이 연구로는 국립 연구소의 건강 중화 프로그램 부처간 계약 # AOD13015-001 지원 되었다. 우리는 그들의 노력과 전문 비디오 제작에 대 한 스테파니 Froberg 피터 허스트를 감사 하 고 싶습니다.

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저자는 공개 없다.
면책 조항:이 문서에 표현 들 저자와 군대의 부, 국방부, 또는 미국 정부의 공식 정책 반영 하지 않습니다. 이 연구는 NIH/NIAID 사이의 USAMRICD, 부처간의 합의 의해 지원 되었고 일부 대학원 연구 참여 프로그램에는 미국 육군 의료 연구 연구소의 화학 방어에 약속에 의해 오크에 의해 관리 릿지 연구소 과학 미국 에너지 부와 USAMRMC 사이의 부처간 합의 통해 교육에 대 한.

본 모델의 HF와 CP 부상 연구에 대 한 심사는 높은 처리량 각 막 상피 세포의 개발에 우리의 방법 및 결과 설명 우리. 우리는 또한 HF 부상에 대 한 기본 siRNA 화면에서 결과 제시. TIC 상해 연구를 위한 HTS 모델의 개발에 많은 도전을 했다. 우리가 셀 문화 모델에 HF, HFA 또는 CP의 연구와 관련 된 문학에서 찾을 수 있는 방법은 작은 도움의 이었다. 불 소 이온에 대부분 생체 외에서 연구 구강 세포?...

Toxicant 유도 눈 부상 화학 빠르게 상당한 조직 손상을 입힐 잠재력을가지고 있기 때문에 진정한 눈 비상 이다. 불행 하 게도, 아니 특정 치료제가이 부상 치료 존재에 toxicant 유발 각 막 부상에 대 한 치료 일반적으로 지원만 있습니다. 현재 치료 전략 일반적인 주로 같은 윤활제, 항생제, 국 소 치료 요법을 포함 하 고 cycloplegics 뒤에 소염제 (예를 들어, 스테로이드) 한 번 각 막1 epithelialized 다시 있다 ,2. 최고의 현재 치료 치료 옵션을 사용할에 불구 하 고 장기적인 예 후는 일반적으로 진보적인 각 막 흐려 및 neovascularization2,3가난.
동물 모델 화학 독성을 조사 하 고 상해의 메커니즘을 이해를 전통적으로 사용 되어 왔습니다. 그러나, 동물 연구는 시간이 소모 되 고 비싸다. 노력을 줄이기 위해 동물 실험도 있다. 예를 들어 유럽 연합에 도달 법안 (EC 1907/2006)를 동물 실험을 줄이기 위한 규정 있다. 규정 회사 동물 테스트 및 동물에 제안 된 테스트를 수행 하기 전에 유럽 화학 제품 기관에서 승인을 얻는 피하기 위하여 데이터 공유 요구 사항이 포함 됩니다. 규정에 따라 동물 실험 최후의 수단 이어야 한다. 또한 단계적으로 동물에서 화장품의 테스트 유럽 화장품 규정 (EC 1223/2009)이 있다. 동물 연구를 실시 하는 경우 그들이 3Rs의 원칙에 의해 인도 된다 (구체화, 감소, 및 교체), 더 자비 롭 동물 연구를 수행, 사용, 동물의 수를 감소 및 비 동물 대안을 사용 하 여 프레임 워크를 제공 하는 가능한 경우. 이러한 이유로 독물학 분야는 독성의 분자 메커니즘에 대 한 통찰력을 제공할 수 있습니다 하 고 높은 처리량4에서 행 해질 수 있다 생체 외에서 분석을 채택 하고자 결정 했다. 이것은 기능 독극물 접근 어디 유독 전적으로 그들의 화학에 의해 보다는 오히려 그들의 기능에 의해 정의 됩니다. 단계는 또한, 기능 toxicogenomics 추구 특정 유전자 유독5의 효과에서 재생 역할을 이해 하. SiRNA 기술을 응용, 유독에 분자와 세포질 응답에 유전자 기능을 조사 하기 위해 화면 높은 처리량에 수행할 수 있습니다. siRNA는 19-25의 뉴클레오티드 긴 포스트 transcriptional 유전자 입을 통로 모든 포유류 세포6에 활용 하는 두 배 좌초 된 RNA 분자. 이 종합적으로 만들어지고 특정 유전자를 대상으로 설계 된. 셀에 도입 하는 경우는 siRNA 처리 되 고 한 가닥, 가이드 물가 RNA 유도 입을 복잡 한 (RISC)에 로드 됩니다. siRNA는 mRNA 분자에 상호 보완적인 영역에 RISC 지시 하 고 있는 RISC 저하는 mRNA. 특정 유전자의 표정 감소 결과. 특정 유전자의 표현의 결과 감소는 toxicant에 세포질 응답에 그 유전자의 기능을 확인 toxicant 노출된 셀에 다음 공부 수 있습니다. 이러한 접근은 더욱 CYP1A17,8의 a h R-종속 유도 리 민감성의 메커니즘 이해 하 사용 되었습니다.
화학 테러 위험 평가 (CTRA) 목록 및 독성 산업용 화학 물질 (TIC) 목록 선택 화학 물질의 독성과 테러, 전쟁, 또는 산업 재해 이벤트9중 출시 될 가능성에 따라 항목별로 나 누이고 있다. 우리는 siRNA 높은 처리량 검열 (HTS) toxicogenomic 접근 테러 사건에서 사용의 높은 위험에 확인 된 CTRA 목록 이용과의 연구에 적용 하 고 있다. 화학 생물;에 있는 불리 한 효과 이해 하고자 하는 전통적인 독물학 그러나, 우리는 더 욕망의 치료제 및 치료 접근을 가능 하 게, 개발을 알리는 목적으로 상해의 메커니즘을 이해 하는 분자 치료 개발에 대 한 대상으로 지정할 수 있는 발견. 몇 가지 방법으로이 노력 유사한 높은 처리량 siRNA 심사 및 약 발견 과정10에서 셀 기반 분석의 사용을 고려할 수 있습니다. 주요 차이점은 우리의 접근 방식에 다소 가능성이 크다 toxicant 노출의 치료에 대 한 높은 치료 가치와 단일 대상 될 것 이다 반면 그 약물 발견 일반적으로 치료 발견에 대 한 단 수 목표를 추구 될 것 이다. 우리 예상 어떤 효과적인 치료 패러다임 toxicant 노출에 대 한 높은 치료 가치를 달성 하는 다각적인 접근 방식이 필요로 하 고 toxicogenomic 데이터 극히 효과적인 치료 패러다임을 알릴 수 있습니다.
벤치탑 자동화 실험실 제약 또는 생명 공학 산업 밖에 서 높은 처리량 방법론을 제공합니다. 우리 연구소에서 생체 외에서 연구 역사적으로 낮은 처리량11,,1213전통적인 분석 되었습니다. 지난 몇 년 동안, 우리의 실험실 벤치탑 높은 처리량 siRNA 심사를 수행 하기 위해 로봇의 사용에 전환 했다. 여기, 우리가 현재 눈 셀 모델의 세련미와 체 외에서 의 개발 수소 불 화물 (HF)와 chloropicrin (CP) 높은 처리량 siRNA 심사 적합에 대 한 노출 방법. 우리의 목표는 이러한 유독 응답에 세포질 상해를 조절 하는 분자를 식별 하는. 우리는 선택 하는 siRNA 도서관의 목표는 G 단백질 결합 된 수용 체, 단백질 kinases, 프로 테아 제, 가수분해, 이온 채널, 그리고 다른 잠재적으로 druggable 대상 포함 됩니다. HF와 CP 선정 됐다 연구에 대 한 상호 CTRA 목록 에이전트 산업 사고의 ToxNet 보고서를 통해 증기 노출9,14눈 상해의 가장 중대 한 모험을 선물 하는 그를 찾을 수 있습니다. WWI15에 최 루 가스 CP (Cl3CNO2화학식, CAS 번호 76-06-2) 원래 사용 했다. 현재16nematicide, 살 균 제, 살충제는 농업 fumigant 및 기능으로 사용 됩니다. 수소 불 화물 (HF)는 정유 및 화학 fluorination 유기 화합물17의 알을 포함 하 여 프로세스에서 사용 됩니다. HF (화학식 HF, CAS 번호 62778-11-4)는 가스 이지만 수성 형태로 소산 (HFA, CAS 번호 7664-39-3). 따라서, 우리가 우리의 셀 HFA를 사용 하 여 선출 노출 모델. SV40 큰 T 항 원 SV40 HCEC 연구에 대 한 선정 되었다 인간 각 막 상피 세포 선 불후. 세포질 상해에 관련 된 목표 세포 죽음과 선 동적인 응답에 반영 해야 하기 때문에 세포 생존 능력 및 염증 성 마커 IL-8 끝점으로 선정 됐다. 특히, 대상 toxicant 노출에서 보호 역할을 한다면, 세포 죽음 또는 염증 성 cytokine 생산 증가 한다 때 대상 식 siRNA에 의해 저해. 반대로 부정적인 역할을 하는 대상에 대 한 사실 것입니다. 또한, 만성 염증 후 노출, 및 세포 죽음 통로 있는 내정간섭에 임상 결과2,18이 향상 시킬 수 있습니다 각 막 병 리에 역할을 나타납니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:879px;" width="880">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo liquid handing platform</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">G5409A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo plate shaker</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96LT disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 178</td>
			<td>96-channel large tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96ST disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 177</td>
			<td>96-channel small tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384ST disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 179</td>
			<td>384-channel small tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96 250 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19477-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384 30 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19133-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384 70 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19133-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">EnSpire multimode plate reader</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td style="width:119px;">2300-0000</td>
			<td>AlphaLISA assay detector with high power laser excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">IL-8 (human) AlphaLISA Detection Kit&#160;</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td style="width:119px;">AL224F</td>
			<td>no-wash bead-based assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">ProxiPlate-384 Plus white 384-shallow well microplates</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td>6008359</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Lipofectamine RNAiMAX</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td style="width:119px;">13778500</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Opti-MEM 1 Reduced Serum Medium</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">TrypLE Express</td>
			<td style="width:247px;">Gibco or equivalent</td>
			<td style="width:119px;">12605010</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">IncuCyte Zoom</td>
			<td style="width:247px;">Essen Instruments or equivalent</td>
			<td style="width:119px;">ESSEN BIOSCI 4473</td>
			<td>Incubator-housed automated microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chloropicrin</td>
			<td style="width:247px;">Trinity Manufacturing or equivalent</td>
			<td style="width:119px;">N/A</td>
			<td>Acute toxicity and irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DMEM-F12 cell culture medium</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>11330-057</td>
			<td>Contains HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Fetal bovine serum</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>1891471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Human epidermal growth factor (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>E9644-.2MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Recombinant human insulin (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>12585-014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Penicillin-Streptomycin solution (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Hydrocortisone (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td>H0888-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Glucose&#160; (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">PBS&#160; (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">P5493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">siRNA</td>
			<td style="width:247px;">Dharmacon or equivalent</td>
			<td style="width:119px;">various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiazolyl blue tetrazolium bromide</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">M5655</td>
			<td>MTT assay substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">siRNA buffer</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">B002000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">96-well cell culture plates</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">T150 cell culture flasks</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS430825</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">BSL-2 cell culture hood</td>
			<td style="width:247px;">Nuaire or equivalent</td>
			<td style="width:119px;">NU-540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">300 mL robotic reservoirs</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">12-565-572&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">96 baffled automation reservoirs</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">1064-15-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">500 mL sterile disposable storage bottles</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS430282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Microplate heat sealer</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">AB-1443A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Microplate heat sealing foil</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">AB-0475</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Cardamonin</td>
			<td style="width:247px;">Tocris or equivalent</td>
			<td style="width:119px;">2509</td>
			<td>Anti-inflammatory, used as positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">SKF 86002&#160;</td>
			<td style="width:247px;">Tocris or equivalent</td>
			<td style="width:119px;">2008</td>
			<td>Anti-inflammatory, used as positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DMSO</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">48% hydrofluoric acid</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">339261</td>
			<td>Corrosive and acute toxicity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">1000 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">200 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">20 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">1000 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">200 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17013810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">20 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17013808</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 1000 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17002920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 200 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014294</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 20 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17002928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chemical fume hood</td>
			<td style="width:247px;">Jamestown Metal Products</td>
			<td style="width:119px;">MHCO_229</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">384-well sample storage plates</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">262261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sodium chloride</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">S6191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">50 mL conical tubes</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">14-959-49A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 50 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 25 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 10 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-574</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 5 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-573</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">SV40-HCEC immortalized human corneal epithelial cells</td>
			<td style="width:247px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>These cells are not commercially available, but can be obtained from the investigators cited in the article</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sceptor Handheld Automated Cell Counter</td>
			<td style="width:247px;">Millipore or equivalent</td>
			<td style="width:119px;">PHCC20060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">GeneTitan Multi-Channel (MC) Instrument</td>
			<td style="width:247px;">Affymetrix or equivalent</td>
			<td style="width:119px;">00-0372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Affymetrix 24- and 96-array plates</td>
			<td style="width:247px;">Affymetrix or equivalent</td>
			<td style="width:119px;">901257; 901434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger tube HF</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">8103251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger tube CP</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">8103421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger pump</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">6400000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Clear Plate seals</td>
			<td style="width:247px;">Resesarch Products International or Equivalent</td>
			<td style="width:119px;">202502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Reagent reservoirs</td>
			<td style="width:247px;">VistaLab Technologies or equivalent</td>
			<td style="width:119px;">3054-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Xlfit</td>
			<td style="width:247px;">IDBS or equivalent</td>
			<td style="width:119px;">N/A</td>
			<td>Excel add-in used for automated curve fitting</td>
		</tr>
	</tbody>
</table>

high throughput sirna screening for chloropicrin and hydrogen fluoride-induced cornea epithelial cell injury

Toxicant 유도 눈 부상 화학 빠르게 상당한 조직 손상을 입힐 잠재력을가지고 있기 때문에 진정한 눈 비상 이다. 아무 특정 치료제가이 부상 치료 존재 toxicant 유발 각 막 부상에 대 한 치료는 일반적으로 지원. 치료 및 노출에 대 한 관심에 치료제를 개발 하는 노력, 그것은이 상해의 분자 및 세포 메커니즘을 이해 하는 것이 중요 수. 있습니다 우리는 높은 처리량 작은 금지 RNA (siRNA) 심사의 활용 화학 각 막 상피 상해의 분자 메커니즘을 더 빠르게 명료를 도울 수 있는 중요 한 도구가 될 수 있습니다 제안 합니다. siRNA는 siRNA에 상 동을가지고 있는 19-25의 뉴클레오티드 긴 mRNA를 저하 하는 통로 입을 post-transcriptional 유전자를 활용 더블 좌초 RNA 분자는. 특정 유전자의 표현의 결과 감소는 toxicant에 세포질 응답에 그 유전자의 기능을 확인 toxicant 노출된 셀에 다음 공부 수 있습니다. 개발 및 체 외에 노출 모델 및 수소 불 화물 (HF) 및 chloropicrin (CP) 유도 눈 부상 (HTS)을 상영 하는 높은 처리량 방법의 유효성 검사는이 문서에 표시 됩니다. 하지만 우리는 이러한 두 가지 유독 선택, 우리의 방법 toxicant 노출 프로토콜에 사소한 수정 다른 유독의 연구에 적용 됩니다. SV40 큰 T 항 원 SV40 HCEC 연구에 대 한 선정 되었다 인간 각 막 상피 세포 선 불후. 세포 생존 능력 및 IL-8 생산 심사 프로토콜에서 끝점으로 선정 됐다. Toxicant 노출의 개발와 관련 된 몇 가지 과제 고 셀 문화 방법 HTS 연구에 대 한 적합 한 표시 됩니다. 이 유독 HTS 모델 설립 화학 눈 부상에 대 한 잠재적인 치료에 대 한 화면을 상해의 메커니즘을 이해 하기 더 연구에 대 한 수 있습니다.

노출 방법 개발
우리는 세련 하 고 HTS 연구에 사용 하기 위해 인간 각 막 상피 세포 선 SV40-HCEC의 적합성을 평가. SV40 HCEC SV 40 큰 T 항 원을 사용 하 여 불멸 하 게 했다 고 Dhanajay Pal23에서 선물 했다. 간결 하 게, 여기 제시 노출 방법론 개발에 탐구 하는 너무 많은 변수가 있었고 그래서, 우리가 생각 ...

Watch this Scientific Journal Video about High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury at JoVE.com

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

높은 처리량 작은 금지 RNA 검사 화학 각 막 상피 상해의 분자 메커니즘을 더 빠르게 명료를 도울 수 있는 중요 한 도구입니다. 여기, 우리는 개발 및 노출 모델 및 각 막 상피 부상 수소 불 화물 그리고 chloropicrin 유도의 높은 처리량 검열 방법의 유효성 검사 제시.

Chloropicrin 및 수소 불 화물 유발 각 막 상피 세포 상해에 대 한 높은 처리량 SiRNA 심사

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

high-throughput-sirna-screening-for-chloropicrin-hydrogen-fluoride

Research

JoVE Journal

Biology

5.8K 조회수.  US Army Medical Research Institute of Chemical Defense. 높은 처리량 작은 금지 RNA 검사 화학 각 막 상피 상해의 분자 메커니즘을 더 빠르게 명료를 도울 수 있는 중요 한 도구입니다. 여기, 우리는 개발 및 노출 모델 및 각 막 상피 부상 수소 불 화물 그리고 chloropicrin 유도의 높은 처리량 검열 방법의 유효성 검사 제시.

비디오: High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

형광등과 높은 처리량 검사 및 Biosensing C. 엘레간스 레인스

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

연구

생물학

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

높은 처리량 효모 플라스미드 Overexpression 화면

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

에서 곰팡이 Endoglucanase 활동의 높은 처리량 검사 대장균</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

안쪽 정류기의 칼륨 채널의 소형 분자 변조기에 대한 높은 처리량 검사

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

높은 처리량 검열을 사용 키나제 기판 쌍의 식별


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

멀티 효소 높은 처리량 유전자 효소 검사 시스템을 사용하여 상영

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.