This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

1. 사전에 준비 <ol><li> 37 ° C에 따뜻한 멸균 인산 완충 식염수 (PBS). </li><li> 10 % 소 태아 혈청 (FBS)이 보충 된 이글 배지 (DMEM) 및 최소 필수 매체 (MEM)의 따뜻한 둘 베코 변형, 100 IU / ml의 페니실린, 100 ㎍ / ml의 스트렙토 마이신을 37 ° C이다. 참고 :이 각각 RAW와 헬라 세포 정상적인 성장 미디어 (NGM)를 나타냅니다. </li></ol> 세포의 2. 준비 <ol><li> NGM에 70-90%의 포화 상태에 RAW 264.7 세포를 성장. <ol><li> 가습 95 % 공기 / 5 % CO 2 분위기에서 37 ° C에서 세포를 유지한다. </li></ol></li><li> NGM에 70-90%의 포화 상태에 헬라 세포를 성장. <ol><li> 가습 95 % 공기 / 5 % CO 2 분위기에서 37 ° C에서 세포를 유지한다. </li></ol></li><li> 수집하기 전에, 멸균 PBS로 한 번 세포 단층을 씻는다. </li><li> 멸균 원뿔 튜브에 미리 합류 세포를 수집합니다. <ol><li> 부드럽게 RAW 세포를 긁어PBS에서 고무 경찰관으로, 반복 피펫으로 세포 집계를 분산한다. </li><li> 육안 검사는 문화면에서 해리가 표시 될 때까지 0.25 % 트립신 솔루션 헬라 세포를 분리합니다. 예를 들어, T-75 플라스크에 대해 2 내지 3 ml의 사용. 트립신 솔루션이 전체 성장 표면을 커버하기 위해 그에 따라 볼륨을 조정합니다. <ol><li> NGM 10 % FBS를 함유하는과 해리 반응을 켄칭. </li><li> 세포 집합체를 분산시키기 반복 피펫 팅으로, 트립신 NGM의 적어도 두 배 볼륨을 사용. </li></ol></li></ol></li><li> 가볍게 4 분 900 XG에서 원심 분리하여 세포 펠렛. </li><li> 멸균 PBS를 사용하여 트립신 / 종결 용액과 동일한 볼륨에 재현 탁. </li><li> 입자 또는 혈구 계수기를 사용하여 세포를 카운트. </li><li> 가볍게 4 분 900 XG에서 원심 분리하여 세포 펠렛. </li><li> 5.5 × 106 6.0 × 106 개의 세포 / ㎖의 PBS를위한 충분한 부피 재현 탁. 참고 : 예를 들어, 하나의 T-75 플라스크 approxim을 얻을 것입니다러면 7.5 × 10 6 세포 (29). </li><li> 전기까지 얼음에 세포 현탁액을 유지합니다. </li></ol> 일렉트로 3. 준비 <ol><li> 얼음 사전 냉각 전기 큐벳 (0.4 cm 간격). </li><li> 전기 장치의 전원을 켜고 0.3 kV의에 전압을 설정합니다. 참고 : 용량 및 저항 (제조업체가 10 μF 600 Ω로 설정) 우리의 electroporator에 설정 조정하지 않았다. </li><li> NGM과 복구 판을 작성하고 37 ℃에서 가습 95 % 공기 / 5 % CO 2 분위기에서 평형 </li></ol> 4. Electroporation에 <ol><li> 사전 냉장 큐벳에 세포 현탁액 400 μl를 놓고 베트 (/ ㎖ 50 μg의) 선택된 단백질 20 μg의를 추가합니다. </li><li> 톡 베트는 부드럽게 ~ 10 배 세포를 손상시키지 않고 혼합합니다. 참고 : 베트는 잘 혼합 여러 번 반전 될 수 있지만, 최대 피펫하지 않고 아래로 또는 소용돌이 세포의 손상을 방지 할 수 있습니다. </li><l난&gt; 종이 타월 또는 다른 와이퍼와 큐벳의 드라이 외부는 electroporator에 전기 불꽃을 방지 할 수 있습니다. </li><li> 1.5-1.7 밀리 0.3 kV의에서 Electroporate 샘플. 참고 :이 연구에 대한 전형적인했다. </li><li> 즉시 전기 플릭 베트 후 부드럽게 ~ 10 배는 잘 혼합합니다. </li></ol> 세포의 5. 도금 <ol><li> 준비가 될 때까지 얼음에 electroporation하여 세포 보관 큐벳 복구 플레이트에 배치합니다. </li><li> 대부분의 다운 스트림 분석을 위해, 외부 이펙터 단백질을 제거하기 위해 미리 예열 NGM으로 세포가 1 배 씻는다. <ol><li> 분석을 위해 세포를 제거하고 NGM의 3-5 ml의 일시 중지합니다. </li><li> 4 분 동안 900 XG에서 원심 분리하여 세포 펠렛. </li><li> 원하는 판 크기 (예 : 35mm 접시 2 ml)에 대한 NGM의 적절한 볼륨을 Resuspend. </li></ol></li><li> 다운 스트림 분석을위한 세포의 적당량을 제거합니다. <ol><li> 예 1 : 현미경 분석을위한 유리 바닥 요리에 판. </li><li> 예 2 : 판 INT친 화성 - 정제를 같은 단백질 분석 용 O 세포 배양 플라스틱. </li></ol></li><li> 세포는 적어도 4 시간 동안 37 ° C에서 95 % 가습 된 공기 / 5 % CO 2 분위기하에 평형화 플레이트에서 복구 할. </li></ol> 6. 현미경 분석 6.1) 고정 / 면역 형광 염색법 <ol><li> 워시 세포는 4 시간의 회복 기간 후 멸균 PBS로 1 배. </li><li> 실온에서 2 분 동안 100 % 메탄올에 세포를 고정. 완전히 세포 (예를 들어, 2 ml의 35mm의 요리)를 충당하기 위해 충분한 메탄올을 사용합니다. </li><li> 멸균 PBS로 세척 배. </li><li> 15 분 동안 PBS에서 0.4 % 트리톤 X-100 세포를 Permeabilize 하시려면. 예를 들어, 35mm 직경 플레이트 된 1 mL의 사용. <ol><li> 에피토프 및 표적 단백질의 위치에 따라 트리톤 X-100 투과성으로 강도의 길이를 조정합니다. 참고 : 최상의 결과를 그러나 위의 조건은 대부분의 C에 적합해야한다, 경험적으로 각 대상에 대해 결정해야합니다ytosolic 목표. </li></ol></li><li> RT에서 1 시간 동안 PBS 중 5 % 소 혈청 알부민 (BSA)으로 차단. 예를 들어, 35mm 직경 플레이트 된 1 mL의 사용. </li><li> PBS로 세척 배. </li><li> 도 4에서 밤새 항체 용액에 결합 차 항체 (PBS 중 0.1 % 트리톤 X-100, 1 % BSA)을 부화 &#160; 부드러운 락과 C를 °. 예를 들어, 35mm 직경 플레이트를 0.5ml를 사용한다. <ol><li> 항체 희석에 대한 제조업체의 권장 사항을 따르십시오. 주 : 200 희석 : PKN1 항체 1에 사용 된 반면, 1000 희석 : 예를 들어, 스트렙 타비 딘 - 결합 펩티드 태그 (SBP-Tag)을 1 항체를 사용 하였다. </li></ol></li><li> PBS로 세척 4 배. </li><li> 빛으로부터 보호 실온에서 1 시간 동안 항체 결합 액에 적합한 형광 접합 된 이차 항체와 함께 배양한다. <ol><li> 예를 들어, 실온에서 1 시간 동안 또는 알렉사 488 알렉사 647을 사용한다. <ol><li> 개미에 대한 제조업체의 권장 사항을 따르십시오ibody 희석. (예를 들어 약 1 : 1,000 본 연구). </li></ol></li><li> 예를 들어, 필요에 따라 다른 얼룩을 추가, 5 μM 밀 배아 agglutinnin (WGA) 실온에서 1 시간 동안, 제조업체의 권고에 따라 알렉사 647 또는 DAPI에 결합. </li></ol></li><li> 이미지에 대한 준비가 될 때까지 빛으로부터 보호 PBS 4 ° C에서 저장 씻을 5 배. </li></ol> 6.2) 공 초점 현미경 및 이미지 분석 <ol><li> 63X 기름 침지 목표를 사용하여 반전 공 초점 현미경 이미지 샘플. <ol><li> 492-542 nm의 방출 사이의 대역폭과 아르곤 레이저의 488nm의 라인을 이용하여, 화상을 녹색 채널. </li><li> 640-718 nm의 방출 사이의 대역폭을 가진 633 nm의 다이오드 레이저를 사용하여 적색 이미지 채널. </li><li> 407-453 nm의 방출 사이의 대역폭을 가진 405 nm의 다이오드 레이저를 사용하여 청색 이미지 채널. </li><li> Z-스택 세포 볼륨의 전체를 커버 여러 채널을 확인합니다. </li></ol></li><li> 해당 이미지 처리 소프트웨어로 이미지를 처리​​. </li></ol> 7. 선호도 정화 <ol><li> 4 시간의 회복 기간 후 4 ° C PBS로 두 번 일렉트로 세포를 씻으십시오. </li><li> 와를 Lyse ~ 용해 완충액 얼음 (PBS에서 프로테아제 억제제 칵테일 및 포스파타제 억제제와 1 % 트리톤 X-100)의 1.0 ml의. 제조업체의 지침에 따라 억제제를 사용합니다. 참고 : 예를 들어, 모두 본 연구에 사용 된 프로테아제 및 포스파타제 억제제 (100X)에 공급하고 있지만, 다른 제제는 동일하게 잘 작동한다. </li><li> 즉시 고무 경찰관과 세포를 긁어 원뿔 튜브에 수집합니다. </li><li> 활발한로 소용돌이 초음파 처리를 Lyse. 참고 : 다른 용해 방법은 똑같이 잘 작동 할 수 있지만 효율이 경험적으로 분석 요구 사항의 적합성 여부를 결정해야합니다. <ol><li> 간헐적 와동와 3 × 30 초 초음파 처리. </li></ol></li><li> 4 ° C에서 10 분 동안 10,000 XG에 원심 분리기 수집세포 파편과 불용성 집계; 뜨는을 저장합니다. </li><li> 엔드 오버 엔드 회전 4 ° C에서 하룻밤 스트렙 타비 딘 아가로 오스 수지 현탁액 50 μL와 일렉트로 세포 용 해물 같은 부피 (~ 1.0 ml)에 결합합니다. 참고 :이 수지는 스트렙 타비 딘 결합 펩타이드 친 화성 태그를 통해 일렉트릭 단백질 (및 관련 단지)를 캡처합니다. </li><li> 2 분 동안 2,500 XG에 원심 분리기 및 상층 액을 제거한다. </li><li> 40 침대 볼륨 (~ 1 ml)을 PBS로 두 번 세척 할 것. </li><li> 30 μl의 4 배 LDS 로딩 버퍼 (141 mM 트리스베이스, 2 % LDS, 10 % 글리세롤, 0.51 mM의 EDTA, 0.22 mM의의 Serva 블루 G, 빨간 0.175 mM의 페놀, 산도 8.5), 20 μL DIH 2 O, 1 μL의 추가 일정량 허용 0.5 M 트리스 (2- 카르복시 에틸) 포스 핀 (TCEP)는 비드 2340. </li><li> 10 분 동안 95 ° C에서 열 및 얼음에 멋진. </li><li> 스핀 5 분 4 ° C에서&gt; 10,000 XG. </li><li> 상층 액을 수집합니다. </li><li> 적절한 항체를 참고하여 웨스턴 블롯을 수행 그는다시, 안티 PKN1 차 항체가 사용됩니다. </li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Electroporation+Is+a+Highly+Efficient+Method+for+Introducing+Restriction+Endonucleases+into+Cells.">Cell Electroporation Is a Highly Efficient Method for Introducing Restriction Endonucleases into Cells.</a> Mutation Research. 225, 49-53 (1989).</li><li>Cortes, F., Ortiz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+damage+induced+by+restriction+endonucleases+recognizing+thymine-rich+DNA+sequences+in+electroporated+CHO+cells.">Chromosome damage induced by restriction endonucleases recognizing thymine-rich DNA sequences in electroporated CHO cells.</a> International Journal of Radiation Biology. 61, 323-328 (1992).</li><li>Cortes, F., Ortiz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+chromosomal+aberrations+in+the+CHO+mutant+EM9+and+its+parental+line+AA8+by+EcoRI+restriction+endonuclease:+electroporation+experiments.">Induction of chromosomal aberrations in the CHO mutant EM9 and its parental line AA8 by EcoRI restriction endonuclease: electroporation experiments.</a> Mutation Research. 246, 221-226 (1991).</li><li>Baron, S., Poast, J., Rizzo, D., McFarland, E., Kieff, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+antibodies,+DNA,+and+other+macromolecules+into+cells:+a+highly+efficient+method.">Electroporation of antibodies, DNA, and other macromolecules into cells: a highly efficient method.</a> Journal of Immunological Methods. 242, 115-126 (2000).</li><li>Lewis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+edges+toward+clinic+for+both+gene+therapy+and+drug+delivery.">Electroporation edges toward clinic for both gene therapy and drug delivery.</a> Genetic Engineering and Biotechnology News. 17, (1997).</li><li>Kotzamanis, G., 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=CFTR+expression+from+a+BAC+carrying+the+complete+human+gene+and+associated+regulatory+elements.">CFTR expression from a BAC carrying the complete human gene and associated regulatory elements.</a> Journal of Cellular and Molecular Medicine. 13, 2938-2948 (2009).</li><li>Chakrabarti, R., Wylie, D. E., Schuster, S. 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K., Horwitz, J., De Lanerolle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+electroinjection+method+for+introducing+proteins+into+living+cells.">Evaluation of the electroinjection method for introducing proteins into living cells.</a> The American Journal of Physiology. 260, C355-C363 (1991).</li><li>Prasanna, G. L., Panda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation:+Basic+principles,+practical+considerations+and+applications+in+molecular+biology.">Electroporation: Basic principles, practical considerations and applications in molecular biology.</a> Bioprocess Engineering. 16, 261-264 (1997).</li><li>Weaver, J. C., Chizmadzhev, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+electroporation:+A+review.">Theory of electroporation: A review.</a> Bioelectrochemistry and Bioenergetics. 41, 135-160 (1996).</li><li>Weaver, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+theory.+Concepts+and+mechanisms.">Electroporation theory. Concepts and mechanisms.</a> Methods in Molecular Biology. 55, 3-28 (1995).</li><li>McAteer, J. A., Douglas, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer+culture+techniques.">Monolayer culture techniques.</a> Methods in Enzymology. 58, 132-140 (1979).</li><li>Ho, T. D., 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=Identification+of+GtgE,+a+novel+virulence+factor+encoded+on+the+Gifsy-2+bacteriophage+of+Salmonella+enterica+serovar+Typhimurium.">Identification of GtgE, a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium.</a> Journal of Bacteriology. 184, 5234-5239 (2002).</li><li>Niemann, G. S., 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=Discovery+of+novel+secreted+virulence+factors+from+Salmonella+enterica+serovar+Typhimurium+by+proteomic+analysis+of+culture+supernatants.">Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants.</a> Infection and Immunity. 79, 33-43 (2011).</li><li>Spano, S., Liu, X., Galan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+targeting+of+Rab29+by+an+effector+protein+distinguishes+the+intracellular+compartments+of+human-adapted+and+broad-host+Salmonella.">Proteolytic targeting of Rab29 by an effector protein distinguishes the intracellular compartments of human-adapted and broad-host Salmonella.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 18418-18423 (2011).</li><li>Haraga, A., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Salmonella+type+III+secretion+effector+interacts+with+the+mammalian+serine/threonine+protein+kinase+PKN1.">A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.</a> Cellular Microbiology. 8, 837-846 (2006).</li><li>Zinchuk, V., Grossenbacher-Zinchuk, O., 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=Quantitative+colocalization+analysis+of+confocal+fluorescence+microscopy+images.">Quantitative colocalization analysis of confocal fluorescence microscopy images.</a> Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et al]. 4 (Unit 4.19), (2011).</li><li>Kimple, M. E., Sondek, J., 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=Overview+of+affinity+tags+for+protein+purification.">Overview of affinity tags for protein purification.</a> Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et al]. 9 (Unit 9.9), (2004).</li></ol>

저자가 공개하는 게 없다.

병원성 박테리아로부터 분비 이펙터 숙주 세포 환경 내에서 작동하도록 진화하고, 따라서 호스트 내에서 (in situ)을 연구하기 위해 유용하다. 숙주 세포에 특정 관심 이펙터의 도입은 관련 병원체 호스트 상호 작용이 다른 세균 단백질의 간섭없이 독립적으로 연구 할 수 있습니다. 목표는 형질 감염 또는 형질 도입시켜과 관련된 몇 가지 문제를 피하고, 진핵 생물 숙주 세포 내로 박테리아 ?...

많은 그람 음성균 직접 숙주 세포를 1-5로 (이펙터라고 함) 병원성 관련 단백질을 주입하도록 전문화 분비 시스템을 사용한다. 숙주의 면역 억제, 세포 골격 변화, 세포 내 신호 전달 및 매매, 전사 변화 수정 및 호스트 테아 변경 6-9 : 이러한 이펙터 포함한 생물학적 기능의 넓은 범위를 갖는다. 일부 이펙터 기능하지만 호스트 대상 외 다수의 생화학 적 조치 (들)이 결정될 수 남아 알려져있다. 야생형 및 재조합 박테리아 감염을 비교하는 세포 독성 이펙터 메커니즘을 연구하는 유효한 방법이지만, 상기 숙주 세포에 도입 개별 이펙터 종종 유리하다. 따라서, 숙주 세포의 컨텍스트에서 세균 효과기 단백질을 도입하고 특성화 간단한 방법이 매우 바람직하다. 실험 분석 위스콘신 단순화하나의 이펙터가 중요 번째 다른 이펙터는 반대 또는 중복 기능을 가질 수 있기 때문이다. 이 단순화를 달성하기 위해, 연구진은 이전에 긁어은 12, 13로드, 바이러스 성 전달 (10), 미세 주입법 (11) 등 다양한 방법에 의해 세포로 거대 분자를 도입, 화학적으로 유도 미세 주입 (14), 독점 단백질 &quot;형질 전환&quot;시약 (15), 인산 칼슘 침전와 세포 융합 (16), 및 전기 17-20. 도입 된 분자는 세포 내 표적 (21, 22)에 대한 단백질, 세포 투과성 염료 및 항체 DNA, RNA, RNAi의 종을 포함하여 핵산 이르기까지 다양합니다. 일부 방법은 도입 될 수있는 고분자의 종류를 포함한 한계가 있고, 특히 하류 분석 높아 세포 독성 작용의 손상 기전, 효능이 낮고 또는 도입 효율이 제한 될 수있다. 형질, ofte또한 대 식세포 및 일차 전지와 같은 몇 가지 중요한 숙주 세포 유형, 형질 향해 특히 내성을 겪고 제한, 포유 동물 세포에서 세균의 유전자를 발현하는 방법을 위해 사용되는 N. 이 외에도, 또한 외래 DNA의 도입시 생성 된 박테리아 단백질의 수준을 조절하는 것이 곤란하다. 많은 작품은 일반적인 실험실 기술로 모두 세균 및 포유 동물 세포에 핵산의 전기를 설립했다; 그러나, 생리 학적 조건하에 단백질을 세포 내로 전달하기위한 최선의 방법에 대한 지속적인 연구가있다. 단백질 형질 전환에 보고서는 유망하지만, 고가의 시약 및 최적화를 필요로한다. 최소한의 비용으로 셀 대상의 다양한에 잠재적으로 독성 세균 이펙터를 소개하는 욕망은 생체 내에서 단백질을 연구하는 방법으로 전기를 고려하는 우리를 이끌었다. 단백질 전기 23-25는 만족입니다또한 전기 형질 전환 또는 전자 주입 (26)로 알려진 electropermeabilization를 통해 살아있는 세포로 단백질을 소개 HOD. 이 방법은 세포막에 구멍을 만드는 고강도 전기 펄스를 사용한다. 이 가역적 모공은 일반적으로 세포 내 공간에서 제외됩니다 거대 분자가 세포를 입력 할 수 있습니다. 외부 전계를 제거하여, 상기 멤브레인은 셀이 통과 구멍 (27, 28)의 분자를 보유 할 수 있도록, 다시 봉인 할 수있다. 표준 electroporator 일관 마우스 대 식세포 유사 세포 및 인간 상피 세포에 모두 세균 이펙터를 도입이 연구에 사용 하였다. 이 방법은 세포 생존 능력의 뚜렷한 감소와 신속하고 효율적이며 저렴하다. 도입 된 단백질은 면역 형광 현미경을 통해 시각 또는 기능 분석에 사용할 수 있습니다. 이뿐만 아니라, 비 독성 표준으로 녹색 형광 단백질 (GFP)을 사용하여 입증되었다두 살모넬라 이펙터 단백질과 SspH1 GtgE. 우리는 진핵 숙주 세포에서 세균 독성 단백질의 기능 연구의 레퍼토리에 추가 도구로 단백질 전기를 제안한다.

<table border="0" cellpadding="0" cellspacing="0" width="1132">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">Material or Equipment</td>
			<td style="width:177px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:315px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.25% Trypsin-EDTA Solution</td>
			<td>Cellgro</td>
			<td>25-050-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.4cm Gap-disposable electroporation cuvettes</td>
			<td>Bio-Rad</td>
			<td>165-2088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Methanol</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Flammable, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4919</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Counting Apparatus - Hemocytometer or Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>Model Z1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell Culture Incubator</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Humidified 95% air/5% CO2 atmosphere at 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Culture Plastic</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Cell culture flasks/plates, pipets, tubes, rubber policeman</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modification of Eagle&#8217;s Medium (DMEM)</td>
			<td>Cellgro</td>
			<td>10-013</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electroporator</td>
			<td>Bio-Rad</td>
			<td>E. coli Pulser</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-016-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent confocal microscope&#160;</td>
			<td>Ziess</td>
			<td>Model&#160; LSM 710&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Bottom Dishes for Microscopy</td>
			<td>Wilco Wells</td>
			<td>HBSt-3522</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HALT Protease Inhibitor Cocktail</td>
			<td>Pierce</td>
			<td align="right">78430</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HeLa Cell Line</td>
			<td>ATCC</td>
			<td>ATCC CCL-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LDS 4X Loading Buffer</td>
			<td>Invitrogen</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimal Essential Medium (MEM)&#160;</td>
			<td>Cellgro</td>
			<td>10-010</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RAW 264.7 Cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primary Antibody Against Target of Interest</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Secondary Antibody Conjugated to Fluorophore</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Chill to 4 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Agarose Resin Suspension&#160;</td>
			<td>Pierce</td>
			<td align="right">20353</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCEP</td>
			<td>Sigma-Aldrich</td>
			<td align="right">646547</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8585</td>
			<td>Irritant, Toxic</td>
		</tr>
	</tbody>
</table>

electroporation of functional bacterial effectors into mammalian cells

살아있는 세포의 맥락에서 단백질 상호 작용의 연구는 현지화, 역학, 그리고 상호 작용 파트너에 대한 중요한 정보를 생성 할 수 있습니다. 이 정보는 호스트 병원체 상호 작용의 컨텍스트에서 특히 유용하다. 많은 병원체 단백질은 세포 내 환경 내에서 숙주의 면역 시스템 및 생존의 회피를 가능 같은 방법의 다양한 숙주 세포 내에서 기능한다. 이러한 병원체 단백질 호스트 세포 상호 작용을 연구하기 위해, 여러 가지 접근 방법이 일반적으로 포함하여, 사용됩니다 생체 내 감염에 형질 전환 또는 형질 도입을 통해 태그 또는 돌연변이 단백질 또는 병원체 유전자의 도입을 발현하는 균주. 이러한 방식은 각각 이점과 단점을 갖는다. 우리는 직접 세포에 외래 단백질을 소개 할 수있는 방법을 모색하고 있습니다. 일반적으로 일렉트로 세포 내로 핵산을 도입하기 위해 사용되지만, 생물 리 학적 근거가 정확히 동일하지만 드물게 단백질에 적용되지 않았다.표준 electroporator는 포유 동물 세포에 친 화성 태그가 세균 이펙터를 소개하는 데 사용되었다. 상피 및 마우스 대식 세포는 전통적인 분리 방법에 의해 배양하고, 관심 외인성 세균 병원체의 단백질 (예를 들어 살모넬라 Typhimurium의 GtgE) 0.4 cm 갭 전기 천공 큐벳에 넣었다. 전기 (0.3 kV의) 짧은 (4 시간) 회복 기간 후, 세포 내 단백질은 형광 친 화성 태그를 통해 단백질을 라벨 및 공 초점 현미경으로 공간과 시간 분포를 조사하여 확인 하였다. 일렉트로 단백질은 세포 내부의 세포 내 작용 및 올바른 거래 및 단백질 - 단백질 상호 작용을 할 수있는 것으로 나타났다. 외래 단백질이 세포의 표면 상에 축적되는 경향이 있지만, 일렉트로 샘플 단독 배양 세포 내 이펙터 농도에 대하여 크게 증가했다. 이 프로토콜은 AP에서 할 수 간단하고 빠른만큼타겟팅 및 병원성 세포 내 단백질의 기능을 포함하는 숙주 세포에서 단백질의 병원체 높은 처리량 특성을 허용 arallel 패션.

개념의 초기 증거로서, 정제 된 녹색 형광 단백질을 이용하여 성공적으로 전기 포유 동물 세포에 도입 하였다. GFP, 대략 27 kD의 분자량 단백질은 일반적으로 세포 독성이없는 중요한 분자 생물학 도구로 (일반적으로 플라스미드 DNA로부터 발현) 포유 동물 세포 내로 도입된다. 헬라 세포 (그림 1A)를 배양 또는 형광 GFP 신호를 확인하기 위해 면역 형광 공 초점 현미경을 25 ㎍ / ㎖의 GFP와 ...

Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

포유 동물 세포에 기능 세균 이펙터의 전기

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

electroporation-of-functional-bacterial-effectors-into-mammalian-cells

Research

JoVE Journal

Immunology and Infection

10.0K 조회수.  Pacific Northwest National Laboratory. Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells. 

비디오: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

세균성 병원체에 의해 인간의 세포의 침략

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

연구

면역학 및 감염병학

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

포유류의 상피 세포에 박테리아의 접착 시험 관내 분석

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

심장 기능 면역 세포의 분리

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

골수 세포 역학의 실시간 영상<em&gt; APC<sup&gt; 최소 / +</sup</em&gt; 소장 종양 디스크 공 초점 현미경을 회전시켜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

요오드 산 쉬프 염색 말초 혈액 단핵 세포에서 글리코겐을 감지

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

세균 인공 염색체 : RNA 바이러스 양성 가닥의 연구에 대한 기능 유전체학 도구

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

투과성 막 삽입 기반 감염 시스템의 구현은 포유 동물 숙주 세포에서 분비 세균 독소의 효과를 연구하기

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

효모 두 하이브리드 화면을 사용하여 비 경작 식물 병원체로부터 세균 이펙터의 기능을 해결했습니다

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...