Name: silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells
Uploaded: 2015-08-12T14:00:00.000+00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

This work was supported by FIRB-Merit grants RBNE08YFN3_005 and RBNE08HWLZ_012, and the Italian Ministry of Economy and Finance to the CNR for the Project &#8220;FaReBio di Qualit&#224;&#8221;.

1. siRNA의 솔루션을 준비 <ol><li> (50 μm의 최종 siRNA의 농도)의 RNA 분해 효소가없는 물을 100 ㎕에 BRCA2 siRNA를 건조 펠렛의 5 스크램블 (CTRL)의 nmol의 5 nmol의를 재현 탁. 이는 siRNA를 스톡하고 10 ㎕의 분취 액으로 -20 ℃에 보관해야한다. </li><li> siRNA를 재고 10 μL의 나누어지는을 타고 10 μm의 siRNA를 작업 솔루션을 얻을 수의 RNase가없는 물을 40 μl를 추가합니다. 이 희석 사용할 때까지 -20 ℃에서 저장해야합니다. 참고 : siRNA의 작업 솔루션 / 냉동을 거쳐 3 번 이상 해동 안된다. </li></ol> 인간 상피 세포주 2. 시드 <ol><li> 37 ° C, 5 % CO 2의 가습 된 배양기에서 조직 배양 플레이트에서 세포 단층으로 성장하는 인간 상피 세포주에서 성장 배지를 제거한다. </li><li> 한 번 RT PBS로 접시에 세포 (100mm 판 PBS로 10 ml)에 씻으십시오. </li><li> 판에서 세포를 분리,0.25 % 트립신 2 ㎖ / 0.53 mM의 EDTA 용액을 추가하고 세포 유형에 따라 3-5 분 동안 배양한다. </li><li> 분리 된 세포를 수집하기 전에, 10 % 소 태아 혈청을 함유하는 완전 성장 배지 2 ㎖를 첨가하여 트립신을 중화. </li><li> 15 ML 튜브에 세포를 전송합니다. </li><li> 스윙 버킷 회 전자에 4 ℃에서 3 분 동안 320-330 XG에서 세포를 원심 분리기. </li><li> 항생제 배지 5ml에 세포 펠렛을 재현 탁. </li><li> BURKER 챔버 현미경 또는 제조업체의 지시에 따라 자동 카운팅 시스템을 사용하여 세포를 카운트. </li><li> 6- 웰 플레이트에 세포가 24 시간 (도 0.5-1 X10 각 배지 2 ㎖로 106 세포) 후 약 80 % 컨 플루 언트 될 시드. 최적의 세포 수는 특정 세포주 및 성장률에 따라 변할 수있다 시딩한다. 참고 : 항생제에 부정적인 transfe의 효율성을 방해하기 때문에이 시점에서 매체에 항생제를 추가하지 마십시오ction의. 그것은 BRCA2 siRNA를 형질의 고효율을 달성하기 위해 낮은 배양 통로 (&lt;10)에서의 셀은 매우 중요하다. </li></ol> siRNA를 3. 형질 세포 <ol><li> 24 시간 세포를 도금 한 후, 형질 전환의 첫 번째 라운드를 진행합니다. <ol><li> 감소 된 혈청 배지 130 μL 지질 계 10 siRNA를 형질 특정 시약 6 μL 희석. </li><li> 감소 된 혈청 배지 130 ㎕를 10 μM의 siRNA (30 pmol의) 3 μl를 희석. </li><li> 희석 된 형질 전환 시약 희석 된 siRNA를 결합하고 실온에서 5 ~ 10 분 동안 품어. </li><li> 배양하는 동안, 세포로부터 배지를 제거하고 새로운 배지 (항생제없이), 37 ℃에서 예열 된 2 ㎖를 추가한다. </li><li> 세포에 siRNA를 형질 감염 시약 착물 250 μL (이 양은 6- 웰 플레이트의 웰에 대해 하나의 경우)에 추가. </li><li> (37)에서 가습 된 배양기에서 세포를 부화76; 5 % CO 2 C. </li></ol></li><li> 24 시간 후, 단계 3.1.2에 대해 다음 수정과 3.1.1-3.1.5 단계를 반복하여 형질 전환의 두 번째 라운드를 진행 : 감소 혈청 배지 130 ㎕를 10 μM의 siRNA (50 pmol의)의 5 μl를 희석. <ol><li> 분석하기 전에 37 ℃에서 1~2 일 동안 세포를 품어. 참고 : 형질과 두 번째 라운드에서 높은 siRNA를 / 형질 시약 비율의 사용의 두 라운드 BRCA2 침묵의 높은 효율을 얻을 필수적이다. </li></ol></li></ol> 면역 분석 4.를 Lyse 세포 <ol><li> 비이 온성, 비 변성 세제 octylphenoxypolyethoxyethanol의 1 %, 5 mM의 피로 인산 나트륨, 1 mM의 나트륨 오르토 바나 데이트, 10 mM의 불소화 나트륨, 2 mM 페닐 메틸 술 포닐 플루오 리드, 10 μg의 함유 PBS (PH 7.4)로 신선한 용해 버퍼를 준비 / ㎖ 류 펩틴, 10 μg의 / ㎖ 아프로 티닌. 얼음에 보관하십시오. </li><li> 세포 및 W에서 매체를 제거접시에 차가운 PBS (2 ㎖ /도)로 한 번에 화산재. 어떤 PBS 이월을 방지하기 위해, 완전히 판에서 제거합니다. </li><li> 각 웰에 용해 버퍼 200 μl를 추가합니다. 주 : 세포가, 다른 분석을 위해 사용될 세포를 Trypsinize 2.1-2.6 단계에서 설명한 바와 같이 용도에 따라 적절한 버퍼 / 배지에서 세포를 재현 탁 될 수있는 경우에 사용된다. </li><li> 조심스럽게 균일하게 용해 버퍼를 배포하고 회 전자에 4 ° C에서 15 분 동안 배양 접시를 회전 할 수 있습니다. </li><li> 셀 스크레이퍼를 사용하여 얼음 마이크로 원심 튜브에 세포 용 해물을 수집. </li><li> 4 ° C에서 25 분 동안 17,900 XG에 원심 분리기와 새로운 튜브에 뜨는를 수집합니다. </li><li> 제조업체의 지시에 따라 시판중인 단백질 분석법을 이용하여 단백질 농도를 측정한다. </li></ol> 5. BRCA2의 면역 분석 <ol><li> 20 배 트리스 - 아세테이트 SDS 실행 B의 50 mL로 희석하여 런닝 버퍼를 준비uffer (50 mM의 트리 신, 50 mM 트리스 자료, 0.1 % SDS, pH가 8.24) 증류수 950 mL를. </li><li> 다음과 같은 샘플을 준비한다 (20)의 최종 부피 15 총 세포 용 해물의 μg의, 4X 샘플 완충액 5 ㎕의 pH를 8.4에서 리튬 도데 실 설페이트를 함유하는, 10 배 샘플 환원제 (0.5 M DTT) 2 μL 및 용해 완충액을 추가 μL. 참고 :이 프로토콜은 (5 μg의 아래로) 또한 총 단백질의 낮은 금액을 사용하여 정상 세포 라인에 BRCA2의 검출 할 수있다. </li><li> 10 분 동안 55 ° C에서 샘플 변성. 주 : 55 ° C를 사용하는 것이 중요하다. BRCA2 단백질이 열성 (11)이기 때문에, 더 높은 변성 온도는 그대로 전체 길이 (390 kDa의) BRCA2 단백질의 일관된 손실이 발생할. </li><li> 제조업체의 지시에 따라 전기 영동 챔버를 설정한다. </li><li> 샘플 및 precasted 젤에 고 분자량 사전 묻은 단백질 표준의 10 μl를 (트리스 - 아세테이트 3~8%)로드 및 AB 120 V에서 실행밖으로 2 시간 12. 55 kDa의 파란색 마커가 precasted 젤을 떠나기 전에 전기 실행을 중지합니다. </li><li> 20 배 전사 완충액 50 ㎖, 100 % 메탄올 100㎖ 및 850 ml의 물을 전송 버퍼를 준비한다. </li><li> 5 분 동안 100 % 메탄올에 침지하여 PVDF 막 (0.45 ㎛의 공극 크기)을 활성화하고 증류수로 헹구고, 적어도 5 분 동안 전송 버퍼로 평형화. 사용까지 4 ° C에서 전송 버퍼의 전송 장치의 스폰지를 적시 게. </li><li> 전기 영동 실행이 종료되면, 다음 순서 (상향식)에 전송 스택에 샌드위치 조립 : 전송 버퍼 포화 세 스폰지 번 3MM 급 용지는 전사 완충액, 겔, 활성 PVDF 멤브레인 번 3MM 포화 전송 버퍼에서 포화 고급 종이 시트는, 세 스폰지는 전송 버퍼 (13, 14)에서 포화. 4 ℃에서 하룻밤 mA의 4 ° C에서 180에서 4 시간 동안 350mA의 장치 및 전송 단백질에 스택을 놓습니다. 주 : 고 분자량 단백질의 완전한 전송을 보장하기 위해 4 시간 또는 밤새 행 (제조 및 가장 전송 프로토콜에 의해 제안 된 바와 같이) 존재 BRCA2 매우 큰 단백질,이를 1 시간에서 전송 시간을 연장하는 것이 중요하다. 또한, 인해 긴 전송 시간에, 그 과열을 최소화하고 샌드위치 분해를 촉진하기 위해 4 ℃에서 냉장실에 블 롯팅을 수행하는 것이 필수적이다. 세미 드라이 전송 시스템은 큰 단백질을 전송하기위한 좋은하지 않습니다. </li><li> 전송 한 후 (30 μL 방지 BRCA2 토끼 폴리 클로 날 항체로 4 ℃에서 하룻밤 5 % 탈지 분유와 프로브를 포함하는 TBS-트윈 0.25 %에서 실온에서 1 시간 (TBS-T)를 위해 그것을 차단, TBS로 막을 세척 [: 300 (v / v)로 희석 한] 9 mL를 TBS-T + 5 % 탈지 분유로 희석 μg의 200 / ㎖). </li><li> 다음 TBS 10ml에 희석 한 서양 고추 냉이 퍼 옥시 다제 - 접합 된 항 - 토끼 이차 항체에 1 μL로 1 시간 동안 필터를 부화, TBS-T로 세 번 막 (각 10 분)을 씻어-T + 5 % 탈지 분유. 그 후, TBS-T로 막 세 번 (10 분마다)을 씻는다. </li><li> 강화 된 화학 발광 (ECL) 웨스턴 블로 팅 검출 시약을 사용하여 제조업체의 지침에 따라 BRCA2 단백질을 공개 ECL에 의해 면역을 수행합니다. 높은 해상도 ECL 필름에 immunochemiluminescent 밴드를 시각화. </li><li> 10 항 튜 불린 항체의 1,000 (10 μL : β-tubulin에하는 단일 클론 항체를 이용하여 1 희석을하여 부하 제어와 같은 필터에, β - 튜 불린 (55 kDa의)처럼, 하우스 키핑 유전자에 대한 항체와 면역을 수행 실온에서 1 시간 동안 ml의 TBS-T + 5 % 무 지방 분유). 고추 냉이 퍼 옥시 다제 - 복합 항 마우스 대신 항 - 토끼 차 항체의 사용을 제외하고, 세척 및 BRCA2 (5.10-5.11)에 기재 한 바와 같이 차 항체를 품어. </li><li> (immunochemiluminescent 밴드 감동 필름의 디지털 이미지를 획득 전용 이미지 분석 소프트웨어에 의한 밴드 강도를 분석 예., ImageJ에). </li></ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+killing+of+BRCA2-deficient+tumours+with+inhibitors+of+poly(ADP-ribose)+polymerase.">Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.</a> Nature. 434, 913-917 (2005).</li><li>Ihnen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+the+poly(ADP-ribose)+polymerase+inhibitor+rucaparib+for+the+treatment+of+sporadic+human+ovarian+cancer.">Therapeutic potential of the poly(ADP-ribose) polymerase inhibitor rucaparib for the treatment of sporadic human ovarian cancer.</a> Mol Cancer Ther. 12, 1002-1015 (2013).</li><li>Jeong, J. H., Jo, A., Park, P., Lee, H., Lee, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brca2+Deficiency+Leads+to+T+Cell+Loss+and+Immune+Dysfunction.">Brca2 Deficiency Leads to T Cell Loss and Immune Dysfunction.</a> Mol Cells. , (2015).</li></ol>

The authors have nothing to disclose.

암수 유방암, 난소 암, 전립선 암, 췌장암 및 흑색 종 3,4- 포함한 여러 암 종류의 위험 증가 BRCA2 유전자 납 생식계 돌연변이는, 다수의 연구는 생물학적 기능을 이해하고 수행 되었기 때문에 BRCA2 단백질의. 이러한 연구의 대부분은 인해 BRCA2 같은 거대한 단백질을 분석하는 기술적 인 문제에 유전 적 기반 주로. BRCA2 단백질 발현을 침묵하고 분석하기위한 여기에 설명 된 방법은 생물?...

BRCA2 (유방암 감수성 유전자-2)의 유전자 재조합 효소 RAD51 (1)의 기능을 조절하여 DNA 이중 가닥 나누기를 복구하는 데 중요한 역할을하는 종양 억제 단백질을 암호화한다. BRCA2는 또한 전사의 조절 및 세포주기 조절에 관련이있다. BRCA2 유전자의 생식 세포 돌연변이는 다른 암 유형 3,4에 상 염색체 우성 남성의 유방과 여성에서 난소 암과 전립선 암에 대한 감수성뿐만 아니라 경향을 유도한다. 그러나, 억제 나 DNA 손상의 원인 5-7 화학 치료제로 암세포 더욱 취약 할 수있다 BRCA2 렌더링 기능을 줄이고 암, BRCA2 돌연변이 현상의 위험 증가에도 불구. 산발적 암은 BRCA2 단백질을 제안, BRCA2 단백질의 감소 수준은 일부 암 종류에서 발견 된하지만 BRCA2 돌연변이 (&lt;3 %)의 낮은 비율을 나타내비 돌연변이에 의존하는 메커니즘 7,8 통해 산발적 암에서 종양 발생시 손실 될 수 있습니다. 따라서, 완전히 암 생물학의 맥락에서뿐만 아니라 다른 생물학적 설정에서 BRCA2 기능을 이해하는 것이 중요하다. 포유류 세포에서 유전자의 신규 한 기능을 식별하는 강력한 도구는 그 발현을 침묵하는 것이다. 일례로서, 정상 상피 세포의 다양한 사일런 BRCA2 발현은 최근 anoikis 저항성 암세포 침습 및 전이 능력 (9)의 획득 동안 중요한 단계의 레귤레이터 등 BRCA2의 신규 기능의 확인하게되었다. 유전자 침묵은 작은 셀 간섭 (SI) RNA 또는 특정 유전자를 대상으로 짧은 헤어핀 (SH) RNA 분자 중 하나에 도입함으로써 달성 할 수있다. 고의 siRNA의 효능과 사용의 용이성이 중요한 생물학적 과정에 새로운 통찰력을 확보하기위한 실험을 입을 것이에게 특혜 도구를 만들ND 새로운 치료 목표를 식별합니다. 그러나, 유전자 발현을 효율적으로 녹다운 모든 유전자를 쉽게 달성되지 않을 수 있고, 세포의 종류에 따라 매우 가변적 일 수있다. 세포 내 단백질 수준에서의 감소는 조사중인 가장 관련된 표현형이기 때문에, 유전자 생성물의 분석에 의해 면역 블 유전자 침묵을 정량화하는 것이 중요하다. 이와 관련하여, BRCA2 단백질은 더 도전을 제공합니다 : 큰 단백질 (약 390 kDa의를)되고, 기술적 인 문제가 면역을 포함하여 기존의 생화학 적 분석을 위해 존재한다. 우리는 여기서 인간 상피 세포 라인에 BRCA2의 효율적인 침묵과 분석을 면역 블에 의해 넉다운 BRCA2 단백질의 신속하고 성공적으로 감지에 최적화 된 프로토콜을보고합니다.

<table><tbody><tr><td>BRCA2 siRNA</td><td>Dharmacon</td><td>L-003462-00</td><td>SMARTpool: ON-TARGETplus BRCA2 siRNA</td></tr><tr><td>Ctrl siRNA</td><td>Dharmacon</td><td>&amp;nbsp;D-001810-10-05</td><td>ON-TARGETplus Non-targeting Pool</td></tr><tr><td>Lipofectamine RNAiMAX Reagent&amp;nbsp;</td><td>Life Technologies</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/13778075&quot;&gt;13778075&lt;/a&gt;</td><td>Transfection Reagent</td></tr><tr><td>OPTI-MEM I Reduced Serum Medium</td><td>Life Technologies</td><td>31985-070</td><td>Medium for transfection procedure</td></tr><tr><td>NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast</td><td>Life Technologies</td><td>EA0378</td><td>Gel for protein electrophoresis</td></tr><tr><td>NuPAGE LDS Sample buffer&amp;nbsp;</td><td>Life Technologies</td><td>NP0007</td><td>Reagent for protein electrophoresis</td></tr><tr><td>NuPAGE Sample Reducing agent (10x)</td><td>Life Technologies</td><td>NP0004</td><td>Reagent for protein electrophoresis</td></tr><tr><td>NuPAGE Antioxidant</td><td>Life Technologies</td><td>NP0005</td><td>Reagent for protein electrophoresis</td></tr><tr><td>HiMark Pre-stained protein standard</td><td>Life Technologies</td><td>LC5699</td><td>Prestained marker for gel electrophoresis</td></tr><tr><td>XCell SureLoc Mini-Cell&amp;nbsp; System</td><td>Life Technologies</td><td>EI0002</td><td>Equipment for protein electrophoresis/transfer</td></tr><tr><td>NuPAGE Tris-Acetate SDS Running Buffer</td><td>Life Technologies</td><td>LA0041</td><td>Reagent for Western blotting</td></tr><tr><td>PVDF membrane</td><td>Millipore</td><td>IPVH00010</td><td>Reagent for Western blotting</td></tr><tr><td>NuPAGE Transfer Buffer</td><td>Life Technologies</td><td>NP0006-1</td><td>Reagent for Western blotting</td></tr><tr><td>BRCA2 H-300 rabbit polyclonal antibody</td><td>Santa Cruz Biotechnology</td><td>sc-8326</td><td>1:300 dilution</td></tr><tr><td>Beta-tubulin monoclonal antibody</td><td>Sigma Aldrich</td><td>T4026-100UL</td><td>1:1,000 dilution</td></tr><tr><td>Skim Milk powder</td><td>Sigma Aldrich</td><td>70166</td><td>Reagent for Western blotting</td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>P1379</td><td>Reagent for Western blotting</td></tr><tr><td>SuperSignal West Pico/Femto Chemiluminescent Substrate</td><td>Pierce Thermo Scientific</td><td>34080/34096</td><td>Chemiluminescence system</td></tr><tr><td>PNT1A cells</td><td>SIGMA Aldrich (for ECACC)</td><td>95012614</td><td>PNT1A human, normal prostate epithelium immortalized with SV40</td></tr><tr><td>Nthy cells</td><td>SIGMA Aldrich (for ECACC)</td><td>90011609</td><td>Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells</td></tr><tr><td>Cell Proliferation Kit I (MTT)</td><td>Roche</td><td>11465007001</td><td>Kit for cell proliferation assays</td></tr></tbody></table>

silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells

종양 억제 단백질 BRCA2와 기존의 생화학 적 분석에 의해 그 검출 침묵은 인간의 BRCA2 단백질 (약 390 kDa의)의 큰 크기 때문에 큰 기술적 도전을 나타냅니다. 우리는 인간의 상피 세포 라인에 각각 내생 BRCA2 단백질을, 인간의 BRCA2를 침묵과 감지 표준 siRNA를 형질 감염 및 면역 프로토콜의 수정 사항을보고한다. 주요 단계는 형질 전환 시약 비율이 높은 siRNA와 같은 실험 내에서 siRNA를 형질의 두 후속 라운드를 포함한다. 안티 BRCA2 항체와 분석을 면역 블 판단으로 우리가 지속적으로 인간 BRCA2 유전자의 70 % 이상 침묵을 달성 표준 프로토콜이 다른 수정을 사용. 또한, 대신에, 종래의 70 ~ 100 °의 C 및 면역 과정의 다른 기술적 최적화 55 ° C에서의 세포 용 해물의 변성은 그대로 BRCA2 단백질의 검출도 가능 WH출발 물질은 매우 낮은 양의 도중에 가능 또는 BRCA2 단백질 발현 수준이 매우 낮은 경우. 인간의 세포에서 BRCA2의 효율적인 침묵은 종양 세포를 포함 그대로 BRCA2와 세포에서 BRCA2 기능을 방해 할 수있는 새로운 분자 경로 및 종양에서 병원성 BRCA2 돌연변이에 의해 영향을받을 수 세포 기능을 조사하기 위해 가치있는 전략을 제공합니다. 효율적인 침묵과 BRCA2와 같은 다른 &#39;큰&#39;단백질의 분석을 위해이 프로토콜의 적응은 쉽게 달성 할 수 있어야합니다.

세포 기능에 BRCA2 사일런의 효과를 조사하기 위해 생물학적 / 생화학 적 분석을 진행하기 전에 첫 번째 단계는 스크램블링 된 siRNA (siRNA의 비는 타겟팅) BRCA2의 siRNA와 나란히 (도 1a를 이용하여 소음의 특이성을 확인하는 것이다 ). 그것은 BRCA2 사일런의 높은 효율을 얻기 위해 높은 siRNA의 / 형질 비율의 siRNA 형질 감염의 두 번째 라운드를 수행하는 것이 중요하다. 실제로, si...

Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

의 침묵<em&gt; BRCA2</em&gt; 배양 된 인간 세포에 소설 BRCA2 규제 생물학적 기능을 확인하는

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

silencing-brca2-to-identify-novel-brca2-regulated-biological

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

9.0K 조회수.  National Research Council. Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. 

비디오: Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

기능 유전체학에게 검사를 활용하여 암 세포 회전 타원체 문화에 잠재적으로 소설 약 표적을 식별하는

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

연구

암 연구학

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

CRISPR 중재 베이스 편집기사용 BRCA1 변종 기능 평가

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

세포 및 조직 샘플에서 인간 유방암 줄기 세포의 분리 및 기능 평가

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

유방암 세포에서 에스트로겐 조절 마이크로 RNA의 프로파일

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

의학

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

암 약물 과민 반응의 효과를 평가<em&gt; 체외</em&gt; 및<em&gt; 생체</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

내생 와일드 타입 BRCA1을 표현 세포를 사용하는 동종 재조합에 BRCA1 변이의 효과를 확인

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

생물학

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

In Vitro (시험관 ) 표적 후성유전학적 침묵을 위한 엔지니어링된 전사 억제제 선택

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

생체공학

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비​​퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

의 전체 순서의 NGS 분석을위한 Transposase 기반 기술에 의해 gDNA를 농축<em&gt; BRCA1</em&gt;<em&gt; BRCA2</emDNA 손상 복구에 참여&gt;, 9 유전자

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

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기능 유전체학에게 검사를 활용하여 암 세포 회전 타원체 문화에 잠재적으로 소설 약 표적을 식별하는

CRISPR 중재 베이스 편집기사용 BRCA1 변종 기능 평가

세포 및 조직 샘플에서 인간 유방암 줄기 세포의 분리 및 기능 평가

유방암 세포에서 에스트로겐 조절 마이크로 RNA의 프로파일

암 약물 과민 반응의 효과를 평가

내생 와일드 타입 BRCA1을 표현 세포를 사용하는 동종 재조합에 BRCA1 변이의 효과를 확인

In Vitro (시험관 ) 표적 후성유전학적 침묵을 위한 엔지니어링된 전사 억제제 선택

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

의 전체 순서의 NGS 분석을위한 Transposase 기반 기술에 의해 gDNA를 농축

면역형광에 의한 DNA 수리 단백질 상호작용의 시각화

의 침묵

요약

더 많은 비디오 탐색

이 비디오의 챕터

기능 유전체학에게 검사를 활용하여 암 세포 회전 타원체 문화에 잠재적으로 소설 약 표적을 식별하는

CRISPR 중재 베이스 편집기사용 BRCA1 변종 기능 평가

세포 및 조직 샘플에서 인간 유방암 줄기 세포의 분리 및 기능 평가

유방암 세포에서 에스트로겐 조절 마이크로 RNA의 프로파일

암 약물 과민 반응의 효과를 평가

내생 와일드 타입 BRCA1을 표현 세포를 사용하는 동종 재조합에 BRCA1 변이의 효과를 확인

In Vitro (시험관 ) 표적 후성유전학적 침묵을 위한 엔지니어링된 전사 억제제 선택

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비​​퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

의 전체 순서의 NGS 분석을위한 Transposase 기반 기술에 의해 gDNA를 농축

면역형광에 의한 DNA 수리 단백질 상호작용의 시각화

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영