이 작품은 국립 과학 재단 (부여 번호 MCB - 0447750)에서 부여에 의해 투자되었다.

<ol>
	<li>Clark, K. A., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+An+improved+high-throughput+method+for+generating+tissue+samples+in+96-well+format+for+plant+genotyping+(Ice-Cap+2.0).">Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0).</a> Plant Methods. 3, 8-8 (2007).</li><li>Krysan, P. I. c. e. -. c. a. p. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+capturing+plant+tissue+samples+for+genotype+analysis.">A high-throughput method for capturing plant tissue samples for genotype analysis.</a> Plant Physiol. 135, 1162-1169 (2004).</li><li>Jander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+map-based+cloning+in+the+post-genome+era.">Arabidopsis map-based cloning in the post-genome era.</a> Plant Physiol. 129, 440-450 (2002).</li><li>Liljegren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHATTERPROOF+MADS-box+genes+control+seed+dispersal+in+Arabidopsis.">SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis.</a> Nature. 404, 766-770 (2000).</li><li>Buechel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+A-type+ARABIDOPSIS+RESPONSE+REGULATORS+in+meristem+maintenance+and+regeneration.">Role of A-type ARABIDOPSIS RESPONSE REGULATORS in meristem maintenance and regeneration.</a> Eur. J. Cell. Biol. 89, 279-284 (2010).</li><li>Krysan, P. J., Jester, P. J., Gottwald, J. R., Sussman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mitogen-activated+protein+kinase+kinase+kinase+gene+family+encodes+essential+positive+regulators+of+cytokinesis.">An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis.</a> Plant Cell. 14, 1109-1120 (2002).</li><li>Bush, S. M., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iTILLING:+a+personalized+approach+to+the+identification+of+induced+mutations+in+Arabidopsis.">iTILLING: a personalized approach to the identification of induced mutations in Arabidopsis.</a> Plant Physiol. 154, 25-35 (2010).</li><li>Comai, L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING:+practical+single-nucleotide+mutation+discovery.">TILLING: practical single-nucleotide mutation discovery.</a> Plant J. 45, 684-694 (2006).</li><li>Henikoff, S., Till, B. J., Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING.+Traditional+mutagenesis+meets+functional+genomics.">TILLING. Traditional mutagenesis meets functional genomics.</a> Plant Physiol. 135, 630-636 (2004).</li><li>McCallum, C. M., Comai, L., Greene, E. A., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+induced+local+lesions+IN+genomes+(TILLING)+for+plant+functional+genomics.">Targeting induced local lesions IN genomes (TILLING) for plant functional genomics.</a> Plant Physiol. 123, 439-442 (2000).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기에 제시 아이스 캡 방법은 하루에 각 식물의 수백, 또는 수천의 조직 샘플을 수집하고 효율적으로 genotyping 반응에 사용하기 위해 이러한 샘플에서 게놈 DNA를 추출 한 과학자 수 있습니다. 개별 과학자 시간의 짧은 기간에 모종의 수천을 유전자형 수있는 능력을 제공함으로써, 아이스 캡 방식은 다르게 수행하는 실용적 않을 유전자 실험의 숫자를 촉진시킬 잠재력이있다. 몇 가지 예제가 아래에 제공됩니다. <ol><li> 유전자 매핑. 연구원은 염색체 3 따라 특정 간격 내에 재조합 이벤트를 식별할 수있다면 미세 규모의 유전자 매핑을 빠른 수 있습니다. 이 간격이 매우 짧은 유전자 거리를 포함한다면, 재조합 이벤트 segregating 매핑 인구 비교적 드문 것입니다. 이런 이유로, 그것은 원하는 다시 찾을 개별 식물의 화면 수천해야 할 수도 있습니다combinant 개인. 이 과정은 크게 얼음 모자의 사용을 통해 빠른 수 있습니다. 심지어 게놈 와이드 다시 시퀀싱의 시대에 직접 주어진 돌연변이가 관심의 표현형 직접 책임이있다는 것을 입증하는 여전히 필요합니다. 이러한 경우에는, 그것은 후보 변이 실제로 원인이되는 것입니다 증명하기 위해 다른 밀접하게 연결된 돌연변이 떨어져 관심의 변이를 분리하기 위해 필요합니다. 이러한 상황에서는, 아이스 캡은 밀접하게 연결된 한 쌍의 변이 사이의 연동을 깨는위한 효율적인 방법을 제공합니다. </li><li> 멀티 돌연변. 그것은 기능 중복 4-6을 극복하기 위해 하나의 개인으로 몇 가지 별개의 loci에서 돌연변이 대립 유전자를 조합하여 Arabidopsis를 공부하고 연구에 대한 일반되었다. 예를 들어, 하나는 유전자 횡단을 통해 단일 ​​공장으로 네 가지 loci에서 돌연변이 대립 유전자를 결합하고자 사건을 고려하십시오. 이 프로젝트에 대해, 그것은을 생성하는 데 필요한 것입니다횡단 계획의 일환으로이 네 loci에서 heterozygous있다 N 개별. 이 사중 - heterozygote 세트의 씨앗이, 사중 homozygote는 자손의 결과 인구 256의 밖으로 하나의 비율로 존재 것으로 예상하는 경우. 전통적인 방법으로이 드문 개인에 대한 선별하는 것은 매우 노동 집중 것입니다. 비교함으로써,이 프로젝트 얼음 모자를 사용하면 하나의 세대에 희귀한 사중 homozygote 식별을 위해 효율적인 전략으로 과학자를 제공할 것입니다. </li><li> iTILLING. 저희 연구실은 최근 Arabidopsis 7의 관심 유발 변이 유전자의 존재를 검사하기위한 새로운 접근 방식을 설명했다. 이 방법은 화학 mutagen 80-10로 치료 개인의 주문 인구의 변이를 찾을 ​​사용되는 TILLING라는 설립된 기술에 대한 변화이다. 그것이 산업 있습니다 TILLING의 개별 버전이기 때문에 우리는 우리의 방법 iTILLING 이름을 붙여 놨ividual 과학자는 전통적으로 완료하는 데 큰 연구팀 필요가 무언가를 달성할 수 있습니다. 아래에 설명된대로 아이스 캡은 iTILLING 방법 7의 중요한 부분을 형성한다. </li></ol> iTILLING 뒤에 아이디어는 전통적인 TILLING 프로젝트 만든 내구성 돌연변이 인구 대비 서 임시 돌연변이 인구를 생산하는 것입니다. iTILLING 들어, bulked M2 인구의 종자가 50-100 얼음층 블록에 sown하고 있으며, DNA는 아이스 캡 방법을 사용하여 추출됩니다. Arabidopsis 모종은 다음 4에 배치됩니다 ° C 돌연변이 검사는 아이스 캡 DNA를 사용하여 수행하는 동안 preps. 토마토 모종은 12 ° C.에 저장됩니다. 모종 운반 원하는 돌연변이가 확인되고 나면, 그들은 아이스 캡 접시에서 회복하고 흙으로 전송됩니다. iTILLING는 화면 전체 연구 커뮤니티에 대한 지속적인 돌연변이 인구를 제공하기위한 것이 아닙니다. 대신, iTILLING는 전략을 제공하는과학자 한사람 효율적으로, 그리고 상대적으로 낮은 비용으로 신속하게 유전자의 소수의 돌연변이에 대한 사용자 지정 돌연변이 인구를 스크린 수 있습니다. 우리는 Arabidopsis, 토마토, 그리고 쌀을 재배하고 genotyping을위한 효과적인 방법으로 얼음 모자를 발견했습니다. 우리는 식물의 다른 종류도 아이스 모자 의무가되어야한다는 기대합니다. 아이스 캡에 의해 수용 수있는 수종의 다양한 한 제한은 종자의 크기입니다. 96 - 웰 플레이트의 우물에 들어갈만큼 작은 씨앗과 식물만이 현재 구성에서 아이스 모자와 호환됩니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> GenoGrinder </td><td> Spex / CertiPrep, Metuchen, 뉴저지 </td><td> 2000 제노 / 그라인더 </td><td> DNA 추출을위한 루트 번호판을 수수 혐의로 머신 </td></tr><tr><td> ABgene 써모 - 실러 </td><td> ABgene 미국 </td><td> AB - 0384 </td><td> 조직 중단하기 전에 루트 판하는 데 사용되는 수동 열 씰링 기계 </td></tr><tr><td> 스윙 microplate 캐리어가 장착된 원심 분리기 </td><td> 베크 만 보습 바로 앞에 달린 풀베는 날 </td><td> Allegra 25R </td><td> 스윙 microplate 캐리어와 함께 모든 원심 분리기는 적절한 것입니다 </td></tr><tr><td> MicroFill Microplate 디스펜서 </td><td> BioTek www.biotek.com </td><td> AF1000A </td><td> 충전 microplates에 대한 자동화된 액체 디스펜서 </td></tr><tr><td> Bialetti 브랜드17x11 인치 대형 상업 쿠키 시트 </td><td> 대상 백화점 </td><td> www.bialetti.com </td><td> 특정 쿠키 시트는 얼음 모자를위한 이상적인 깊이를 가지고 </td></tr><tr><td> 사용자는 금속 랙을 제작 </td><td> 철물점 </td><td></td><td> 전체 설명에 대한 텍스트를 참조하십시오 </td></tr><tr><td> 33 리터 플라스틱 저장 박스 </td><td> 대상 백화점 </td><td> Sterilite 브랜드 </td><td> 아이스 캡 분수하십시오. 17x11 인치 쿠키 시트를 고정할 정도의 커다란 플라스틱 저장 박스 </td></tr><tr><td> 시간 분수 펌프 당 30갤런 </td><td> Sunterra </td><td> 104,506 </td><td> 정원 공급 상점에서 사용 가능한 소형 잠수정의 분수 펌프 </td></tr><tr><td> 분수 펌프를위한 호스 </td><td> 모든 실험실 공급 회사 </td><td></td><td> 분수 펌프와 호환 호스 </td></tr><tr><td> 플라스틱 클램프 </td><td> 철물점 </td><td></td><td> 쿠키에 호스를 잡아 고정시트 </td></tr><tr><td> 팔콘 Microtest 평면 바닥 96 - 웰 폴리스티렌 접시에서 투명한 플라스틱 뚜껑 </td><td> BD 팔콘 www.bdbiosciences.com </td><td> 351,172 </td><td> 아이스 캡 묘목 플레이트를 덮고위한 투명한 플라스틱 뚜껑 </td></tr><tr><td> 3M Micropore 외과 테이프 </td><td> 피셔 과학 </td><td> 19-027-761 </td><td></td></tr><tr><td> 3/32-inch 직경 스테인레스 스틸 공 </td><td> 하트 포드 테크놀로지, 록키 힐, CT </td><td> 034-006 - 1K </td><td> 수확 후 뿌리 조직을 방해하는 데 사용 </td></tr><tr><td> 3 인치 탄성 헤어 밴드 </td><td> 지역 식료품 가게 </td><td></td><td> 묘목 플레이트에 루트 플레이트를 확보하는 데 사용 </td></tr><tr><td> 4mm 직경 나무 꼬치 </td><td> 지역 식료품 가게 </td><td></td><td> 이러한 꼬치 일반적으로 kabobs를 준비하는 데 사용됩니다 </td></tr><tr><td> 피셔 과학 Nunc 브랜드 프릿없이 1 - ML 필터 플레이트 </td><td> 피셔 과학 </td><td> 278,012 </td><td> 바닥에 구멍이있는 96 - 웰 플레이트 묘목. </td></tr><tr><td> Fisherbrand 96 - 잘 크고 - 굴뚝 PCR 플레이트 </td><td> 피셔 과학 </td><td> 14-230-242 </td><td> 96 - 웰 플레이트 루트 </td></tr><tr><td> 한천 - 세포 배양 테스트 </td><td> 시그마 - 올드 리치 </td><td> A1296 </td><td></td></tr><tr><td> 4 - Morpholinoethanesulfonic 산 (MES) </td><td> 시그마 - 올드 리치 </td><td> M2933 </td><td></td></tr><tr><td> Murashige와 Skoog (MS) 기초 소금 혼합물 </td><td> 시그마 - 올드 리치 </td><td> M - 5524 </td><td></td></tr><tr><td> 호일을 밀봉 쉽게 벗겨 열 </td><td> ABgene 미국 </td><td> AB - 0745 </td><td> 조직 중단하기 전에 루트 번호판을 씰링을위한 열 씰링 필름 </td></tr><tr><td> 오리 브랜드 엑스트라 와이드 포장 테이프, 3.00 &quot;X 54.6 야드 </td><td> OfficeMax www.officemax.com </td><td> 아무 항목 없습니다. 21242139 </td><td> 접착제 테이프스토리지를위한 DNA 추출물을 포함하는 씰링 microplates에 대한 </td></tr><tr><td> 실링 롤러 </td><td> 바이오 방사선 </td><td> MSR - 0001 </td><td> 손 microplates에 접착 씰링 테이프를 누르면위한 압연 도구를 개최 </td></tr><tr><td> 96 - 웰 금속 열 차단 </td><td> 콜 - Parmer </td><td> 36400-66 </td><td> 조직의 수확의 루트 번호판을 동결하는 데 사용 </td></tr><tr><td> 워트 먼지 필터 용지 </td><td> 워트 먼지 국제 </td><td> 1002 090 </td><td> 표면 살균 씨앗에 사용 </td></tr><tr><td> 일회용 파스퇴르 피펫 </td><td> 피셔 과학 </td><td> 13-678 - 6A </td><td> 5 어 &quot;일회용 파스퇴르 피펫은 묘목 플레이트에 씨앗을 전송에 사용 </td></tr><tr><td> Pyrex 베이킹 접시 (20cm X 30cm) </td><td> 대상 백화점 </td><td></td><td> 드라이 아이스 / 에탄올 목욕을 구축하는 데 사용됩니다. </td></tr></tbody></table>

ice-cap: a method for growing arabidopsis and tomato plants in 96-well plates for high-throughput genotyping

그것은 식물이 다수의 genotyping을 필요로 프로젝트를 개발하기 위해 식물 과학자에 대한 일반지고 있습니다. 모든 genotyping 프로젝트의 첫 번째 단계는 각각의 공장에서 조직 샘플을 수집하는 것입니다. 이 작업에 대한 전통적인 접근 방식은 샘플 식물 하나에서 - - 시간입니다. 개인의 유전자형 수백 또는 수천 한 소망 그러나, genotyping 파이프라인에 상당한 병목 현상이 전략 결과를 사용하는 경우. 우리가 여기서 설명하는 아이스 캡 방식은 과학자 한사람 하루 1,2에서 수천 모종의 조직을 수집할 수 있도록 허락해하여이 도전하는 높은 처리량 솔루션을 제공합니다. 처리량이 수준은 조직이 아니라 하나에서 - - 시간보다, 식물의 96시 - 시간을 수확있다는 사실에 의해 가능하게됩니다. 아이스 캡 방법은 묘목의 성장, 조직 수확 및 DNA 추출을 수행하기위한 통합 플랫폼을 제공합니다. 얼음 모자의 기초는 seedli의 성장입니다96 - 웰 플레이트의 스택 쌍의에 ngs. 상단 접시의 우물 개별 모종은 발아되는 한천 성장 미디어의 플러그가 포함되어 있습니다. 뿌리는 한천 매체를 통해 구멍을 통해 상부 판을 종료하고, 낮은 플레이트가 포함된 물에 통과 아래로 성장합니다. 상단 플레이트에있는 묘목은 실온에서 유지하면서 DNA 추출, 뿌리 조직을 포함하는 낮은 접시에 물이 조직을 수확하는 것은 급속 냉동입니다. 상부 판은 다음 얼음과 96 가능한 모종 한 판에 얼어 붙 어서 96 루트 조직 샘플 한 접시를 항복, 멀리 낮은 접시에서 벗겨 있습니다. 그것은 루트 조직을 캡처하는 얼음을 사용하기 때문에이 기술은 &quot;아이스 모자&quot;라는 것입니다. 묘목을 포함한 96 - 웰 플레이트 다음 호일에 싸여과 낮은 온도로 전송하실 수 있습니다. 이 과정은 모종의 추가 성장을 정직 시키실지만, 그들의 생존에 영향을주지 않습니다. 유전자형 분석이 완료되면 원하는 유전자형과 모종은 96 잘 P에서 전송할 수 있습니다자세한 전파를 위해 토양에 늦었어요. Arabidopsis thaliana 우리는, 토마토, 그리고 쌀 모종을 사용하여 아이스 캡 방식의 유틸리티를 증명하고있다. 우리는 방법도 96 - 웰 플레이트의 우물에 들어갈만큼 작은 씨앗과 식물의 다른 수종에 적용되어야한다고 기대합니다.

Watch this Scientific Journal Video about Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping at JoVE.com

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping | Biology | Video

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

아이스 캡 방식은 하나가 각 묘목의 96 - 웰 플레이트 및 비 destructively 수확 루트 조직에 식물을 성장 할 수 있습니다. 이 루트 조직에서 추출한 DNA는 genotyping 반응에 사용될 수 있습니다. 우리는 얼음 모자가 잘 작동하는지 발견<em&gt; Arabidopsis thaliana</em&gt;, 토마토, 쌀 모종.

얼음 총액 : 성장을위한 방법 Arabidopsis높은 처리량 Genotyping을위한 96 - 웰 플레이트에와 토마토 식물

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

ice-cap-method-for-growing-arabidopsis-tomato-plants-96-well-plates

Research

Education

JoVE Journal

JoVE Core

Biology

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

Plant Biology

Plant Reproduction

SCIENTISTS IN ACTION

22.7K 조회수.  University of Wisconsin-Madison. 아이스 캡 방식은 하나가 각 묘목의 96 - 웰 플레이트 및 비 destructively 수확 루트 조직에 식물을 성장 할 수 있습니다. 이 루트 조직에서 추출한 DNA는 genotyping 반응에 사용될 수 있습니다. 우리는 얼음 모자가 잘 작동하는지 발견 Arabidopsis thaliana, 토마토, 쌀 모종.

비디오: Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing : 정확한 Genotyping을위한 간단한 방법

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

연구

생물학

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

높은 처리량 Zebrafish의 정자 Cryopreservation에 대한 방법과 체외에서 수정

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

인간 사전 mRNA에 매핑 Ribonucleoproteins에 대한 신속한 하이 스루풋 방법 (RNPs)

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

측정 Caenorhabditis 엘레간스</em96 웰 플레이트에 Microtiter> 수명

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

의식 쥐의 사구체 여과율의 측정을위한 높은 처리량 방법

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

측정 공간적 칼슘<sup&gt; 2 +</sup애기 장대 식물에서&gt; 신호

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

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 ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

의 산화 스트레스 저항을 측정<em&gt; 예쁜 꼬마 선충</em&gt; 96 웰 마이크로 타이 터 플레이트에

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag 분석 - 높은 처리량, Sc2.0 유전형에 대한 실시간 PCR 분석

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

토마토 털이 뿌리의 생성에 의한 높은 처리량 CRISPR 벡터 건설 및 DNA 수정의 특성

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...