처리 유도 된 DNA 손상과 분리 될 수있는 모든 조직에 적용 할 수있다. 우리의 연구에서는 골수 7,9, 위 (6), 신장 (9), 방광 (9), 폐 <s...

알칼리 혜성 분석은 단일 세포 수준에서 DNA 가닥 나누기를 측정한다. 단일 세포 현탁액은 현미경 슬라이드에 아가에 매립하고 세포의 DNA를 포함하는 수퍼 코일 루프 nucleoids을 형성 용해. 양극으로 마이그레이션하는 DNA의 해제 가닥으로 가닥 나누기를 포함하는 DNA 루프에서 수퍼 코일의 손실 산도&gt; (13) 결과에서 전기 영동, 형광 현미경으로 관찰 할 수 혜성 같은 구조를 생성. 단편화 된 DNA는 DNA 파괴 하나를 정량화하는데 사용될 수있는 단편의 크기 및 총 강도 (머리와 꼬리)에 비해 혜성 꼬리의 상대적 형광에 기초하여 &quot;꼬리&quot;에 혜성의 &quot;헤드&quot;에서 마이그레이션 2. 분석은 단순 구분, 다양한, 빠른, 1 상대적으로 저렴하다. DNA를 손상 에이전트에 의한 단편화 된 DNA의 검출은 세포의 DNA 손상을 정량화하기위한 분석을 사용 또는에서 핵을 격리 INDIVI잠재적 유전 독성 물질 (들)로 처리 동물의 이중 조직. 장점으로 인해 생체 내 혜성의 분석은 현재의 가늠자 조정에 국제 회의 (ICH) (3)과 유럽 식품 안전청에서 제품의 안전성 평가를 수행하기위한 (생체 내 소핵 분석과 쌍) 두 번째 생체 내 유전 독성 분석 (EFSA로 추천 ) 4 규제 지침. 우리가 실험실에서, 우리는 음식 재료, 의약품, 나노 물질 5-10에 의한 생체 내 DNA 손상을 평가하기위한 분석을 사용했다. 쥐 간 이러한 프로토콜의 예로서 사용되지만, 혜성 분석은 실험 동물의 다른 조직 / 기관만큼 조직으로부터 단리 할 수​​있다 그대로 단셀로 수행 될 수있다. DNA 손상의 특정 타입은 염기성 알칼리 혜성 시험을 수정하지 않고 DNA 가닥 나누기로 검출하기 어렵다. 산화성 DNA 손상의 경우에, 스트랜드 Breaks은 같은 8 oxoguanine (8 oxoGua) 및 메틸 fapy - 구아닌 (11)에서 휴식을 만들어 인간의 8-oxoguanine-DNA-N-글리코 1 (hOGG1, 같은 수리 효소로 소화에 의해 DNA의 산화 적 병변에서 만들 수 있습니다. 또한, 효소 III (엔도 III)의 산화 피리 미딘 1 주로 휴식을 생성한다. 따라서, 효소 분해 단계의 추가가 분석을 생체 (12)의 산화 적 DNA 손상을 측정하기위한 구체적이고 민감한 방법을한다. 이러한 분석을 활용, 우리는 증명하고있다 쥐와 생쥐 6-8의 간에서 쥐 (10)의 중심부에 독성 물질에 의한 산화 적 DNA 손상. 시험관 내에서 민감한에 의해 식별 genotoxins에 대한 생체 분석에서 후속는 3,13 테스트로 1), 2) 여러 조직에서 생체 이물에 의한 DNA 손상의 메커니즘을 평가하기 : 알칼리 혜성 분석은 유전 독성과 인간 생체 모니터링에 많은 응용 프로그램이 있습니다 14 3)하면 조사발암 물질, 4), 5) 사람의 질병 직업적 노출 (16)을 조사하고 DNA 손상을 복구 (15)을 평가하기 위해 유전자 독성 또는 동작의 비 유전 독성 모드 (MOA) (7)를 사용하여 동작 6)에 대한 잠재적 인 높은 처리량 스크리닝 검사로서 기관 고유의 유전 독성 17.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Coverslips (No. 1, 24 x 50 mm)&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">12-544-14</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Microscope Slides</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">12-550-123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Dimethylsulfoxide (DMSO)&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">67-68-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">EDTA, Disodium</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">BP120-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Phosphate buffered saline</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">ICN1860454</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">1X Hanks Balanced Salt Solution (HBSS) (Ca++, Mg++ free)&#160;</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:156px;">SH30588.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HEPES</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">BP310-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Low Melting Point Agarose (LMP)</td>
			<td style="width:140px;">Lonza</td>
			<td align="right" style="width:156px;">50081</td>
			<td>NuSieve GTG Agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Normal Melting Agarose (NMA)&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">BP1356-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">pH testing paper strips (pH 7.5-14)</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">M95873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Potassium Cloride</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">7447-40-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Potassium Hydroxide</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">1310-58-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">slide labels, (0.94 x 0.5 in.)</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">NC9822036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Sodium Chloride (NaCl)&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Sodium Hydroxide (NaOH)&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">1310-73-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">SYBR&#8482; Gold&#160;</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:156px;">S11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Triton X-100&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">9002-93-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Trizma Base&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">77-86-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">2.0 mL microcentrifuge tubes</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">05-402-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Cell strainer (40 &#181;m)</td>
			<td style="width:140px;">Fisher</td>
			<td align="right" style="width:156px;">22363547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Endonuclease III (Nth)&#160;</td>
			<td style="width:140px;">New England Biolabs</td>
			<td style="width:156px;">M0268S</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">hOGG1&#160;</td>
			<td style="width:140px;">New England Biolabs</td>
			<td style="width:156px;">M0241S</td>
			<td>Dilution 1:1000</td>
		</tr>
	</tbody>
</table>

in vivo alkaline comet assay and enzyme-modified alkaline comet assay for measuring dna strand breaks and oxidative dna damage in rat liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

생체 내 알칼리성 혜성 분석은 사이 프로 테론 아세테이트 (CPA) (5)로 처리 된 래트의 간에서 직접 및 산화성 DNA 손상을 모두 측정하는 효소 변성 혜성 분석과 함께 수행 하였다. CPA와 함께, 섹스 특정 방식으로 쥐의 간 종양을 유도하는 합성 호르몬 약물 여성 (24)에 비해 남성 쥐에서 간 종양을 유도하는 데 필요한 높은 용량을 5 배. 우리 수컷 및 암컷...

Watch this Scientific Journal Video about In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver at JoVE.com

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

쥐 간에서 DNA 가닥 나누기 및 산화 적 DNA 손상을 측정하기위한 생체 알카라인 혜성 분석 및 효소 수정 알카라인 혜성 분석에서

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Research

JoVE Journal

Biology

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

이미징 불일치 복구 및 DNA의 손상 세포 응답 바실러스 subtilis</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

연구

생물학

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

포유 동물 세포의 DNA 이중 가닥 브레이크 (DSB) 수리 분석

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

비례 생체 분자 비콘을 사용하여 살아있는 세포의 단일 엔지니어링 RNA 성적 증명서의 실시간 영상

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

세포질 DNA 검출 진학의 자극<em&gt; 체외</em&gt;와<em&gt; 생체</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

전신 산화 스트레스의 간접 측정으로 혜성 분석

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

만성 후 마우스 비장 세포에서 DNA 손상 및 복구를 측정<em&gt; 생체</em&gt; 베타와 감마 방사선의 매우 낮은 복용량에 노출

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

애기 장대 단백질 DNA 상호 작용의 식별을위한 염색질 면역 침전 분석<em&gt; 생체</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

레트로 바이러스 통합 사이트의 증폭, 차세대 시퀀싱 및 게놈 DNA 매핑

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

고처리량 DNA 추출 플레이트의 로딩을 위한 수정된 방법은 오염 가능성을 감소시니다.

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...