이 작품은 인간 프론티어 과학 프로그램 보조금 RGP0024에 의해 재정 지원되었다.

1. DNA를 템플릿 준비 및 시험 관내 스크립트 작성에 <ol><li> 관심있는 유전자는 T7 및 / 또는 예 SP6 transcriptional 발기인 아래에 복제됩니다. </li><li> 시험 관내 전사의 내용은 템플릿 DNA가 ORF 정류장 코돈 및 / 또는 mRNA 3 &#39;끝에 스트림 절삭 적절한 제한 효소로 선형있다. 하나는 아가로 오스 겔 전기 영동에 제한 소화 제품을 실행하여 플라스미드 DNA의 완전한 선형화를 확인해야합니다. </li><li> 선형 플라스미드는 시험 관내 전사 반응에 사용됩니다. 템플릿 DNA의 서로 다른 농도는 시험 관내 전사에 필요한 최적의 DNA 농도를 확인하는 테스트를 할 수 있습니다. 일반적으로 Ambion의 MEGAscript 높은 항복 전사 키트 (Ambion / 생활 기술, 그랜드 아일랜드, NY)로, 1 μg 선형 DNA는 mRNA의 40-60 μg를 얻을. 시험 관내 전사에는 제조 업체의 지침 (Ambion / 생활 기술, 그랜드 Islan에 따라 이루어집니다D, NY). </li><li> 시험 관내 전사에 따라 mRNA는 제조 업체의 지침 (Ambion의 MEGAscript 높은 항복 전사 키트)에 따라 염화 리튬의 석출에 의해 정화된다. </li><li> mRNA 무결성이 더욱 아크릴 아미드 또는 아가로 오스 젤에서 전기 영동으로 확인됩니다. </li></ol> 2.에서는 체외 세포 무료 번역 다음 단계를 따르십 R abbit R eticulocyte Lysate, RRL을 (Promega, 매디슨, WI)를 사용하여 체외 번역의 경우 : <ol><li> 체외 번역 반응 다음과 같은 제조 업체의 지침에 100 μl를 준비합니다. 간단히, nuclease 무료 물에 2 μl 아미노산 혼합물 마이너스 메티오닌 (1 ㎜), RNase 억제제 (Invitrogen / 생활 기술, 그랜드 아일랜드, 뉴욕), 방사성의 20 μCi [35 S]-메티오닌 (MP의 Biomedicals 10 단위를 추가, ) 오, 솔론, 토끼 Reticulocyte Lysate (Promega, 매디슨, 위스콘신) 70 μl. </li><li> 일 품어따뜻한 번역 반응을 미리하는 5 분 30 ° C에서 전자 반응. 5 분 30 ° C에서 번역 반응과 부화 mRNA의 2 μg을 추가합니다. </li><li> 얼음 번역 반응을 배치하여 반응을 중지합니다. </li></ol> 3. 체외 번역 반응의 초기 Polypeptides의 분리 <ol><li> 리보솜 (polysome)에 바인딩 초기 폴리펩티드를 분리하려면 번역 반응이 100 밀리미터 KCl, 10 MM MgCI 2를 포함하는, 10 MM 트리스-HCL 버퍼 산도 7.6에서 30 % 글리세롤 4.5 ML 위에 뛰어넘는 세월을 거슬러 올라가, 그리고 100,000 X g에서 centrifuged합니다 4 ° C.에 1 시간을위한 TLA-110 로터 (Beckman 보습 바로 앞에 달린 풀베는 날, Inc의 어딘지, CA)에서 </li><li> 뜨는는 더욱 신중하게 제거됩니다. </li><li> 어렸을때 polypeptides를 포함 polysom​​al 펠릿에는 0.5 MG / ML의 ribonuclease (Invitrogen / 생활 기술, 그랜드 아일랜드, 뉴욕), 그리고위한 incubated를 포함, 1 ㎜ 트리스-HCL 버퍼 산도 7.6의 작은 부피 (10-15 μl)에 resuspended 있습니다 37 30 분 ° C. 순서대로peptidyl-tRNA 에스테르 결합의 가수 분해를 향상시키기 위해, NaOH는 10 mm의 최종 농도에 추가되고, 그리고 부화가 추가로 30 분 동안 계속됩니다. </li></ol> 체외에서 번역 반응 mRNA없이 조립과 동일한 조건 (설명되었습니다대로 초기 펩티드의 분리에 의해 다음) 하에서 수행이 중요한 컨트롤 역할을합니다. 밀도 기울기 원심 분리로 추출한 쓰는 전에 puromycin으로 변환 반응 (들)의 치료는 추가적인 제어 될 수있다. 4. 트리스 - tricine SDS 페이지의 초기 폴리펩티드를 해결 <ol><li> 격리된 초기 polypeptides가 해결 및 트리스 - tricine SDS-PAGE를 분석하고 있습니다. 물질의 양의 단백질 라벨, 단백질 번역과 많은 다른 파라미터에 대한 효율의 효율성에 관한 것이다 의존 겔에로드합니다. 로드된 물질의 양은 bes 수 있도록 경험적으로 조정되어야합니다t 시각화 및 개별 초기 폴리펩티드 사슬의 분리. </li><li> 전기 영동 장치 젤류가 해결된 후, 진공 젤 다이어를 사용하고 autoradiography를 받게 건조. 어렸을때 polypeptides의 분포는 phosphoimaging를 사용하여 관찰하고 있습니다. </li></ol> 5. 대표 결과 소의 감마-B Crystallin은 잘 E.와 같은 토끼 Reticulocyte의 Lysate로 번역되었다 대장균 S30 추출 시스템 (Promega, 매디슨, 위스콘신) 초기 폴리펩티드 사슬의 고립으로 따라갔다. 그림 1은 ribosome 바운드 초기 체인의 분리에 관여 단계를 묘사. tRNAs (때문에 생물 사이의 코돈 바이어스의 차이로 실질적으로 포유 동물의 다른 (RRL)와 prokaryotic (E. 대장균) 시스템으로 알려져 있음) 수의 번역 즉 레퍼토리의 환경을 변경하는 경우에는 현재의 연구에서는 목표, 테스트하는 것이었다 mRNA 따라 ribosome 운동을 변경하고 번역을 일시 중지 사이트의 분포에 영향을.변경된 ribosome 운동은 번역뿐만 아니라 그들의 상대적인 농도 동안 축적 초기 polypeptides의 크기 분포의 변화로 연결되어야합니다. 밴드의 상대 농도는 일시 정지 기간을 반영합니다. 어렸을때 polypeptides은 고립과 16.5 % T, 6퍼센트 C 트리스-tricine SDS 페이지 (그림 2)에서 해결이되었습니다. 감마-B Crystallin는 20 kDa 단백질이다. RRL와 E가 아닌 동일한 길이와 농도의 초기 polypeptides의 관찰 대장균 시스템은이 두 시스템의 mRNA를 따라 리보솜의 움직임이 다른 번역 속도론을 따른다는 것을 나타냅니다. 예를 들어, E.에 17.7 kDa의 유명 밴드를 관찰 그것은 E.로 번역되었을 때 mRNA의 3&#39; 엔드 지역 (감마 B Crystallin의 인코딩의 C-말단 지역)에서 추가 translational 일시 사이트가있다는 것을 암시 RRL 시스템에서 대장균의 lysate가 아닌, 대장균 시스템. 우리는 참고 감마-B Crystallin 항만 탠덤 아르헨티나 codons (AGG 일포유류 시스템에서 자주 있지만, E. 극히 드문 천천히 번역된 것으로 알려져 53과 AGA 154) 대장균. E.에 축적 소설 초기 펩타이드 대장균 시스템 (~ 17.7 kDa 그림 2.2) 가능성이 탠덤 희귀 codons에서 일시 중지 ribosome로 인해 발생합니다. 어떤 밴드는 mRNA의 부재 (그림 2.3)에서 관찰되지 않으며 그 puromycin 치료가 polysomes (그림 2.3)에서 초기 사슬의 대부분을 제거 할 수있는 점에 유의하십시오. puromycin (&gt; 15 분)와 장기간 치료는 초기 체인 (데이터가 보이지 않음)를 제거합니다. 이것은 관찰된 폴리펩티드 제품이 오히려 polysom​​es로 cosedimenting mRNPs 이상의 ribosome 관련 초기 체인,한지 확인합니다. 위에서 언급한 바와 같이 코돈 바이어스는 유기체뿐만 아니라 조직마다 다릅니다. 대표적인 예제는 여기에 설명된 분명 tRNAs / 코돈의 번역 즉 레퍼토리의 환경을 변경함을 나타냅니다바이어스는 mRNA 따라 ribosome의 개조 운동으로 이어질 수 있습니다. 결국,이 간단한 방법은 mRNA를 따라 translational 일시 중지 사이트의 신분을 허락할 수 없다뿐만 아니라, 서로 다른 시스템 간의 번역을 일시 중지 사이트의 빠른 비교를 허용합니다. <img alt="그림 1" src="/files/ftp_upload/4026/4026fig1.jpg" /> 1 그림. 도식은 ribosome 바운드 초기 polypeptides를 격리에 관련된 단계를 설명. 소의 감마-B Crystallin는 ORF 시퀀스 (528 기본 쌍) pBSKS에서 하류 T7 프로 모터의 복제되었습니다 +. 시험 관내 전사의 내용은 템플릿 DNA가 EcoRI으로 치료하여 선형되었다. 선형화된 템플릿은 시험 관내 전사에 사용되었습니다. DNA를 템플릿 T7 프로 모터는 하류 감마-B Crystallin 유전자를 transcribes T7 RNA 중합 효소에 의해 인정되고 있습니다. mRNA는 염화 리튬의 석출 방법을 사용하여 정화하고 있습니다. 정화 mRNA는 체외 변환에 사용됩니다. 다음의인큐베이션, 번역 반응은 30 % 글리세롤 용액 위에 뛰어넘는 세월을 거슬러 올라가, 4에서 1 시간 동안 centrifuged되고 ribosome 바운드 초기 polypeptides 격리시킬 100,000 X g에서 ° C에서. <img alt="그림 2" src="/files/ftp_upload/4026/4026fig2.jpg" /> 그림 2. 래빗 Reticulocyte Lysate (RRL)와 E로 번역 소의 감마-B Crystallin 초기 polypeptides의 분리 대장균의 Lysate가. 2 μg의 mRNA는 RRL 또는 E를 포함하는 100 μl 반응에 혼합되었다 5 분 20 μCi [35 S]-메티오닌 30시 incubated ° C와 제조 업체의 지시에 따라 대장균 S30 추출 시스템 (Promega, 매디슨, 위스콘신). 아래 부화는 번역 반응이 100 밀리미터 KCl, 10 MM MgCI 2를 포함하는, 10 MM 트리스-HCL 버퍼 산도 7.6에서 30 % 글리세롤 4.5 ML 위에 뛰어넘는 세월을 거슬러 올라가, 그리고에서 4 ° C와 100,000 X g에서 1 시간 동안 centrifuged되었다 TLA-110 로터 (Beckman 보습 바로 앞에 달린 풀베는 날, Inc의 어딘지, CA). 격리된 polyribosome 펠렛은 열매가 나타나고이었다은 (Invitrogen / 생활 기술, 그랜드 아일랜드, 뉴욕) NaOH (같은 프로토콜 섹션에서 설명)가있는 치료이어서 0.5 MG / ML의 ribonuclease를 포함, 1 ㎜ 트리스-HCL pH는 7.6 20 μl에 spended. 어렸을때 polypeptides은 16.5 % T, 6퍼센트 C 트리스-Tricine SDS PAGE 젤에 해결이되었습니다. 겔은 건조하고 autoradiography에 대해 접했습니다. 노출에 이어 젤은 GE 헬스케어의 태풍 이미징 스캐너를 사용하여 스캔했습니다. 그림 2.1은 번역 반응은 두 가지 다른 시스템 (RRL와 E 대장균)에 완료 이후 격리 및 해결 어렸을때 polypeptides로 젤을 보여줍니다. 그림 2.2는 겔은 이미지 양의 TL에 의해 분석되었다 . 번역 후 SDS 페이지를 격리 및 해결 어렸을때 polypeptides : v2005 소프트웨어와 두 시스템에 축적 초기 polypeptides의 분자량과 강도를 쉽게 분석하고 비교할 수 있도록 표시하는 예제로 여기에 사용되는 그림 2.3 컨트롤의 두 세트를 보여줍니다 반응이 모였습반응은 원심 분리에 노출되기 전에 mRNA 및 / 또는 후에없이 번역 반응 puromycin (5 분, 마지막 conc. 1 ㎜)로 치료되었다.

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Biochem. 166, 368-379 (1987).</li><li>Shirole, N., Balasubramanian, S., Yanofsky, C., Cruz-Vera, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Translating+Ribosomes+Containing+Peptidyl-tRNAs+for+Functional+and+Structural+Analyses.">Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses.</a> J. Vis. Exp. (48), e2498 (2011).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. S., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

시험 관내 전사 및 번역 반응에 사용되는 부품의 재현성 결과, 품질과 집중이 중요하십시오. 현재의 연구에서 우리는 주의깊게 처리하는 경우, 높은 재현성 데이터를 제공 상용 키트와 추출물을 사용했다. 필요한 경우에는 번역 - 관할 추출물은 하나의 선택의 세포에서 작성하실 수 있습니다. mRNA의 품질이 번역에 영향을 미칠 수 있으므로, 그것은 시험 관내 번역하는 동안 그?...

<table border="1"><tbody><tr><td> 시약 / 키트의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> MEGAscript T7 높은 항복 전사 키트 </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> Ribonuclease 억제제 </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> 트랜스 [35 S]-라벨 </td><td> MP의 Biomedicals </td><td> 0,151,006 </td></tr><tr><td> Ribonuclease - </td><td> Invitrogen </td><td> 12,091 </td></tr><tr><td> 래빗 Reticulocyte Lysate 시스템, Nuclease는 치료 </td><td> Promega </td><td> L4960 </td></tr><tr><td> 선형 템플릿에 대한 E. 대장균 S30 추출 시스템 </td><td> Promega </td><td> L1030 </td></tr><tr><td> 원심 분리 </td><td> Beckman 보습 바로 앞에 달린 풀베는 날 </td><td> Optima TLX의 Ultracentrifuge </td></tr><tr><td> 저장 형광체 autoradiographY </td><td> GE 헬스케어 </td><td> 태풍 9,410 가변 모드 영상기 </td></tr><tr><td> 초기 폴리펩티드 분석을위한 소프트웨어 </td><td> GE 헬스케어 </td><td> 이미지 양의 TL, v2005 </td></tr></tbody></table>

isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna

translational 신장의 속도가 아닌 교복이다. 이차 구조, 코돈 사용을 mRNA와 1 참조 관련 단백질 검토를 위해 메시지에 ribosome 운동을 변경할 수도 있습니다 mRNA. 그러나 이제는 널리 동의어 코돈 사용하는 비 균일한 translational 신장 속도 1 차 원인임을 인정있어. 동의어 codons는 동일한 주파수로 사용되지 않습니다. 바이어스가 다른이보다 더 자주 사용하는 일부 codons와 동의어 codons의 사용에 있습니다. 코돈 바이어스는 유기체뿐만 아니라 2,3 구체적인 조직이다. 또한 코돈 사용 빈도가 기원 tRNAs 4의 농도에 직접 비례합니다. 따라서 자주 사용하는 코돈은 더 자주 코돈이 빠르게 드문보다 번역된다는 것을 의미합니다 해당 tRNAs 높은 무리를 가질 것입니다. 따라서, 희귀 codons (잠재 일시 사이트)에 풍부 mRNA의 지역은 원칙적으로 messa에 ribosome 움직임이 느려지며각각의 크기는 5-8의 초기 펩티드의 GE와 명분 축적. 이러한 일시 사이트가 단백질 발현에 대한 기능적 영향을 미칠 수 mRNA 안정성 및 검토를위한 단백질 폴딩 9를 참조하십시오. 실제로, 그것은 같은 일시 중지 사이트의 완화는 mRNA에 ribosome 운동을 변경할 수 있으며, 이후 공동 translational (생체내)을 단백질 폴딩 1,7,10,11의 효율성에 영향을 미칠 수있는 것으로 나타났습니다. 궁극적으로 그것이 translational 연신율 동안 mRNA를 따라 리보솜의 움직임에 대한 코돈 사용 / tRNA 콘텐츠의 영향으로 종합적인 통찰력을 얻기 위해 필수적인 단백질 합성의 과정에 결합되어 세포에서 생체내의 단백질 접힘의 과정을 이해하려면 . 여기에서 우리는 다양한 세포없는 시스템을 6-8로 번역 주어진 mRNA에 대한 주요 번역 일시 사이트를 찾는 데 사용할 수있는 간단한 기술을 설명합니다. 이 절차는 초기 polypeptides accumulati의 고립을 기반으로합니다대상 mRNA의 체외 번역의 도중 리보솜에 NG. 근거는 낮은 주파수 codons에서 해당 규모의 초기 펩티드의 증가 금액 리보솜 결과의 체류 시간 증가. 체외 베꼈는데 mRNA에서 radioactively 라벨이 아미노산의 존재에 체외에서 translational 반응에 사용되고있다는 것입니다 초기 체인의 검출을 허용합니다. ribosome 바운드 초기 폴리펩티드 단지를 분리하기 위해서는 번역 반응은 원심 분리에 의해 다음에 30 % 글리세롤 용액 위에 뛰어넘는 세월을 거슬러 올라가있다. polysom​​al 펠렛의 초기 polypeptides은 더욱 ribonuclease로 치료와 SDS 페이지에 의해 해결된다. 이 기술은 잠재적으로 어떤 단백질에 사용되며 ribosome mRNA를 따라 움직임과 주요 일시 중지 사이트의 검출 분석을 허용 할 수 있습니다. 또한이 프로토콜은 ribosome 운동을 변경할 수 있으며 따라서 잠재적으로 또 일을 변경할 수있는 요인과 조건을 연구하는 데 적용할 수 있습니다전자 기능 / 단백질의 형태.

Watch this Scientific Journal Video about Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA at JoVE.com

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

mRNA의 translational 일시 사이트를 식별하는 기술은 설명되어 있습니다. 이 절차는 denaturing 겔 전기 영동을 사용하여 초기 체인의 크기를 분석하여 다음 타겟 mRNA의 체외 번역의 도중에 변이를 축적 초기 polypeptides의 분리에 근거한다.

Ribosome 바운드 초기 Polypeptides의 분리<em&gt; 체외에서</em&gt; mRNA 함께 Translational 일시 중지 사이트를 식별하려면

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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Biology

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

16.2K 조회수.  Cleveland State University. mRNA의 translational 일시 사이트를 식별하는 기술은 설명되어 있습니다. 이 절차는 denaturing 겔 전기 영동을 사용하여 초기 체인의 크기를 분석하여 다음 타겟 mRNA의 체외 번역의 도중에 변이를 축적 초기 polypeptides의 분리에 근거한다.

비디오: Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR - 클립 - RNA 단백질의 바인딩 Transcriptome 전체 바인딩 사이트를 식별하는 방법

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

연구

생물학

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

분리 및 체외에서</em의> 활성화 Caenorhabditis 엘레간스 정자

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Coregulated 진스 잠재적인 규제 모티브를 식별하기 위해 범위를 사용하여

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribosome 바운드 Polypeptides을 파악하는 도구로 SecM 체포 시퀀스를 사용하여

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

체외 합성에 인간의 세포에서 단백질 발현 유도의 mRNA를 수정의

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

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 ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

포유 동물 세포에서 초기 DNA에 바인딩 단백질의 시험 BrdU의 칩 슬롯 서양 기술을 사용하여

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

유전자를 사용 하 여 프로토콜 변환 연구에 대 한 적절 한 동물 모델을 식별 하기 위해 농축 분석 설정

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

생체 외에서 스모 E3 리가 활동 공부 SUMOylation 분석 결과

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...