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>

<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 border="0" cellpadding="0" cellspacing="0">
	<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>&#160;D-001810-10-05</td>
			<td>ON-TARGETplus Non-targeting Pool</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Reagent&#160;</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/13778075">13778075</a></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&#160;</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&#160; 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>1:1000 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

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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

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

기본 교양 Hippocampal 신경에 덴스 코어 Vesicles의 라이브 영상

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

연구

생물학

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

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

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

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

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 ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

유전자 발현의 DNA 상호 작용, 디스 전사 레귤레이터, 그리고 소설 안티 종양 에이전트를 식별 : 단백질을 연구하는 정량 분석

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

배양 된 세포의 소포체 하위 도메인의 시각화

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

캡처 복합 질량 분석 - 소설의 C 디 GMP 이펙터 단백질을 식별 할 수있는 강력한 도구

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebrafish의에서 콩팥 세포 독소 미세 주입은 급성 신장 손상을 모델링하기

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

포도당 섭취량 측정 및 인슐린 자극에 대한 반응<em&gt; In Vitro</em&gt; 배양 된 사람의 주요 근육 세포

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...