이 작품은 버지니아 대학, 버지니아 암 센터의 대학과 토마스 F.와 케이트 밀러 제프리스 기념 트러스트에 의해 지원됩니다. 우리는 세포 라인에 대한 제임스 E. 다넬 랩 (록펠러 대학교) 로버트 로스 연구실 (포드햄 대학) 감사합니다.

계획 칩 실험이 계획되어 하루 전에, 세포가 충분한 숫자가 제공되도록 교양하여야한다. 각 칩 분석이 ~ 1-1.5 X 10 7 세포가 필요합니다 있다고 가정합니다. 항상 부정적인 (IgG)와 긍정적인 (팬 히스톤 항체) 컨트롤과 중복 실험을 모두 할 계획입니다. 팁 - 2FTGH 전지, 각 칩 분석이 80 % confluency 15 약 cm 조직 문화 요리가 필요합니다. 1 부 : 염색질 준비와 칩을 수행 <ol><li> 문화 미디어를 제거와 10 %의 우주 송아지 혈청 (CCS) / 5 NG / ML에서 IFN - 감마를 포함한 DMEM 20 ML로 필요한 시간에 대한 세포를 유도. CCS / DMEM뿐만 아니라 10 % 20 ML과 uninduced 접시에있는 미디어를 교체합니다. </li><li> 퓸 배출 후드에서는 15cm의 접시 미디어 20 ML (1 % 최종 농도)을 540 μL 37 % 포름 알데히드를 추가합니다. 팁 - 틸트 판과 미디어 수영장에 포름 알데히드를 추가 - 다음 바위 플레이트는 균일하게 그것을 확산합니다. </li><li> 실온에서 10 분 진행 crosslinking을 허용합니다. </li><li> crosslinking를 중지 0.125 M 최종 농도 글리신을 추가합니다. </li><li> 미디어를 대기음 진공 10 ML 얼음 추위 인산염과 함께 한 세포 세차 호수 (PBS)를 버퍼. </li><li> ~ 7 ML PBS에 세포 스크레이퍼와 세포를 수집하고 얼음 50 ML 원뿔 관에 동일하게 취급했다 세포를 (6 판에서) 결합. </li><li> 4 1,800 rpm으로 원심 분리기 (베크맨 Coutler 알레 그라 12 - R 원심 분리기) · 5 분 C와 PBS를 대기음. 팁 - 스냅 동결 세포 펠릿 경우 여기를 중지하고 -80 ° C.에 저장 </li><li> 4 ML 부풀어 올랐고 버퍼 (25 MM의 HEPES 산도 7.8, 1.5 MM MgCl 2, 10 MM KCl, 0.1 % NP40. 1 ㎜의 DTT, 완전한 프로 테아제 억제제 및 사용하기 전에 얼음에 진정을 추가)의 세포 펠렛을 Resuspend과 15 전송 ML 원뿔 관. 팁 -이 붓기 버퍼 볼륨 6 접시 풀링 (~ 6-9 X 10 7 세포)에 적합합니다. 더 많거나 적은 버퍼 douncing 효율성을 변경할 수 있습니다. </li><li> 10 분 얼음에 세포 현탁액을 품어. </li><li> 미리 차게 15 ML douncer (위턴)에 정지를 전송하고 유봉 A. 20 스트로크를 다운스 </li><li> 전송 얼음에 15 ML 원뿔 관에 다시 세포를 dounced. </li><li> 4 5 분 2,500 rpm으로 원심 분리기는 ° C와 뜨는 폐기하십시오. </li><li> Sonication 버퍼 3 ML의 핵 펠릿 (6 접시에서 ~ 200 μL) (15 펠릿 볼륨) (50 MM의 HEPES의 산도 7.9, 140 MM NaCl, 1 MM EDTA (에틸렌 다이아 민 테트라 초산), 1 % 트리톤 X - 100, 0.1 %의 나트륨 deoxycholate, Resuspend 0.1 % SDS). 사용하기 직전 얼음 완료 테아제 억제제와 진정을 추가합니다. 팁 -이 sonication 버퍼 볼륨 6 접시 풀링 (~ 6-9 X 10 7 세포)에 적합합니다. 더 많거나 적은 버퍼 sonication 효율을 변경할 수 있습니다. </li><li> microtip 장착된 Misonix의 Sonicator 3000을 사용하여 10 사이클 (20초 8 설정 on/40 초 ON / OFF 진폭)에 얼음 핵 서스펜션을 Sonicate. 크기 200-1000 BPS 평균 염색질 조각이 일반적으로 발생합니다. 팁 - sonicators이 달라집니다 때문에 sonication 효율이 경험적으로 결정되어야합니다. 이렇게하려면, sonication 전에 각 sonication 사이클 이후 20 μL의 aliquots 가져가라. 4 시간 동안 65 ° C에서이 샘플을 가열. DNA 로딩 버퍼를 추가하고 DNA 조각을 시각화하기위한 기준 1퍼센트 아가로 오스 / TBE 젤을 실행합니다.) </li><li> 전송 4 20 분 SS34 원심 분리기 튜브와 원심 분리기에 자료를 sonicated ° 일반적으로 세포 파편의 소량 무엇 SS34 로터의 13,000 RPM (Sorvall RC6 플러스 원심 분리기)에서 펠렛을 C. </li><li> 조심스럽게 얼음 15 ML 원뿔 관에 뜨는을 전송하기만하면됩니다. </li><li> 360 μL 연어 정자 DNA / 단백질 A 또는 G 아가로 오스 비즈 슬러리를 추가하고 적어도 30 분 동안 4 ° C에서 락을에서 미리 취소합니다. 팁 -이 단계가 아닌 특정 바인딩 이벤트에서 발생 분석에서 소음을 감소시킵니다. </li><li> 펠렛 아가로 오스 비즈 5 분 동안 4 ° C에서 1,000 rpm으로 원심 분리기. </li><li> 얼음 다른 15 ML 원뿔 관에 뜨는 사전 허가 전송합니다. </li><li> 미리 지워 염색질의 A260 단위를 측정하고 4 A260 단위 (0.2 MG / ML 1 A260 단위 = 0.050 MG / ML 가정)에 Sonication 버퍼로 음량을 조절 190 μL DDW 10 μL sonication 버퍼와 빈 분광 광도계, - 팁 A260 단위를 측정하려면, 190 μL DDW의 사전 허가 염색질 10 μL를 희석. </li><li> 20 입력 샘플 및 저장소로 염색질의 75 μL (4 A260 단위에서) 저장 ° C crosslinks을 뒤집을 준비까지. 팁 -이 단계는 데이터 분석을 위해 매우 중요합니다. 입력 나누어지는을 위해 게을리하지 마십시오. </li><li> I에서 1.7 ML의 microfuge 튜브로 나누어지는가 준비 염색질CE 1 ML의 aliquots로. 팁 - 여기 중지하는 경우, -80에 드라이 아이스와 저장소에 준비된 염색질을 동결 스냅 ° C. NB가 : 가능하면 저장된 염색질은 덜 최적의 결과를보다 생산 저하시킬 수 있으므로 피하십시오. </li><li> 유도와 uninduced 샘플에 칩 등급 항체를 추가하면 중복으로 설정합니다. 유도 및 uninduced 샘플 세트로 부정과 긍정적인 컨트롤로 IgG (2 μg)과 히스톤 H3 이동 항체 (15 μL)를 추가합니다. 팁 - 사용하는 항체의 금액은 경험적으로 결정 또는 제안 금액에 대한 공급 업체의 제품 시트를 참조해야합니다. 칩 학년 항체의 일반적으로 1-2 μg 잘 작동합니다. 적절한 볼륨이 혈청으로 제공하는 항체에 대한 경험적으로 결정되어야합니다. </li><li> 4 박 모두 튜브를 록 ° C. </li></ol> 2 부 : IMMUNOCOMPLEXES를 수집 <ol><li> 모든 튜브에 연어 정자 DNA / 단백질 A 또는 단백질 G 아가로 오스 비즈 슬러리의 60 μL를 추가합니다. 팁 사용 단백질 마우스 단클론 항체에 대한 토끼 polyclonal 항체 및 단백질 G위한. </li><li> 적어도 60 분 4 ° C에서 록. </li><li> 4 2,0​​00 rpm으로 부드럽게 microfuging하여 펠렛은 immunocomplexes (아가로 오스 비즈) · 3 분 C. </li><li> 뜨는을 대기음. </li><li> 4 ° C. 로킹 10 분 동안 다음 씻어 버퍼의 각 1 ML로 immunocomplexes 와시 4 2,0​​00 rpm으로 3 분 Microfuge · 각 씻고 뜨는을 대기음 사이에 C. 얼음에 냉장 샘플 보관하십시오. 사용하기 직전 PMSF를 추가합니다. <ul><li> 낮은 소금 워시 - 0.1 % SDS, 1 % 트리톤 X - 100, 2 MM EDTA (에틸렌 다이아 민 테트라 초산), 20 MM 트리스 - CL, 산도 8.0, 150 MM NaCl, 1 MM PMSF </li><li> 고 염분 워시 - 0.1 % SDS, 1 % 트리톤 X - 100, 2 MM EDTA (에틸렌 다이아 민 테트라 초산), 20 MM 트리스 - CL, 산도 8.0, 500 MM NaCl, 1 MM PMSF </li><li> LiCl 워시 - 0.25 M LiCl, 1 % NP - 40, 1 %의 나트륨 deoxycholate, 1 MM EDTA (에틸렌 다이아 민 테트라 초산), 10 MM 트리스 - CL, 산도 8.0, 1 ㎜ PMSF </li><li> TE 워시 - TE 버퍼 -10 MM 트리스 - CL, 산도 8.0, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 1 MM PMSF 두 씻는다 </li></ul></li><li> 최종 TE 버퍼 세척을 제거하고 구슬을 500 μL 용출 버퍼 (50 MM 트리스 - CL, 산도 8.0, 10 MM EDTA (에틸렌 다이아 민 테트라 초산), 1 % SDS)을 추가합니다. 팁 - 용출 버퍼가 신선하게 준비한다. </li><li> 65 30 분 용출 버퍼와 더위에 아가로 오스 비즈를 섞어 소용돌이 ° C. 팁 -이 삼십분 동안 각각의 튜브는 최소한 10 분 간격 와동. </li><li> 2,000 rpm으로 30 초 동안 Microfuge 새로운 microfuge 튜브에 eluent를 전송합니다. 팁 - 용출하는 동안 귀하의 입력 샘플을 해동하고 각 500 μL 용출 버퍼를 추가합니다. 앞으로 모든 단계에서 이러한 샘플을 포함합니다. </li><li> 모든 샘플 및 와동 200 mm의 최종 농도 5 M NaCl을 추가합니다. </li><li> 65 품어 ° C 숙박 (또는 적어도 4 시간). </li></ol> 3 부 : 정화 ChIP&#39;d 및 입력 DNA <ol><li> 뚜껑에 결로를 수집하는 30 초 동안 모든 샘플을 Microfuge. </li><li> 15 분 동안 상온에서 각각의 튜브와 부화 RNAse의 1μL (10 U / μL) 추가합니다. </li><li> 10 μL 0.5 M EDTA (에틸렌 다이아 민 테트라 초산), 40 μL 0.5 M 트리스 - CL 산도 6.5, 2 μL Proteinase K (10 MG / ML) 추가 45에서 60 분 ° C 품어. 팁이 시약의 마스터 믹스를 준비하고 각 튜브 52 μL를 추가합니다. </li><li> 페놀 / 클로로포름 / isoamyl 알콜 추출하고 클로로포름 / isoamyl 알콜 농도는 샘플을 추출합니다. </li><li> 2 μL 글리코겐 (25 μg / μL)와 3M 나 아세테이트 산도 5.2과 0.1의 소용돌이 볼륨을 추가합니다. </li><li> 100 % 에탄올과 와동 2.5 볼륨을 추가합니다. </li><li> ° C 하루 20 알을 품다. </li></ol> 4 부 : 정화 ChIP&#39;d 및 입력 DNA 계속해서 <ol><li> 4 13,000 rpm으로 20 분 최소 Microfuge 샘플 ° C DNA / 글리코겐 알약을 촉진합니다. </li><li> 70 % 에탄올 1 ML과 DNA / 글리코겐 알약과 함께 씻으십시오. </li><li> 에어 DNA 사반트 속도 진공 원심 분리기에 알약이나 신속하게 건조 건조. 팁 - 이상의 DNA를 건조하지 마십시오. </li><li> 200 μL TE 버퍼의 DNA / 글리코겐 알약을 Resuspend. </li><li> DNA (1 μL)은 실시간 PCR을 통해 부량 준비가되었습니다. 20 스토어 남아있는 DNA ° C. 팁 - DNA 이상의 1 μL를 사용하여이 실시간 PCR 반응을 억제 수 있으므로주의와 규모. </li></ol> 제 5 부 : 대리인 결과 : 칩 및 입력 DNA 샘플은 관심의 유전자 로커스 특정 primers를 사용하여 리얼 타임 PCR 7,8 (Biosystems 7500 빠른 리얼 타임 PCR 시스템을 적용)과 계량 수 있습니다. 알려진 대상 게놈 시퀀스의 강화 또는 assayed되는 히스톤 수정이 부족하는 것을 Primers는 긍정적이고 부정적인 통제뿐만 아니라 사용할 수 있습니다. 데이터 입력의 비율로 제공됩니다. 칩 절차는 전적으로해야 R세 epeated 생물 학적는 히스톤 수정에 보고된 통계적으로 의미있는 변화된다 보장하기 위해 복제합니다. 뇌관 디자인, PCR 효율, 그리고 입력과 정상화 절차의 %를 계산하는 적절한 방법을 포함하여 DNA를 수치 실시간 PCR를 사용할 때 고려해야 할 몇 가지 중요한 문제가 있습니다. 유전자에 걸쳐 히스톤 수정 인을 프로 파일링 때, PCR 효율은 모든 프라이머 쌍 간의 일치해야합니다. 실시간 PCR 시스템 제조 업체가 필요한 정보와 교육을 제공할 수 있습니다. 그림 1은 염색질의 대표적인 결과는 IFN - γ에 의해 트리거, IRF1 유전자 9 STAT1 활성화하는 동안 immunoprecipitated 보여줍니다. <img src="/files/ftp_upload/2053/2053fig1.jpg" alt="그림 1" /> 그림 1. STAT1, RNA 중합 효소 II와 H3K36me3의 인 IRF1 유전자의 STAT1 활성화하는 동안 동적입니다. (A, B, C) ​​α - H3K36me3, α - STAT1과 2FTGH 세포에서 수집한 염색질의 α - 폴 II와 칩 IFN - γ를위한 30 분 (빨간색 사각형), 1.5 시간 (파란색 원), 5 시간 (로 유도 녹색 삼각형) 또는 uninduced (블랙 다이아몬드). IgG는 대조군 (회색 십자가)입니다. (D) 팬 H3는 긍정적인 제어이며, 유도 (파란색 원)에 로커스 전체 히스톤 인과 uninduced 조건 (블랙 다이아몬드)를 보여줍니다. -5 BP 주변 딥는 전사 시작 사이트에서 발견 nucleosome 고갈로 인한 것입니다.

<ol>
	<li>Levy, D., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling:+Stats:+transcriptional+control+and+biological+impact.">Signalling: Stats: transcriptional control and biological impact.</a> Nature Reviews Molecular Cell Biology. 3, 651-662 (2002).</li><li>Bromberg, J., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+STATs+in+transcriptional+control+and+their+impact+on+cellular+function.">The role of STATs in transcriptional control and their impact on cellular function.</a> Oncogene. 19, 2468-2473 (2000).</li><li>Hu, X., Ivashkiv, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-regulation+of+signaling+pathways+by+interferon-gamma:+implications+for+immune+responses+and+autoimmune+diseases.">Cross-regulation of signaling pathways by interferon-gamma: implications for immune responses and autoimmune diseases.</a> Immunity. 31, 539-550 (2009).</li><li>Campos, E. I., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histones:+annotating+chromatin.">Histones: annotating chromatin.</a> Annu Rev Genet. 43, 559-599 (2009).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+Modifications+and+Their+Function.">Chromatin Modifications and Their Function.</a> Cell. 128, 693-705 (2007).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Wittwer, C. T., Herrmann, M. G., Moss, A. A., Rasmussen, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+fluorescence+monitoring+of+rapid+cycle+DNA+amplification.">Continuous fluorescence monitoring of rapid cycle DNA amplification.</a> Biotechniques. 22 (130-131), 134-138 (1997).</li><li>Higuchi, R., Dollinger, G., Walsh, P. S., Griffith, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+amplification+and+detection+of+specific+DNA+sequences.">Simultaneous amplification and detection of specific DNA sequences.</a> Biotechnology (N Y). 10, 413-417 (1992).</li><li>Kroger, A., Koster, M., Schroeder, K., Hauser, H., Mueller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activities+of+IRF-1.">Activities of IRF-1.</a> J Interferon Cytokine Res. 22, 5-14 (2002).</li><li>Ross, R. A., Spengler, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neuroblastoma+stem+cells.">Human neuroblastoma stem cells.</a> Seminars in cancer biology. 17, 241-247 (2007).</li></ol>

관심 없음 충돌 선언하지 않습니다.

명시된 칩 프로토콜은 분석보다보다 20 가지 히스톤 수정을 위해 실험실에서 성공적으로 사용되었습니다. 또한, 우리는 전사 요소의 숙박 변화를 특징으로 가지고 히스톤 - 수정 IFN - γ 여러 유발 유전자에서 히스톤 수정 및 RNA 효소 II의 전사 기계를 인식 효소, 단백질. 우리는 또한 암 줄기 세포 10 분화 동안 히스톤 수정의 변화를 특징으로 프로 시저를 사용합니다. ?...

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<tr>
<td>37% Formaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP531-500</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform/Isoamyl alcohol</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>AC32715-5000</td>
<td>&nbsp;</td>
<tr>
<td>Complete Mini Protease Inhibitors</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1836153</td>
<td>&nbsp;</td>
<tr>
<td>Cosmic Calf Serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30087.04N</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30243</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D0632</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP118-500</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Pharmco</td>
<td>zp1110EP</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>100149</td>
<td>&nbsp;</td>
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<td>Glycogen</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>901393</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3784</td>
<td>&nbsp;</td>
<tr>
<td>IFN-gamma</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>285-IF-100</td>
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<td>Jackson Immunoresearch</td>
<td> 109-005-003</td>
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<td>MP Biologicals</td>
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<td>L4408</td>
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<td>M8266</td>
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<td>Fisher</td>
<td>BP333-500</td>
<td>&nbsp;</td>
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<td>Deoxycholic Acid Sodium Salt</td>
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<td>Fisher</td>
<td>BP349-100</td>
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<td>NaCl</td>
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<td>Fisher</td>
<td>7647-14-5</td>
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<td>NP40</td>
<td>&nbsp;</td>
<td>US Biological</td>
<td>N3500</td>
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<tr>
<td>Anti-Histone H3, CT, pan</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>07-690</td>
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<td>Phenol/chloroform/isoamyl</td>
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<td>Fisher</td>
<td>108-95-2</td>
<td>&nbsp;</td>
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<td>Phosphate Buffered Saline</td>
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<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>50-230-0316</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500140</td>
<td>&nbsp;</td>
<tr>
<td>RNAse A</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500130</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein A Agarose</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-157</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein G Agaorse</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-201</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP166-500</td>
<td>&nbsp;</td>
<tr>
<td>Tris-Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5941</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>327372500</td>
<td>&nbsp;</td>
<tr>
<td>Anti-K36me3</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab9050</td>
<td>&nbsp;</td>
<tr>
<td>Anti-STAT1</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-345X</td>
<td>&nbsp;</td>
<tr>
<td>Anti-RNA Polymerase II</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-899</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells

세포외 리간드의 다양한 대응, STAT (전사의 신호 변환기 및 활성)이 전사 요소 빠르게들은 하나의 티로신 잔기 1 인산화에 의해 활성화됩니다 세포 표면 수용체에 세포질에서 숨겨진 상태에서 모집하고 있습니다. 그들은 다음 성장, 분화, 항상성 및 면역 반응에 영향을 미치는 대상 유전자의 전사를 드라이브에 핵을 dimerize 및 이동시키다. 되지 놀랍게도, 정상적인 세포 프로세스에서 광범위한 참여에 따라, STAT 기능의 dysregulation 특히 암 2 면역 질환 3, 인간의 질병에 기여하고 있습니다. 그것은 잘 녹음이 4,5 염색질 템플릿 변경에 의해 규제되어 설정됩니다. 이러한 변경은 ATP에 의존 단지뿐만 아니라 공유 결합 히스톤 수정 및 DNA의 methylation 6 활동을 포함합니다. 유전자 표현의 STAT 활성화가 신속하고 과도 모두이기 때문에, 그것은 STAT - 의존 유전자 loci modulating에서 염색질 템플릿에 대한 구체적인 메커니즘이 필요합니다. 이러한 메커니즘을 정의하려면, 우리는 히스톤 수정 및 STAT 신호에 응답 유전자 loci에서 그들을 생성하는 효소 활동을 특징. 이 프로토콜은 염색질 immunoprecipitation, STAT가 활성 유전자 발현의 염색질에 신호의 연구에 대한 가치있는 방법을 설명합니다.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells at JoVE.com

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

이 프로토콜은 염색질 immunoprecipitation (칩)가 신호 전달 경로에 의해 유도 전사 조절 염색질 템플릿 동적 변경을 연구하는 데 사용됩니다 방법을 설명합니다.

인간의 세포에서 활성 유전자 발현의 어세 동적 히스톤 수정을 염색질 Immunoprecipitation (칩)

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

chromatin-immunoprecipitation-chip-to-assay-dynamic-histone

Research

JoVE Journal

Biology

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

28.5K 조회수.  University of Virginia. 이 프로토콜은 염색질 immunoprecipitation (칩)가 신호 전달 경로에 의해 유도 전사 조절 염색질 템플릿 동적 변경을 연구하는 데 사용됩니다 방법을 설명합니다.

비디오: Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

인간 배아 줄기 세포에서 염색질의 Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

연구

생물학

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

로보틱스 및 식물 조직에서 유전자 발현의 연구 동적 이미지 분석

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

유전자 협회의 기능 기초를 테스트하는 Allele 특정 유전자 발현 분석

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenic 분석 : 자기편 셀

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

사용 염색질 Immunoprecipitation (CHIP) Drosophila 조직

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

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 ...