Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

대 식세포 세포주 1. 유지 보수 <ol><li> RAW264.7 성장 및 DMEM에서 J774A.1 세포주 5 % CO 2 존재하에 37 ºC에서 10 % 소 태아 혈청 (FBS)으로 보충. , 불임을 유지하는 데 도움이 (이 반드시 필요한 것은 아니지만) 미디어에 1 % 페니실린 / 스트렙토 마이신 (펜 / 연쇄상 구균)를 추가합니다. </li><li> 중요한 단계 : 대 식세포는 효율적인 형질과의 siRNA에 의한 유전자 최저의 건강한 상태로 성장하고 있는지 확인합니다. 이들 대 식세포 라인 가장 형질 효율을 발휘하고,이 단계에서의 강력한 후 형질 생존을 유지하는 것처럼, 신선한 해동 후 유로 (3) 및 (10) 사이의 세포를 사용하여; 양이 크게 감소 녹다운 형질 전환 효율과 유전자로서, 높은 통로 번호 (&gt; 20)을 사용하지 않는다. </li><li> 세포가 더 이상 80 % 합류점에 도달하지 않을 때까지 최적화 된 siRNA 형질 감염의 경우, 플레이트 20 % 컨 플루 언시 세포는 하루 또는 이틀 성장. 자란 세포는 effic 트랜하지 않습니다iently. </li></ol> 2. 형질의 96 웰 셔틀 시스템 프로그래밍 <ol><li> 셔틀 버스에 연결된 컴퓨터의 96 웰 셔틀 소프트웨어를 시작합니다. 왼쪽 상단의 파일 메뉴에서 새로운 매개 변수 파일을 선택합니다. </li><li> 표시 96 웰 플레이트 회로도에 형질 전환 될 우물을 강조 표시합니다. 녹색 상자가 선택되어있는 우물 표시됩니다. </li><li> 개념도 96 웰 플레이트 아래에있는 (J774A.1에 대한 RAW264.7 또는 DS-130 DS-136)를 사용하고 적용을 선택 대식 세포주에 해당하는 프로그램을 선택합니다. 프로그램을 선택한 후, 회로도 접시에 우물이 어두운 푸른 색이 될 것이라고합니다. 빈 동그라미는 프로그램이 할당되지 않았으며 충격되지 않습니다 우물을 나타냅니다. </li></ol> 3. 형질에 대한 시약 준비 <ol><li> SF 세포주 혼합하여 작업 Nucleofector ™ 솔루션을 준비 96 웰 Nucleofector ™ 솔루션동봉 보충 관의 전체 내용. 혼합 시약은 사용하기 전에 실온에서 평형을 허용합니다. 나중에 사용하기 위해 최대 3 개월까지 4 ºC에서 초과 완전한 Nucleofector ™ 솔루션을 저장합니다. </li><li> 사전 따뜻한 형질 대한 대 식세포를 제조하기 전에 37 ºC에서 DMEM, RPMI-1640, 0.25 % 트립신 / EDTA, 인산염 완충 식염수 (PBS). </li><li> 얼음에 해동에 의해 형질 전환에 대한 siRNA를 준비. 된 siRNA의 동결 녹아서의 수를 최소화하기 위해, 분취 그들이 사용되는 처음 새로운 siRNA를 동결. </li><li> 초기 실험에서, (20 μM 종목에서 1:10 희석) 2 μM의 비교적 높은 투여 량의 siRNA를 사용한다. 일반적으로 더 높은 투여 량은 다른 세포 유형에 비해 대 식세포에서 효율적인 유전자 녹다운 필요한되지만이어서, 저용량까지 넉다운 강력한 유전자 유도 된 siRNA를 적정한다. 참고 : siRNA를 여러 소스에서 사용할 수 있습니다; 매우 강력한 유전자 녹다운을 얻을 수있다각각의 유전자를 대상으로 네 개의 siRNA를 풀을 수 있습니다 siGenome의 SMARTPOOLs를 사용. 음성 대조군으로, 다양한 비 대상으로 s​​iRNA를 풀 또는 siRNA를의 사용. </li><li> , 형질 전환 효율을 모니터링 siRNA를 실험에 평행 pmaxGFP 제어 (Amaxa 키트에서 제공) 플라스미드 또는 형광 표지 된 siRNA를 형질한다. GFP 플라스미드의 30 % ~ 50 %의 형질 전환 (현미경으로 분석 또는 형질 전환 후 하루 유동 세포 계측법)과 (형질 전환 및 복구 후 세포 계측법 즉시 흐름에 의해 분석) 형광 표지 된 siRNA의&gt; 99 %의 형질 전환을 기대합니다. </li></ol> 4. 형질의 대 식세포를 준비 <ol><li> 트립신으로 세포 배양 접시에서 대 식세포를 분리합니다. 미디어를 대기음 10 ml의 PBS로 두 번 세포를 씻으십시오. PBS를 제거한 후, 함께 세포 배양 세포를 분리하기 시작 때까지 1-2 분 동안 0.25 % 트립신 / EDTA (1 ㎖ / 10cm 플레이트를) 미리 예열. 조심스럽게 접시를 누르고 SOLU 이동배양시 기는 트립신을 극대화한다. </li><li> 배양 후, 멸균 튜브에 피펫 팅하여 세포를, 9 ml의 DMEM + FBS 미디어를 추가 위아래로 부드럽게 혼합하고 수집합니다. </li><li> 혈구를 사용하여 고립 된 대 식세포의 수를 계산합니다. 10cm 접시 당 약 1000 만 대 식세포를 기대합니다. 필요한 식세포의 총 수를 계산; 잘 96 - 웰 셔틀에 각각 20 만 셀을 필요로한다. 피펫 손실을 허용하는 세포의 몇 가지 추가 우물 &#39;의 가치를 추가해야합니다. </li><li> 원심 분리 튜브에 세포의 수를 계산 피펫. 실온에서 150 XG에서 10 분 동안 세포를 원심 분리기. 이 대 식세포의 건강과 형질 전환 효율을 감소하므로 너무 빨리 원심 분리하지 마십시오. </li></ol> siRNA의와 대 식세포의 5 Nucleofection <ol><li> (시약 손실을 최소화하면서 혼합을 용이) 멸균 96- 웰 둥근 바닥 플레이트를 사용하여 각 웰 될 TRANSF 각각의 siRNA의 2 μl를 추가반사 된. 주 : siRNA를 수에 따라, 이것은 식세포 원심 분리 단계 동안 달성 될 수있다. </li><li> 원심 분리하여 세포에서 미디어를 대기음. 부드럽게 펠렛 200,000 세포 당 20 μl를 이용하여, (보충 용액 함유) Nucleofector ™ 솔루션 SF에서 세포를 재현 탁. 재현 탁하면, 최적의 형질 즉시 세포를 사용합니다. </li><li> 멸균 구유에 재현 탁 세포로 이동; siRNA가 들어있는 96- 웰 플레이트의 각 웰에 전사 재 부유 세포 혼합액 20 μL를, 멀티 채널 피펫을 사용. 부드럽게 피펫 아래로 섞는다. </li><li> 이전 96 웰 Nucleocuvette ™ 플레이트에 세포 / siRNA의 혼합물의 20 μl의 중요한 단계 :.이 Nucleofection 동안 오류를 유도 할 수있는 전송 단계에서 기포가 발생하지 않도록. </li><li> 형질 전환 플레이트에서 실험 모든 샘플을 배치 한 후, 제공된 뚜껑과 수직로드 96- 웰 플레이트 커버Y는 부드럽게 샘플에서 모든 거품을 제거하기 위해 딱딱한 표면에 누릅니다. </li><li> 프로그램 화면의 오른쪽 아래에 &#39;업로드 시작&#39;96 웰 Nucleofector 셔틀에 뚜껑이있는 96 웰 플레이트를 놓고 선택합니다. Nucleofection 이전에 결과를 저장하기 위해 프로그램의 프롬프트를 따르십시오. 참고 : Nucleofection 과정에서 성공적으로 tranfected 우물 더하기 기호가있는 녹색 원으로 화면의 96 웰 회로도에 묘사됩니다. 마이너스 (-) 기호가있는 빨간색 원으로 인해 기포 나 잘못 피펫 팅 시약의 올바른 볼륨을, 가장 가능성이 오류를 나타냅니다. </li></ol> 6. 복구 및 대 식세포의 도금 <ol><li> 즉시 Nucleofection 후, 멀티 채널 피펫을 사용하여, 각 웰에 RMP1-1640 미디어를 예열 (37 ° C)의 80 μl를 추가합니다. 미디어가 천천히이 단계에서 매우 섬세하고있는 형질 전환 된 세포와 혼합 할 수 있도록, 각 웰의 측면에 미디어를 추가합니다. 이 같이, 너무 빨리 미디어를 추가하지 마십시오크게 가능성을 감소시킨다. </li><li> 2 분 5 % CO 2와 37 ºC 배양기로 형질 전환 된 샘플 및 RPMI-1640 96 웰 플레이트를 놓고; 이 세포가 조작을 발전하기 전에 복구 할 수 있도록하는 것이 중요합니다. </li><li> 인큐베이터에서 96 웰 플레이트를 제거하고 신중 멀티 채널 피펫을 이용하여 96- 웰 조직 배양 플레이트에 각 웰로부터 혼합물을 전송. </li><li> 멀티 채널 피펫을 사용하여, 각 웰에 예열 (37 ° C) DMEM + FBS 배지 100 μl를 추가합니다. </li><li> 24 ~ 36 시간 동안 5 % CO 2 37 ºC 배양기에서 샘플을 96- 웰 조직 배양 플레이트 (이 시간 범위는 일반적으로 잘 작동하지만, 개개의 siRNA를위한 정확한 타이밍을 최적화) 부화. </li></ol> LPS와 대 식세포의 7 자극 <ol><li> 24 ~ 36 시간 후 Nucleofection 배양 한 다음, 새로운 미디어에 LPS (또는 다른 PAMPs가)를 준비합니다. 모든 우물에 대한 충분한 신선한 매체를 준비합니다 (200 μL의 media / 웰의 96 웰 플레이트 플러스 피펫에 대한 약간의 추가). DMEM + FBS 20 ng를 / ml의 최종 농도 LPS를 희석. </li><li> 96- 웰 플레이트에서 조심 흡 미디어는 세포에 LPS를 함유하는 신선한 미디어를 추가. </li><li> 자극 후 여러 번 LPS 또는 다른 PAMPs가에 대한 응답을 모니터링합니다. 6 시간에는 IL-6, TNFα와 같은 사이토 카인에 대한 강력한 염증성 사이토 카인 응답을 생성하기에 충분하다. </li></ol> 8 대 식세포에서 유도 타고난 면역 반응, 유전자 넉다운,과 생존을 모니터링 <ol><li> LPS 유도 된 사이토 카인 생성을 모니터하기 위해, 96- 웰 저장 플레이트에 세포로부터 상등액을 피펫. 사이토 카인 생성 후 ELISA에 의해 측정 될 수있다. </li><li> 상등액을 제거한 후에, 형광 디 아세테이트를 사용하여 세포 생존을 모니터 (생균 셀룰러 에스 테라 제에 의해 절단 될 때,이 화합물은 형광성된다). T를 사용하여 세포 생존을 조절하는 유전자를 대상으로 s​​iRNA가를 확인그의 접근 방식. 주 : 제대로 수행 한 경우 형질 전환 과정 자체가 생존에 영향을 거의 미치지 않는다. <ol><li> (: PBS 100 최종 희석 1) 각 웰에 플루오 레세 인 디 아세테이트 (아세톤 5 ㎎ / ㎖ 주식을)를 추가합니다. </li><li> 다섯 분에 한 시간 동안 실온에서 인큐베이션 한 후, 플레이트 판독기 (460 nm의 여기, 515 nm의 발광)를 사용하여 세포 내의 형광을 모니터링한다. </li><li> 선택 사항 : 우물의 형광 균일 성을 확보하기 위해, 예를 RIPA로 버퍼를 사용하여를 Lyse 세포; 일부 플레이트 판독기가 웰의 작은 부분만을 모니터링하도록 구성되므로 이것은 잘 균일 한 형광 신호를 제공한다; 세포가 균일하게 도금되어 있지 않은 경우, 플루오 레세 인 디 아세테이트는 부정확 한 판독을 줄 수 있습니다. </li></ol></li><li> 세포 생존을 모니터링하는 대신에, 부착 세포로부터 유래 된 RNA를 사용하여 넉다운 qPCR의 siRNA에 의한 유전자를 모니터링. <ol><li> 상등액을 세포로부터 제거 된 후, 광고 (상기 단계 8.1)를 용해에 직접 부착 세포 (용해 및 RNA의 보존에 사용되는) D RLT 버퍼. </li><li> 이러한 RNeasy 미니 키트로서 RNA 조제 키트를 사용하여 RNA를 분리하고, 유전자를 siRNA의 처리를 제어하는​​ 넉다운 대해 모니터링하는 유전자 특이 적 프라이머를 사용 qPCR에. 정상화 Βactin에 프라이머 또는 GAPDH를 사용합니다. </li></ol></li><li> 다른 대안은 선천성 면역 반응의 다른 측면을 모니터링 할 때, FITC 표지 된 E. 추가하여 대 식세포의 탐식 기능을 모니터링 직접 키트를 사용하여 접착 세포 상 대장균 입자. 몇 시간 밖에 소요 제조자의 프로토콜을 따라, 플레이트 판독기 또는 현미경을 사용하여 식균 흡수를 모니터링. </li></ol>

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S., Choi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+methodologies+for+the+functional+study+of+signaling+molecules.">RNAi methodologies for the functional study of signaling molecules.</a> PloS one. 4, (2009).</li><li>Carralot, J. P., 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=Automated+high-throughput+siRNA+transfection+in+raw+264.7+macrophages:+a+case+study+for+optimization+procedure.">Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.</a> Journal of biomolecular screening. 14, 151-160 (2009).</li><li>Alper, 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=Identification+of+innate+immunity+genes+and+pathways+using+a+comparative+genomics+approach.">Identification of innate immunity genes and pathways using a comparative genomics approach.</a> ProcNatl Acad Sci USA. 105, 7016-7021 (2008).</li><li>De Arras, L., 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=An+evolutionarily+conserved+innate+immunity+protein+interaction+network.">An evolutionarily conserved innate immunity protein interaction network.</a> J. Bio. Chem. , 1967-1978 (2013).</li><li>Yang, I. V., 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=Novel+regulators+of+the+systemic+response+to+lipopolysaccharide.">Novel regulators of the systemic response to lipopolysaccharide.</a> Am J Respir Cell Mol Biol. 45, 393-402 (2011).</li><li>Yang, I. V., 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+novel+innate+immune+genes+by+transcriptional+profiling+of+macrophages+stimulated+with+TLR+ligands.">Identification of novel innate immune genes by transcriptional profiling of macrophages stimulated with TLR ligands.</a> Molecular immunology. 48, 1886-1895 (2011).</li><li>Yang, I. V., 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+novel+genes+that+mediate+innate+immunity+using+inbred+mice.">Identification of novel genes that mediate innate immunity using inbred mice.</a> Genetics. 183, 1535-1544 (2009).</li><li>Raschke, W. C., Baird, S., Ralph, P., Nakoinz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+macrophage+cell+lines+transformed+by+Abelson+leukemia+virus.">Functional macrophage cell lines transformed by Abelson leukemia virus.</a> Cell. 15, 261-267 (1978).</li><li>Ralph, P., Moore, M. A., Nilsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysozyme+synthesis+by+established+human+and+murine+histiocytic+lymphoma+cell+lines.">Lysozyme synthesis by established human and murine histiocytic lymphoma cell lines.</a> The Journal of experimental medicine. , 1528-1533 (1976).</li><li>Zumbansen, 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=First+siRNA+library+screening+in+hard-to-transfect+HUVEC+cells.">First siRNA library screening in hard-to-transfect HUVEC cells.</a> Journal of RNAi and gene silencing. 6, 354-360 (2010).</li><li>Lu, Y. C., Yeh, W. C., Ohashi, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS/TLR4+signal+transduction+pathway.">LPS/TLR4 signal transduction pathway.</a> Cytokine. 42, 145-151 (2008).</li><li>Aliprantis, A. 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=Cell+activation+and+apoptosis+by+bacterial+lipoproteins+through+toll-like+receptor-2.">Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2.</a> Science. 285, 736-739 (1999).</li><li>Alexopoulou, L., Holt, A. C., Medzhitov, R., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+double-stranded+RNA+and+activation+of+NF-kappaB+by+Toll-like+receptor+3.">Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.</a> Nature. 413, 732 (2001).</li><li>Conforti, R., 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=Opposing+effects+of+toll-like+receptor+(TLR3)+signaling+in+tumors+can+be+therapeutically+uncoupled+to+optimize+the+anticancer+efficacy+of+TLR3+ligands.">Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands.</a> Cancer Res. 70, 490-500 (2010).</li><li>Fernandez-Botran, R., V&#283;tvi&#269;ka, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cellular Immunology. 8, 8 (2001).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다. 이 문서는 론자, Inc.에서 후원

수많은 연구하는 개별 유전자가 쥐의 대 식세포에 siRNA를 표적이되어 발표되었다. 지질 - 매개 형질 감염이 개별적으로 대 식세포 세포주에 siRNA를 전달하기 위해 사용되었지만, 이러한 방법은 유전자의 유전자 생존 제한된 유전자 녹다운 및 변동에 미치는 잠재적 영향 겪는다. 중간 또는 높은 처리량 방식으로 유전자를 대상으로 사용할 수있는보다 강력한 분석을 개발하기 위해, 우리는 유전자 ?...

이 프로토콜에서, 우리는를 사용하여 siRNA를 마우스 대식 세포주에서 유전자를 억제하고 선천성 면역 반응에 대한 이러한 치료의 효과를 모니터링하는 효율적인 방법을 설명한다. 이러한 절차는 중간 또는 높은 처리량 방식 RNAi의 스크린을 허용 96- 웰 포맷에서 수행된다. 감염에 응답하여, 인간은 즉각적인 선천성 면역 반응과 더 느리지 만 특정 적응성 면역 반응을 탑재. 이 빠른 선천성 면역 반응은 대 식세포 (1)을 포함하여 식세포 선천성 면역 세포의 모집 및 활성화를 포함한다. 클래식 활성화 대 식세포는 급성 염증 반응에 관여하는 항균 단백질과 화합물, 사이토 카인과 케모카인 및 현재 항원 2,3를 생산하고 있습니다. 또는 활성화 된 대 식세포는 허용 오차, 조직 복구를 유지, 면역 조절에 중요한 역할을하고, 4-8 상처 치유. 때문에 기능의 그들의 다양한의대 식세포는 죽상 동맥 경화증, 관절염, 암 (9)을 포함하여 다수의 질병에서 역할을 할 수있다. 따라서, 대 식세포의 연구는 질병 다양한 분야에서 오랫동안 연구의 핵심 영역이었다. 선천성 면역 반응에서 그 중요성에도 불구하고, 대 식세포와 함께 작동하도록 세포를 도전 할 수 있습니다. 특히, 관련된 독성 10,11없이 식세포 지질 시약을 사용하여 형질 전환을 효율적으로 얻는 것이 곤란하다. 또한, siRNA를 효율적 식세포로 전달되는 경우에도, 종종 넉다운 RNAi를 유도 유전자의 견고성 상당히 온건 할 수 있고, 유전자로 유전자로부터 변할 수있다. 이러한 기술적 과제를 극복하기 위해, 우리는 두 마우스 대식 세포주 RAW264.7 17 J774A.1 18 형질 감염 및 녹다운 기술 12-16을 최적화했다. 이 방법은 형질 전환을위한 Amaxa Nucleofector 96 웰 셔틀 시스템을 사용합니다; 이 시스템96- 웰 포맷 (19)에서 세포를 형질 감염 할 특수 시약 및 전기의 조합을 사용한다. 형질 전환에 따라, 우리는 방법 세포 복구 및 생존 능력을 극대화하기 위해 이후의 siRNA에 의한 유전자 최저을 극대화하기 위해 설명합니다. 이 접근 방법의 유용성을 설명하기 위해 리포 폴리 사카 라이드 (LPS)로 이들 세포를 자극하고, 여러 가지 전 염증성 사이토 카인의 생산 수준에서의 선천성 면역 반응을 모니터링, 이들 대 식세포 세포주에 대한 siRNA의 전달을위한 프로토콜을 기술한다. 우리는 우리가 회원 LPS와 다른 병원체 관련 분자 패턴 (PAMPs가)를 감지, 선천성 면역을 조절하는 수신자 같은 수용체 (TLR) 가족을 대상으로하는 샘플 데이터를 제공합니다.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

using rna-interference to investigate the innate immune response in mouse macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

이 방법을 사용하여 형질 감염 효율을 입증하기 위해, 우리는 플로우 사이토 미터 (도 1)를 사용하여 FITC 표지 된 siRNA에의 흡수를 모니터링. 선천성 면역 반응을 모니터링하는 우리의 접근 방법의 유용성을 설명하기 위해, RAW264.7 대식 세포주에 공지 선천성 면역 조절 유전자를 타겟팅 된 siRNA를 형질 LPS로 세포를 자극 한 다음 - 염증성 사이토 카인의 생산을 모니?...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

마우스 대 식세포의 타고난 면역 반응을 조사하기 위해 RNA 간섭을 사용하여

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

using-rna-interference-to-investigate-innate-immune-response-mouse

Research

JoVE Journal

Immunology and Infection

10.6K 조회수.  National Jewish Health and University of Colorado School of Medicine. In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

비디오: Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

진드기의 RNA 간섭

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

연구

면역학 및 감염병학

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

의 기생충 말벌에 대한 소개<em&gt; Drosophila</em&gt; 그리고 Antiparasite 면역 반응

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2 - 광자 현미경을 사용하여 마우스 Thymus의 Intravital 영상

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

면역 반응과 수면에서의 양적 측정<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

제브라 피쉬의 선천성 면역 건강의 지표로 호흡 버스트 응답의 정량화

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

사이토 카인 생산의 바이러스 성 회피에 타고난 면역 신호를 해부

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

웨스트 나일 바이러스 돌연변이 균주의 감염에에서 MyD88 매개 세포 면역 반응의 생체 외 분석

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

의 Zebrafish의 애벌레 모델의 선천성 면역 반응의 비 침습적 영상<em&gt; 연쇄상 구균 iniae</em&gt; 감염

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

사용 하 여 계통 발생 분석은 진 핵 유전자 원산지 조사

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Lipopolysaccharide 취급 마우스 간에 면역 반응 동안 백혈구의 2 광자 Intravital 이미징

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...