We thank Sue Lindquist, Aaron Gitler, and Martin Duennwald for kindly sharing reagents. Our studies were supported by: an American Heart Association Post-Doctoral Fellowship (M.E.J); NIH Director's New Innovator Award DP2OD002177, NIH grants R21NS067354, R21HD074510, and R01GM099836, a Muscular Dystrophy Association Research Award (MDA277268), Packard Center for ALS Research at Johns Hopkins University, Target ALS, and an Ellison Medical Foundation New Scholar in Aging Award (J.S.).

1. 도서관 생성 <ol><li> 도메인 별 오류가 발생하기 쉬운 PCR 사용 Hsp104의 라이브러리를 구성하려면 먼저 오류가 발생하기 쉬운 DNA 중합 효소 (20) 관심의 영역을 증폭. </li><li> 겔을 추출하여 PCR 생성물을 정제 하였다. </li><li> 표준 부위 지정 돌연변이 유발 프로토콜을 사용 megaprimer 확장 단계를 수행하여 PCR 완충액 50 ng의 템플릿 플라스미드 250 겨 megaprimer, 200 μM의 dNTPs 및 고성능 DNA 중합 효소를 결합하고, PCR 등급의 물 (20)과 50 ㎕의 총 부피로 희석 . 표준 PCR 프로그램을 실행합니다. NOTE : 증폭되는 유전자의 특정 지역에 따라 달라질 사용될 특정 프라이머. <ol><li> PCR에 이어, 37 ° C에서 2 시간 동안 1 ㎕의 DPN I 제한 효소로 부모 주형 DNA를 소화. </li></ol></li><li> ultracompetent E.에서 전기 또는 다른 수단에 의해 라이브러리 변환 대장균 미니 프렙하여 정제하고. 참고 : 라이브러리 생성실질적으로 주어진 실험의 목적에 따라 달라집니다. Hsp104 매우 큰 단백질이므로 우리가 특정 도메인 에러 유발 PCR을 이용하는 이유는 전체 유전자를 랜덤 비실용적이다. 또한 Hsp104의 구조는 저조한 21 도전 관한 라이브러리 디자인을 이해 남아. 라이브러리 유일한 제한은 템플릿 플라스미드 백본 스트로스 미디어 -5- FOA 카운터의 선택을 허용하는 URA3 유전자를 포함해야한다는 점 이외에는, 돌연변이 유발에 관한 랜덤 접근 방식을 사용하여 구성 될 수있다. </li></ol> Hsp104 도서관 2. 변환 <ol><li> 표준 리튬 아세테이트 / PEG 변환 프로토콜 (22)를 사용하여 W303aΔ의 hsp104 효모에 질병 관련 기판을 통합 할 수 있습니다. 하나의 식민지를 선택하고, 기판 (23)을 관련 질병의 높은 독성을 가진 균주를 분리 독성을 화면. 참고 : 통합 변형 및 솔라를 사용하여모든 셀에 동일한 발현을 보장하기 위해 단일 ​​콜로니 테. pAG416GAL 플라스미드 (우라실 마커) (24)로 우라실 이외 마커 (우리는 히스티딘 사용) Hsp104 라이브러리 하에서 유전자의 통합을 허용하는 플라스미드로 질병 기판을 복제. 5-FOA의 counterselection을 허용하기 위해 라이브러리가 우라실 마커와 플라스미드로부터 발현되어 있는지 확인합니다. 다른 효모 균주도 사용될 수있다. 우리는 W303a 모두 WT와 Δ hsp104 배경에 BY4741 효모 균주 (MEJ과 JS, 관찰되지 않은)을 사용하여 유사한 독성 억제 표현형을 언급했다. </li><li> 동일한 리튬 아세테이트 / PEG 변환 프로토콜 (22)을 사용하여이 균주에 Hsp104 라이브러리 변형. 라이브러리의 시퀀스 공간을 유지하고 예측 된 라이브러리 크기를 유지하기 위해 적절히 변형 배율 확대. </li><li> 충분히 판을 사용하여 noninducing, 선택적 플레이트 상에 변환 혼합물 (예 : SD-그의 우라)를 플레이트콜로니 다수의 성장을 보장한다. 필요한 접시의 수를 최소화하기 위해 큰 배양 접시 (150mm)를 사용합니다. 플레이트를 사용하여 형질 전환 체를 복구하는 것은 변환 효율을 평가 할 수 있습니다. </li><li> 병렬 Hsp104 WT 및 벡터 음성 대조군 변환. </li></ol> Proteotoxicity의 억제를위한 3. 심사 <ol><li> 피노 오스 보충 드롭 아웃 미디어 (예 : SRaff-그의 우라)를 사용하여 판 오프 식민지를 씻으십시오. 접시에서 식민지를 풀어 혈청 학적 피펫 멸균 나무 어플리케이터를 사용합니다. 50 ML 원뿔 튜브와 소용돌이에 액체 세척을 이동하는 것은 철저히 세포의 덩어리를 분리합니다. 약간 흐린 문화, OD 600 ~ 0.025로 희석. 참고 : 여기 라 피노 오스 따라서 갈락토스 매개 유도 세포를 프라이밍, 유도의 포도당 매개 억압의 세포를 완화하는 역할을한다. </li><li> 3에서 진탕 피노 오스 드롭 아웃 미디어에서 하룻밤 희석 문화를 성장0 ºC. 병렬 Hsp104 WT 및 벡터 제어를 성장. </li><li> 다음날 아침, 개인 갈락토스 위에 농도의 범위를 판 (예 SGal-그의 우라) 판 (400 μL 총 볼륨 문화를 집중 2 ml의 1 μL). 농도의 넓은 범위를 도금하는 것은 접시가 하나의 식민지있을 것이라는 점을 보장합니다. 필요한 실제 농도는 관심있는 질병 기판의 독성에 의존한다. </li><li> 라이브러리의 OD 600 벡터 및 Hsp104 WT 문화를 정상화하고 컨트롤 라이브러리 상대적인 성장을 비교하는 같은 부피를 접시. </li><li> 선택의 엄격 성을 평가하기 위해, 미디어 (억압) 포도당에 라이브러리를 접시. 식민지 (그림 2) 나타날 때까지, 30 ºC 2-3 일 동안 번호판을 품어. 그 결과 식민지를 평가하고 컨트롤에 비교합니다. NOTE : 종종 모두 크고 작은 콜로니가 수득되지만, 콜로니 크기와 행동 사이에 상관 관계의 ivity가 관찰된다. 시퀀싱 이월 가양 최소화 -5- FOA 카운터 선택 기술 (단계 4)를 사용하여 이러한 잠재적 히트 화면. </li></ol> 4. 5-FOA Counterselection 스포팅는 오판 가능성을 제거하기 위해 <ol><li> 단일 및 이중 드롭 아웃 매체에 중복 된 단일 콜로니 (예 : SD-그의 우라과 SD-그의 판) 30 ºC에서 하룻밤 성장 밖으로 행진. 벡터 및 Hsp104 WT 컨트롤을 반복합니다. 도금 SD-그의 종래 5-FOA들은 5-FOA에 도금되는 경우 세포 Hsp104 플라스미드를 상실한 가능성을 증가시켜 5-FOA 단계의 효율을 증가시킨다. </li><li> 5 FOA 화면을 수행하는 동안 마르지 판을 방지하기 위해, 4 ºC에서 SD-그의 우라의 파라 필름에 접시와 저장소를 포장. 이 판은 결국 시퀀싱에 사용됩니다. </li><li> 행진에서 식민지 SD-그의 5-FOA 플레이트에 하나의 식민지 판 (YPD 미디어 + 1g / L 5-FOA) 및 (30)에서 1~2 일 동안 배양# 186; C는 하나의 식민지가 나타날 때까지. 여기서, 5-FOA는 URA3 유전자를 포함하는 세포에서의 독성 생성물 (5-fluorodeoxyuridine)로 변환된다. 따라서, 여전히 Hsp104 플라스미드를 보유하는 모든 세포는 죽을 것이다. </li><li> 행진 SD 우라과 SD-그의 접시 위에 중복의 5-FOA 판에서 하나의 식민지. 각 테스트 3 식민지는 각각의 히트에 대해 적어도 하나의 식민지가 Hsp104 플라스미드를 상실 할 가능성을 증가했다. 30 ° C에서 1~2 일 동안 품어. SD-번호판하지만 SD 우라 판에 성장합니다 Hsp104 플라스미드를 잃은 식민지. </li><li> 분석을 안보에 대한 Hsp104 플라스미드를 잃은 균주​​를 성장. 진탕 30 ºC에서 하룻밤 (96) DeepWell 2 ml의 판에 (예를 들어 SRaff-그의) 라 피노 오스 드롭 아웃 미디어에서 포화 균주를 성장. 5 FOA 처리 제어 계통을 포함해야합니다. </li><li> 분석을 안보에 대 한 번호판을 준비합니다. 멀티 채널 피펫을 사용하여, 나누어지는 200 μL 피노 오스 드롭 아웃 미디어 (예 : SRaff-그의) 당96 웰 플레이트의 잘, 예약 열 깔끔한 문화 1, 7. 나누어지는 250 열 1에 16 포화 문화의 각 μL 및 플레이트 7. </li><li> 직렬 멀티 채널 피펫 철저하게 혼합, 플레이트의 각 열에 문화 5 배 희석. 각 웰의 최종 부피를 위해 컬럼 (6) 및 (12)로부터 희석 배양의 50 μl를 제거하여 200 μL이다. 이것은 심지어 안보 보장하기 위해 필수적이다. </li><li> 96 볼트 복제 도구 (Frogger와)를 사용하여 SD-그의 및 SGal-그의 판에 중복의 문화를 발견. 2~3일 30 ºC에서 번호판을 품어. </li><li> 컨트롤 유사한 SGal-그의 플레이트에 독성을 나타내는 균주를 시퀀싱을 위해 선택한다. 컨트롤로 유독하지 않음으로 오탐 (false positive) 균주를 폐기하십시오. 5-FOA는 그 자체로 종종 독성, 그래서 혼자 질병 기판보다 SD-그의 접시에 성장 결함 또는 전시 실질적으로 더 큰 독성을 나타내는 균주를 (폐기 <stronG&gt; 그림 3). </li></ol> 5. 식민지 PCR에 의해 Hsp104 변종을 시퀀싱 <ol><li> 다 쳤어요 ~ 10 μL의 PCR 스트립 관에서 3 μL 20 mM의 NaOH를에서 SD-그의 우라 판에서 효모와 동결 - 해동하여 세포를 용해. 다음 10 분 동안 열 순환기에서 99 ºC에서 부화, 10 분 동안 또는 액체 질소 -80 ºC 냉동고에서 스트립 튜브를 놓습니다. PCR 등급 물 100 μL에 균주를 희석. </li><li> PCR 반응 준비 : 5 μL PCR 템플릿, 0.5 μL 100 μm의 각 프라이머, 0.5 μl의 10 mM이의 dNTPs, PCR 버퍼 0.25 μL DNA 중합 효소를, 그리고 PCR 등급 물 25 μL 총 볼륨으로 구성한다. 디자인 프라이머는 무작위 지역 플러스 양쪽 끝에서 약 100 bp의 증폭합니다. 성공적인 증폭의 가능성을 증가시키기 위해 증폭 된 영역의 크기를 최소화한다. 표준 PCR 프로그램을 실행합니다. </li><li> 적절한 SIZ의 PCR 생성물의 증폭을 확인하기 위해, 아가 로스 겔 전기 영동에 의해 시료를 분석전자. 어떤 제품이 존재하지 않는 경우 PCR을 반복한다. NOTE : PCR 장애가 통상적 단계 5.1에서 사용되는 너무 많은 효모 때문이다. </li><li> DNA 시퀀싱 샘플을 준비합니다. 이러한 엑소 뉴 클레아 I와 새우 알칼리 포스 파타 아제 효소로 PCR 정화 또는 PCR 제품 정리 시약을 사용하여 하나 PCR 프라이머를 제거 (ExoSAP-IT)의 단일 가닥 DNA를 분해합니다. 이 시약은 효소 높은 처리량을 허용, 비법 인 프라이머 및 dNTPs를 저하됩니다. </li><li> PCR 제품 시퀀싱. 철저하게 돌연변이 염기 서열 크로마토 그램을 분석해야합니다. 종종 여러 플라스미드 항구 효모로 돌연변이는, 특정 사이트 (그림 4)에서 두 개의 뉴클레오티드의 혼합물로 관찰 할 수있다. </li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+modulation+of+eIF2alpha+phosphorylation+rescues+TDP-43+toxicity+in+amyotrophic+lateral+sclerosis+disease+models.">Therapeutic modulation of eIF2alpha phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models.</a> Nature. 46 (2), 152-160 (2014).</li><li>Ju, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+model+of+FUS/TLS-dependent+cytotoxicity.">A yeast model of FUS/TLS-dependent cytotoxicity.</a> PLoS Biol. 9 (4), (2011).</li><li>Shorter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp104:+a+weapon+to+combat+diverse+neurodegenerative+disorders.">Hsp104: a weapon to combat diverse neurodegenerative disorders.</a> Neurosignals. 16 (1), 63-74 (2008).</li><li>Vashist, S., Cushman, M., Shorter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+Hsp104+to+protein-misfolding+disorders.">Applying Hsp104 to protein-misfolding disorders.</a> Biochem Cell Biol. 88 (1), 1-13 (2010).</li><li>DeSantis, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operational+Plasticity+Enables+Hsp104+to+Disaggregate+Diverse+Amyloid+and+Nonamyloid+Clients.">Operational Plasticity Enables Hsp104 to Disaggregate Diverse Amyloid and Nonamyloid Clients.</a> Cell. 151 (4), 778-793 (2012).</li><li>Jackrel, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiated+Hsp104+Variants+Antagonize+Diverse+Proteotoxic+Misfolding+Events.">Potentiated Hsp104 Variants Antagonize Diverse Proteotoxic Misfolding Events.</a> Cell. 156 (1-2), 170-182 (2014).</li><li>Wittrup, K. D., Verdine, G. L., Wittrup, K. D., Verdine, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 503, 13-14 (2012).</li><li>Boeke, J. D., Trueheart, J., Natsoulis, G., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 154, 164-175 (1987).</li><li>Miyazaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Random+Mutagenesis+Libraries+by+Megaprimer+PCR+of+Whole+Plasmid+(MEGAWHOP).+Directed+Evolution+Library+Creation:+Methods+in+Molecular+Biology.">Creating Random Mutagenesis Libraries by Megaprimer PCR of Whole Plasmid (MEGAWHOP). Directed Evolution Library Creation: Methods in Molecular Biology.</a> eds FrancesH Arnold &amp; George Georgiou. 231, 23-28 (2003).</li><li>Saibil, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone+machines+for+protein+folding,+unfolding+and+disaggregation.">Chaperone machines for protein folding, unfolding and disaggregation.</a> Nat Rev Mol Cell Biol. 14 (10), 630-642 (2013).</li><li>Gietz, R. D., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+yeast+transformation+using+the+LiAc/SS+carrier+DNA/PEG+method.">High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.</a> Nat. Protocols. 2 (1), 31-34 (2007).</li><li>Armakola, M., Hart, M. P., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+toxicity+in+yeast.">TDP-43 toxicity in yeast.</a> Methods. 53 (3), 238-245 (2011).</li><li>Alberti, S., Gitler, A. D., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+suite+of+Gateway&#174;+cloning+vectors+for+high-throughput+genetic+analysis+in+Saccharomyces+cerevisiae.">A suite of Gateway&#174; cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae.</a> Yeast. 24 (10), 913-919 (2007).</li></ol>

The authors have nothing to disclose.

여기에서 우리는 효모 proteinopathy 모델을 사용하여 질병 관련 기판의 독성을 억제하는 효력을 더 Hsp104 변종을 분리에 대한 우리의 접근 방식을 제시한다. 이 방법을 사용하여, 변형의 큰 라이브러리 만 제한 -5- FOA 보조 스크린을 통과 변이체의 개수 인 상태, 높은 처리량 스크리닝 할 수있다. 96 웰 형식으로 다음 단계를 수행함으로써, 우리는 정기적으로 1-2주의 과정을 통해 5-FOA 단계에서 한 번에 2...

효모 proteinopathy 모델은 근 위축성 측삭 경화증 (ALS) 및 파킨슨 병 (PD) 1-3 포함한 단백질 미스 폴딩 장애 개발되어왔다. ALS 환자에서 misfold 단백질 TDP-43 및 FUS,의 발현은, 효모 1, 2에서 세포질 응집체를 형성하는 독성 및 mislocalize 있습니다. 마찬가지로, PD에 연루되어 α - 시누 클레인 (α-SYN)의 표현은, 독성 및 mislocalizes 효모 3 세포질 응집체를 형성 할 수 있습니다. 이러한 기능은 이러한 장애 4,5 환자의 표현형을 요점을 되풀이. 따라서, 효모 모델을 방지하거나 이러한 표현형 2,6-13 역 단백질이나 작은 분자 스크리닝을위한 유용한 플랫폼을 제공합니다. 저자들은 TDP-43, FUS 및 α-SYN에 집계 및 독성을 반전 할 수있는 단백질의 개발에 관심이 있습니다. 우리는 Hsp104, 모두 FR 단백질을 disaggregating의 고유 할 수있는 효모에서 AAA + 단백질에 초점효모에서 톰 비정질 단위 및 아밀로이드는 아직 더 인간의 상동 (14, 15)이 없습니다. Hsp104 미세 내생 효모 프리온을들을 분해하도록 조정하고 일반적으로 16, 17가 발생하지 인간의 신경 퇴행성 질환에 연루 기판을들을 분해하는 제한된 능력을 가지고 있습니다. 따라서, 우리는 efficaciously이 인간의 기판을들을 분해 할 수있는 Hsp104의 향상된 버전을 설계하는 것을 목표로하고 있습니다. Hsp104는 오류가 발생하기 쉬운 PCR을 사용하여 변형이를 위해, 우리는 큰 라이브러리를 구성; 이 라이브러리는 효모 proteinopathy 모델 (17)를 사용하여 검사를 할 수 있습니다. Hsp104 (17) 매우 큰 것처럼 우리는 라이브러리를 구성하고 심사에 도메인 타겟 접근 방식을 채택했습니다. 유사한 접근법이 다른 도메인을 선별하기 위해 사용될 수 있지만 우리는 처음, 중간 도메인 Hsp104 17 (MD)에 집중했다. 예컨대, 나머지는 표면 디스플레이, 같은 대체 기술과 반대로 이러한 모델은, 직접적 disaggregase 활성 스크리닝 에이블18 바인딩 모니터링을위한 사용하기 ricted. 우리의 프로토콜은 두 심사 단계 (그림 1)을 기반으로합니다. 첫째, 효모 질병 기판의 독성을 억제 Hsp104 변이체가 선택된다. 이렇게하려면 Hsp104 변종과 질병 관련 기판은 Δ의 hsp104 효모에 cotransformed된다. 우리는 야생 형의 부재 (WT) Hsp104 17 Hsp104 시퀀스 공간을 탐험 Δ의 hsp104 효모를 사용합니다. 중요한 것은, Hsp104의 삭제는 효모의 α-SYN, FUS, 또는 TDP-43 독성에 영향을주지 않으며, Hsp104 (WT)의 표현은 최소한의 구조 1,13,17을 제공합니다. 효모는 양쪽 단백질의 발현을 유도하는 유도 배양액에 도금된다. 식민지의 질병 관련 기판 협의 성장의 독성을 억제 Hsp104 변종을 품고 효모. 식민지 독성 다이를 억제하지 않는 변종을 유지하면서 이러한 변형은, 추가 분석을 위해 선택됩니다. 그러나,오탐 (false positive)이 화면에 상당한 문제가 있습니다. TDP-43, FUS 및 α-SYN의 식으로 표현되는 Hsp104 변형과 관련이없는 독성의 자발적인 유전자 억제의 모양에 대한 강력한 선택적 압력을 생성하는, 매우 독성이 있습니다. 따라서, 우리는 이러한 비특이적 독성 억제 (17)를 제거하기 위해 상대적으로 높은 처리량입니다 보조 화면을 사용했다. 이 보조 화면에서 선택 효모 Hsp104 플라스미드 (19)에 대한 선택에 대응하기 위해 5 Fluorootic 산 (5-FOA)로 처리된다. 균주 후 기판의 독성 Hsp104 플라스미드의 손실 후 복원 될 수 있도록 분석을 통해 안보 (TDP-43, FUS, 또는 α-SYN) 독성 기판에 대한 평가된다. 따라서, 독성이 보조 화면에 복원되는 효모는 아마도 원래 인해 Hsp104 변종의 존재에 대한 독성 억제를 표시. 이러한 효모 &#39;히트&#39;로 표시되고 Hsp104 플라스미드는되어야회수 Hsp104 유전자 17 (도 1)의 변이를 식별하는 서열. 모든 히트 곡은 다음 독성 억제에 대한 재검사 후 독립적으로 돌연변이를 구성하는 부위 특이 적 변이를 사용하여 재확인해야한다. 이 프로토콜에 대한 잠재적 인 응용 프로그램은 광범위하다. 이러한 방법을 이용하여 단백질의 임의의 타입의 라이브러리가 효모의 독성 어떤 기질 단백질의 독성을 억제하는 변형에 대해 스크리닝 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="766">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:320px;">Table of Specific Materials/Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:132px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneMorphII EZClone Domain Mutagenesis Kit</td>
			<td>Agilent</td>
			<td>200552</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150mm Petri dishes</td>
			<td>Falcon</td>
			<td>351058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Fluorootic Acid</td>
			<td>Research Products International</td>
			<td>f10501-5.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-DeepWell 2mL Plates</td>
			<td>Eppendorf</td>
			<td>0030 502.302</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 bold replicator tool</td>
			<td>V&#38;P Scientific</td>
			<td>vp-404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ExoSAP-IT</td>
			<td>Affymetrix</td>
			<td>78200</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating potentiated hsp104 variants using yeast proteinopathy models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

우리는 중간 영역에서 무작위 Hsp104 변종의 라이브러리를 구축하고 TDP-43 독성 억제를 위해 그것을 상영했다​​. 도서관은 변화와 화면의 엄격 성을 평가하기 위해 포도당과 갈락토스 판 (그림 2)에 도금. 단일 콜로니를 선택하고, 균주 카운터 Hsp104 플라스미드를 제거하기 위해 5-FOA를 사용하여 선정되었다. 이 균주는 그 독성을 확인하기 위해 평가되었다 것은 Hsp104 변종 않고, TDP-43 형...

Watch this Scientific Journal Video about Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models at JoVE.com

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

효력을 더 Hsp104 변종 사용 효모 Proteinopathy 모델을 분리

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

isolating-potentiated-hsp104-variants-using-yeast-proteinopathy-models

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Biology

8.1K 조회수.  Perelman School of Medicine at the University of Pennsylvania. Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

비디오: Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

전기 분무 이온화 질량 분광법을 사용하여 효모 Lipidome의 양적 평가

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

연구

생물학

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

인간의 DNA 수리 단백질의 유전자 연구는 모델 시스템으로 효모를 사용하여

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

효모 콜로니 임베딩 방법

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

뉴 외과 의사에 대한 쥐 외과의 원칙

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

수동 규제와 마우스 및 쥐 일반적인 합성 관리 경로

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

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, ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104의 정화, 단백질 Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

전 · Cryo 키트를 사용하여 마우스 정자 Cryopreservation 및 복구

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

파편을 사용하여 풀링된 장면에서 드문 게놈 변종의 탐지

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

전기 영동 이동성 분석 (EMSA) 및 Shift 키를 사용하여 기능 비 코딩 유전자 변형에 대한 심사 DNA 화성 강수 분석 (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...