우리는이 작품에 대한 자금 제공을위한 오하이오 주립 대학 종합 암 센터에 감사하고 있습니다.

1. 통합 재조합 기판과 세포주 <ol><li> 헬라 세포는 안정 PDR - GFP (M. Jasin, 메모 리얼 슬로언 케터링 암 센터에서 플라스미드 7 선물)로 변환하고 transformants 선정을위한 puromycin 유지했다. </li><li> 세포 10% 태아 소 혈청, 2 MM GlutaMAX, 1 MM 나트륨 pyruvate, 페니실린 / 스트렙토 마이신 (100 U / mL/0.1 MG / ML), 그리고 1.5 UG / ML puromycin 포함된 DMEM / FBS에 passaged 있습니다. </li></ol> 플라스미드, 그리고 안정적​​인 transformants에 PDR - GFP의 재조합 기판은 GFP의 두 대립 유전자 비활성 (그림 1)가 포함되어 있습니다. 한 allele 인해 I - SceI에 대한 18 BP 제한 효소 인식 사이트의 코딩 순서에 포함하는 운영 중지 상태입니다. 이러한 세포에서 I - SceI 표현을 두 번 좌초 DNA의 휴식 (DSB)를 만듭니다. 가이 DNA를 비활성 GFP의 두 번째 allele는하지만, 첫 번째 allele에있는 I - SceI 사이트와 상관의 순서의 부분은 야생 타입입니다. 이 두 번째 GFP의 allele은 DSB의 동종 재조합의 수리를 위해 순서 기증자와 같은 기능을 수 있으며, 쉽게 유동세포계측법에 의해 감지되는 활성 GFP의 allele에 나타날 수 있습니다. 또는, DSB가 nonhomologous I - SceI 제한 사이트를 파괴 될 것이며 GFP - 부정적인 세포가 발생하는 것입니다, 최종 입사하여 수리 수 있습니다. 세포 동종 재조합의 수준은 GFP 양성 세포의 백분율을 계산에 의해 결정됩니다. 이 세포주, pHeLa - DR13 - 9는 제로 배경 GFP 신호와 플라스미드, pCBASce을 표현 I - SceI로 transfection시 GFP 양성 세포의 상대적으로 높은 비율의 모든 클론 중 선정되었습니다. (pCBASce 및 벡터 제어, pCAGGS는 메모 리얼 슬로언 케터링 암 센터의 M. Jasin에 의해 공급되었다 모두.) 헬라 - DR13 - 9 세포주 요청시 저자에서 사용할 수 있습니다. 2. Transfection은 내생 BRCA1을 고갈 및 뒤로 돌연변이 BRCA1를 추가합니다. <ol><li> 첫 번째 transfection (보통 화요일) 전에 하루 : 헬라 - DR13 - 9 세포 최소 90 %의 합류 10cm 요리와 함께 시작합니다. </li><li> 1 ML에 Trypsinize 9 ML DMEM / FBS를 추가하여 반응을 정지, pipetting으로 잘 섞는다. </li><li> trypsinized 세포의 40 μL 당 우물의 DME / FBS 0.5 ML에 24 잘 접시를 추가합니다. </li><li> 1 일 transfections 일 (수) : 훨씬 적은가 다시 시작하면 세포가 ~ 40~50%의 합류해야합니다. 혼합하여 Transfect : 5 pmol의 siRNA + 12.5 μL Opti - 멤에서 플라스미드 DNA을 표현 0.3 UG BRCA1 - 돌연변이 및 12.5 μL Opti - 멤 0.5 μL Lipofectamine 2000. Lipofectamine 프로토콜 (Invitrogen)에 따라 transfection과 함께 진행합니다. </li><li> 나중에 미디어 4~6시간를 교체하십시오. 우리가 BRCA1을 고갈하는 데 사용하는 siRNA는 80,798 (가입 번호 AY273801)로 BRCA1 3&#39; - UTR 시퀀스 80,780을 지정 순서 GCUCCUCUCACUCUUCAGU에 따라 달라집니다. </li><li> 일 2 : 6 잘 접시에 24 잘 접시에서 trypsinizing하여 세포를 전송합니다. </li><li> 3 일 transfections : 25 pmol의 siRNA + 플라스미드 DNA을 표현 0.75 UG BRCA1 돌연변 + - 62.5 μL Opti - 멤와 62.5 μL Opti - 멤 2.5 μL Lipofectamine 2000 년 0.75 UG의 pCBASce (또는 pCAGGS 벡터 제어). 나중에 미디어 4~6시간를 교체하십시오. </li></ol> 3. Transfectants 분석. <ol><li> 일 6 각 우물에서 세포를 trypsinize 1 ML DMEM / FBS에서 중지합니다. GFP의 표현에 대한 세포를 분석하는 것은 일 5 7 일을하지만 우리는 6 작품이 최고의 그날을 찾을 수 있습니다. </li><li> 유동세포계측법에 의해 잘 10,000 세포를 분석할 수 있습니다. 우리는 오하이오 주립 대학 종합 암 센터의 분석 Cytometry 공유 자원에 백톤 디킨슨 FACSCalibur기구를 사용합니다. 싱글, 라이브 세포는 앞으로 측면 산란 매개 변수를 사용하여 문이 있습니다. GFP 양성 세포가 GFP 형광 검출기 (FL1)를 사용 감지하며, 결과는 히스토그램 (그림 2 하단)로 제공됩니다. </li><li> 원하는 경우 왼쪽을 통해 세포는 형광 현미경으로 사진에 대한 replated 수 있습니다. </li></ol> 4. 대표 결과 : BRCA1은 상동 재조합 6,11에 의해 두 번 좌초 DNA 나누기의 복구에 대한 경로를 규정합니다. 상동 재조합에 의해 수리를받은 이들 세포는 쉽게 형광 현미경 (그림 2 위)에 의해 감지됩니다. 검색된 GFP 양성 세포 (그림 2, 오른쪽)의 감소에서 BRCA1 결과의 고갈. FACSCalibur의 출력은 X 축 및 Y 축 (그림 2 하단)에 세포의 수를 셀 당 GFP 강도와 히스토그램이다. 한 실험에서 결과의 전형적인 설정에서 세포의 15.5 %가 I - SceI 표현 및 제어 RNAi 고갈 (그림 3) 후 GFP 양성했다. 비교함으로써, BRCA1과 벡터의 고갈 다시 0.7 %로 GFP - 전환을 감소 추가할 수 있습니다. 추가 다시 야생 형 BRCA1이나 BRCA1 (I21V) 완벽하게 복원된 동종 재조합의로 15~16% GFP 양성 세포를 얻어 발견했습니다. 추가 다시 BRCA1 (M1의8T) 돌연변이 (그림 3, 레인 5) 벡터 컨트롤의 추가 기능 다시와 유사한 효과를했습니다. 이 변종은 내생 단백질 구와 유사한 수준의 표현부터, 우리는 따라서 BRCA1 (M18T) 변종은 상동 재조합에 비 작동 해석한다. 우리는 일반적으로 I - SceI의 표현이 플라스미드 transfecting 후 GFP 양성으로 전환할 세포의 10~20% 있습니다. BRCA1의 고갈은 reproducibly 80-10 대해 접이식하여 GFP 양성 세포의 전환을 줄일 수 있습니다. GFP 양성 세포의 실제 비율은 실험 내에서 제어 고갈에 비해 BRCA1의 고갈 다음과 같은 실험에서 실험, GFP 양성 세포의 비율을 변경할 수 있지만 것은 상당히 일관성이있다. 우리는 100 %와 백분율로 BRCA1의 depletions과 추가 떠밀렸고 표현 결과에 노골적인 전환율을 설정하여 결과를 정상화. 이러한 방법으로, 우리는 여러 실험을 결합 수 있으며, 실험 유사 높지는 않습니다. 지금까지 강력한 결과를 낮은 다시 BRCA1의 돌연변이의 추가 : 각 BRCA1의 변형도 완전 상동 재조합 활동을 복원되었거나 전혀 아무런 영향을 미치지 않았다 있습니다. 이러한 결과를 얻기에 중요한 고려 사항은 우리 transfection 조건이 약 치료 세포의 내생적인 단백질의 농도를 동일 BRCA1 단백질의 수준을 생산한다는 것입니다. BRCA1의도 결함 돌연변이의 overexpression의 너무 높은 몇 가지 긍정적인 상동 재조합 활동에 발생할 수 있습니다. 우리가 극복한다고 문제가 transfection의 독성했다. BRCA1 자체의 고갈은 세포 성장을 늦출 수 있으며, 접시에 세포의 숫자에 비해 Lipofectamine - 2000 및 플라스미드 DNA의 균형은 지속적인 수준에서 실시되어야합니다. 너무 Lipofectamine 또는 플라스미드 DNA는 세포 숫자를 줄일 것이며, 세포의 숫자는 신뢰할 수있는 유동세포계측법 결과를 얻을 부족합니다. transfected 세포의 수를 비교 플라스미드와 Lipofectamine 시약의 균형은 세포의 생존을 유지하기 위해 매우 중요합니다. <img src="/files/ftp_upload/2468/2468fig1.jpg" alt="그림 1" /> 그림 1. 재조합 기판. M. Jasin 7,12의 그룹에 의해 개발되었습니다 전략 그려져 있습니다. 플라스미드 PDR - GFP는 I - SceI 제한 endonuclease 사이트를 포함 하나있는 GFP의 두 대립 유전자 결함을 포함하고 있습니다. 헬라 파생된 세포 선이의 DNA 게놈 9 한 사이트에서 통합 기판, 그리고 GFP 양성 한 allele의 전환이 동종 재조합의 결과에 의해 I - SceI 사이트에서 이중 좌초된 DNA 브레이크의 적극적인 복구가 포함되어 있습니다. <img src="/files/ftp_upload/2468/2468fig2.jpg" alt="그림 2" /> 그림 2. 형광 현미경에 의해 GFP 양성 세포 감지. 세포는이 절차에 따라 테스트 및 제어 - 고갈 전지는 GFP 표현 (왼쪽)과 함께 세포의 높은 비율로 검출, 상동 재조합에 적극적으로 하였다. GFP 양성 세포 (오른쪽)의 개수에 날카로운 감소 RNAi 결과로 BRCA1의 고갈. <img src="/files/ftp_upload/2468/2468fig3.jpg" alt="그림 3" /> 그림 3. BRCA1의 missense의 돌연변이의 추가 다시 결과. 한 실험에서 결과는 본 막대 그래프에 표시됩니다. 플라스미드을 표현 transfected I - SceI의 부재 (레인 1)에서 감지하지 GFP 양성 세포가 없습니다. 레인스 2-6의 결과는 모든 플라스미드 표현 I - SceI이 일 3 transfected어진 세포에서 찍은. 관련성이없는 유전자 (루시페라제)의 고갈 및 벡터 추가 다시와 세포의 15.5 %가 (레인 2) GFP 양성에 있었 세포. BRCA1 및 벡터의 고갈 추가 - 다시 상동 재조합에 BRCA1의 손실의 효과 (레인 3) 보여준다. BRCA1의 고갈과 추가 다시 야생 타입 BRCA1 (레인 4), BRCA1 (M18T) (레인 5) 및 BRCA1 (I21V) (레인 6)의 효과가 표시됩니다. 이러한 결과는 단일 실험에서, 그리고 특정 돌연변이 BRCA1 단백질 지원하는 동종 재조합의 측정을 얻기 위해서는 적어도 두 번 더 반복 실험과 결합됩니다.

<ol>
	<li>Carvalho, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+cancer+risk+associated+with+germ+line+BRCA1+missense+variants+by+functional+analysis.">Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.</a> Cancer Res. 67, 1494-1501 (2007).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+epidemiology+of+BRCA1.">Genetic epidemiology of BRCA1.</a> Cancer Biol Ther. 3, 509-514 (2004).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+functional+effects+of+unclassified+genetic+variants.">Assessment of functional effects of unclassified genetic variants.</a> Hum Mutat. 29, 1314-1326 (2008).</li><li>Tomlinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+breast+cancer+cell+line+derived+from+a+germ-line+BRCA1+mutation+carrier.">Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.</a> Cancer Res. 58, 3237-3242 (1998).</li><li>Ruffner, H., Joazeiro, C. A., Hemmati, D., Hunter, T., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-predisposing+mutations+within+the+RING+domain+of+BRCA1:+loss+of+ubiquitin+protein+ligase+activity+and+protection+from+radiation+hypersensitivity.">Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.</a> Proc Natl Acad Sci U S A. 98, 5134-5139 (2001).</li><li>Moynahan, M. E., Chiu, J. W., Koller, B. H., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brca1+controls+homology-directed+DNA+repair.">Brca1 controls homology-directed DNA repair.</a> Mol Cell. 4, 511-518 (1999).</li><li>Nakanishi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Fanconi+anemia+monoubiquitination+pathway+promotes+homologous+DNA+repair.">Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.</a> Proc Natl Acad Sci U S A. 102, 1110-1115 (2005).</li><li>Pierce, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+breaks+and+tumorigenesis.">Double-strand breaks and tumorigenesis.</a> Trends Cell Biol. 11, 52-59 (2001).</li><li>Ransburgh, D. J., Chiba, N., Ishioka, C., Toland, A. E., Parvin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+breast+tumor+mutations+in+BRCA1+that+abolish+its+function+in+homologous+DNA+recombination.">Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.</a> Cancer Res. 70, 988-995 (2010).</li><li>Easton, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+genetic+assessment+of+1,433+sequence+variants+of+unknown+clinical+significance+in+the+BRCA1+and+BRCA2+breast+cancer-predisposition+genes.">A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes.</a> Am J Hum Genet. 81, 873-883 (2007).</li><li>Snouwaert, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+deficient+embryonic+stem+cells+display+a+decreased+homologous+recombination+frequency+and+an+increased+frequency+of+non-homologous+recombination+that+is+corrected+by+expression+of+a+brca1+transgene.">BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.</a> Oncogene. 18, 7900-7907 (1999).</li><li>Pierce, A. J., Hu, P., Han, M., Ellis, N., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ku+DNA+end-binding+protein+modulates+homologous+repair+of+double-strand+breaks+in+mammalian+cells.">Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.</a> Genes Dev. 15, 3237-3242 (2001).</li><li>Spearman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinically+applicable+models+to+characterize+BRCA1+and+BRCA2+variants+of+uncertain+significance.">Clinically applicable models to characterize BRCA1 and BRCA2 variants of uncertain significance.</a> J Clin Oncol. 26, 5393-5400 (2008).</li><li>Sweet, K., Senter, L., Pilarski, R., Wei, L., Toland, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+BRCA1+ring+finger+variants+of+uncertain+significance.">Characterization of BRCA1 ring finger variants of uncertain significance.</a> Breast Cancer Res Treat. 119, 737-743 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 방법의 장점은 우리가 야생 형 endogenously 표현 BRCA1을 가지고 세포를 사용 상동 재조합에 의해 DNA의 복구를 규제에서 BRCA1 단백질의 기능 연구를 수있다는 것입니다. 우리는 상동 재조합 과정을 공부하고 DNA 수리의 기능을 위해 BRCA1 변종 테스트를위한 소설 방법을 얻기 위해 RNAi - 중재 고갈을 이용한 두 가지 별도의 설립 방식을 채택했습니다. 이 방법의 성공을위한 열쇠는 우리가 GFP 변환의 매우 높은 수준을 얻는 것과 같은 게놈의 재조합 기판과 같은 헬라로 쉽게 transfectable 세포주를 확립하는 것이었다. 우리는 전혀 감지 배경 GFP 신호도 없었을 얻기 위해서는 원래의 변화의 여러 클론을 상영지만, 플라스미드 I - SceI 표현의 transfection에 따라 GFP 변환의 매우 높은 수준을했다했다. 이 방법을 통해, 우리는 이제 동종 재조합의 기능에 대한 다른 구체적인 BRCA1 아미노산 잔류물을 조사하고 있습니다. 이 작품의 생물 학적 통찰력과 함께, 이러한 결론은 임상 암 유전자에 적용할 수 있습니다. 많은 희귀한 변종이 있기 때문에 BRCA1 missense 변이의 높은 비율을 알 수없는 임상 중요성을 가지고 유전자 분리 분석 3,10,13,14하여 암 협회를 결정하기 위해 이들 중 많은 부족한 정보가있다. 이 방법을 사용하여 상동 재조합을 조절에서 BRCA1의 생물 학적 기능의 특정 변형의 결과는 자신의 게놈에서 이러한 변종 중 하나를 수행 여자를 알려하는 데 사용할 수 있습니다.

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11960</td>
<td>&nbsp;</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>9871-5200</td>
<td>&nbsp;</td>
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<td>Invitrogen</td>
<td>N/A</td>
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<td>Invitrogen</td>
<td>11668</td>
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<td>Invitrogen</td>
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<td>Enzo Life Sciences</td>
<td>BML-GR312</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
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<td>Sodium Pyruvate</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11360</td>
<td>&nbsp;</td>
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<td>GlutaMAX I</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>35050</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
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identifying the effects of brca1 mutations on homologous recombination using cells that express endogenous wild-type brca1

missense 변이의 기능 분석은 내생 단백질의 세포에 존재에 의해 복잡하실 수 있습니다. BRCA1의 구조 기능 분석은 전체 길이 BRCA1 단백질과 hypomorphic BRCA1 단백질 1,2,3,4,5을 표현하는 세포 라인 작업 고유의 문제에 대한 강력한 분석의 부족에 의해 복잡하고 있습니다. 우리는 세포의 내생 BRCA1 단백질이 심하게 BRCA1의 mRNA의 3&#39; - UTR을 대상으로 RNAi에 의해 고갈 및 BRCA1의 변형을 표현하는 플라스미드를 공동 transfecting으로 대체되었습니다 약자 시스템을 개발했습니다. 이 절차의 장점 중 하나는 BRCA1과 동시 교체의 급성 입을은 세포가 보조 돌연변이 BRCA1 또는 기능의 손실을 보상하기 위해 시간이 지남에 따라 발생할 수 adaptations없이 성장할 수 있도록한다는 것입니다. 이 고갈 및 추가 기능 다시 절차는 즉시 동종 재조합의 활동에 assayed되었습니다 헬라 유도 세포 라인에서 이루어졌다. 상동 재조합 분석은 재조합 기판은 게놈 (그림 1) 6,7,8,9에 통합되어 의하여 이전에 게시된 방법에 따라 달라집니다. 이 재조합 기판은 비활성 GFP allele의 내부 드문 절감 I - SceI 제한 효소 사이트를 가지고 있으며, 하류 두 번째 비활성 GFP의 allele입니다. 상동 재조합에 의해 수리 수있는 두 번 좌초 휴식에 I - SceI 결과를 표현하고, 상동 재조합은 휴식을 수리 않은 경우는 즉시 GFP 단백질의 표현 유동세포계측법에 의해 채점되어 활성 GFP의 allele를 생성하는 플라스미드의 Transfection . 내생 BRCA1의 고갈은 상동 재조합 활동에 8 - 10 - 배 감소하고, 추가 기능 다시 야생 형 플라스미드 완전 상동 재조합 복원 기능의. 전체 길이 BRCA1의 특정 지점 돌연변이 공동 transfected plasmids에서 표현되었을 때, 특정 missense의 돌연변이의 효과가 득점 수 있습니다. 예를 들어, BRCA1 (M18T) 단백질, 알 수없는 임상 중요성 10 변종의 표현이 이러한 세포로 표현되었다, 그것은 BRCA1 종속 상동 재조합을 복원하지 못했습니다. 또한 알 수없는 의미의 또 다른 변종의 명암, 표현에 의해, BRCA1은 (I21V) 완전 BRCA1에 의존 상동 재조합 기능을 복원. BRCA1 missense 변이의 기능을 테스트의이 전략은 centrosome 기능 (Kais 외, 관찰되지 않은)에 시금 다른 생물 학적 시스템에 적용되었습니다. 전반적으로, 이러한 방식은 recessively 분석해야하는 유전자의 missense의 돌연변이의 분석에 적합합니다.

Watch this Scientific Journal Video about Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1 at JoVE.com

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

우리는 RNAi를 사용하여 세포의 내생 BRCA1 단백질을 파괴하고 코딩 변경을 포함하는 BRCA1 포인트 돌연변로 대체하여 DNA 손상의 동종 재조합의 수리를 위해 조직 문화를 기반으로 분석에서 BRCA1 변종을 테스트하기위한 방법을 제공합니다.

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 ...

identifying-effects-brca1-mutations-on-homologous-recombination-using

Nanyang Technological University

Research

JoVE Journal

Biology

14.6K 조회수.  The Ohio State University. 우리는 RNAi를 사용하여 세포의 내생 BRCA1 단백질을 파괴하고 코딩 변경을 포함하는 BRCA1 포인트 돌연변로 대체하여 DNA 손상의 동종 재조합의 수리를 위해 조직 문화를 기반으로 분석에서 BRCA1 변종을 테스트하기위한 방법을 제공합니다.

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

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

아크릴 아미드 기반 Hydrogels을 사용하여 세포 기능에 매트릭스 강성의 영향을 공부

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

연구

생물학

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

편광 상피 세포에 Plasmids의 Microinjection에 의해 작은 GTPases의 기능을 분석

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

의 야생 - 타입과 장식 품종을 구별하기 PCR 기반 Genotyping 방법 Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

에서 상동 재조합 구문을 Recombineering<em&gt; 초파리</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

에 박테리아의 효과를 측정<em&gt; C. 엘레</em계란 보존 분석을 사용하여&gt; 행동

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

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 ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

펩타이드 배열을 사용하여 단백질 - 단백질 상호 작용 사이트를 확인

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

나의 기능적 특성<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; 선택을 양자가 살해 사용하여 세포 내 구획의 교환기는 플라즈마 막에서 그들을 표현하는

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

야생형과 간암의 시알 산의 결정<em&gt; CMAH</em&gt; 넉 아웃 마우스.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

마우스의 장내 미생물에 대한 박테리오신 효과 평가 방법

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...