우리는 유동세포계측법 그녀 도움 캘리포니아 국립 메이트 연구 센터에서 애비 스피너 감사하고 싶습니다. 이 연구는 국립 알레르기 및 전염병 연구소 (NIAID) 보건 국립 연구소 (NIH) AI073745, AI080799 및 AI078183의 기금에 의해 지원되었다.

<ol><li> 이전 프로토콜을 시작하기 위해서는 실험에 사용되는 모기 세포가 건강하고 ~ 80 %의 합류에서 자기편 경우 (그림 1). 것을 확인하는 것이 중요합니다 자란하거나 건강하지 못한 세포는 낮은 transfection 효율의 결과하고 일관성 결과를 줄 것이다. </li><li> 30 분 28 ° C의 물을 욕조에 사용하기 전에 모든 미디어를 따뜻하게. <ol class="lalpha"><li> 멤, 얼의 W / 글루타민 5 % 열 inactivated FCS, 0.2 % D - 글루코오스, 1 % 페니실린 - 스트렙토 마이신의 항생제,, 1 %가 아닌 - 필수 아미노산 : A. stephensi MSQ43 세포 E5 매체를 필요로합니다. </li><li> 슈나이더의 Drosophila 중간 5 % 열 inactivated FCS,, 1 % 페니실린 - 스트렙토 마이신의 항생제 : A. gambiae SUA5B 전지는 S2 매체를 필요로 </li></ol></li><li> 해동 얼음의 모든 transfection 시약 재료, plasmids, 그리고 oligonucleotides를 저장합니다. </li><li> 모든 절차는 무균 기술을 사용하여 조직 문화 후드에서 수행되어야합니다. </li><li> 자기편 세포 (A. gambiae SUA5B, A. stephensi MSQ43) 세포를 방해하지 않고 이전 미디어를 대기음 들어, 28 예열되어 신선한 미디어의 상당 볼륨을 추가 ° C 다음의 맨 아래에서 세포를 씻어 내 버리지 피펫을 사용하여 플라스크. </li><li> 세포의 농도는 결정과 1x10 6 세포 / ML의 최종 농도 조정해야합니다. transfection 시약 자료를 준비하는 동안 세포는 상온에서 보관하실 수 있습니다. </li><li> 6 잘 플레이트의 각 잘하는 모기 세포 (1x10 6 셀 / ML) 1 ML을 추가합니다. 각 실험 조건 (아래 참조)에 대한 번호판을 설정합니다. 표 나는 일반적인 transfection을위한 pipetting 방식입니다. 표 II는 BFP 특정 LNA 기능을 사용하여 단일 염기의 돌연변이 유발에 대한 성공적인 테스트 다섯 조건을 설명합니다. 첫 번째 두 pGFP 및 pBFP은 각각 양극과 음극을 제어합니다. 나머지는 BFP 특정 LNA 기능의 증가 농도와 pBFPs의 실험 조건입니다. 12 단계 8 각 상황에 따라 시약의 적절한 볼륨을 추가합니다. </li><li> 멸균 1.5 ML의 microcentrifuge 관에 플라스미드 DNA를 전송 및 EC 버퍼의 적절한 볼륨을 추가합니다. <ol class="lalpha"><li> 1 μL 플라스미드 DNA (1 μg / μL)의 경우, EC 버퍼 99 μL를 추가합니다. </li><li> 1 μL pGFP (μL / 1μg)의 경우, EC 버퍼 99 μL를 추가합니다. </li><li> 1 μL pBFP (μL / 1μg)의 경우, EC 버퍼 99 μL를 추가합니다. </li><li> 1 μL pBFP (μL / 1μg) 및 1 μL (μg / μL)의 경우, EC 버퍼의 98 μL를 추가합니다. </li><li> 1 μL pBFP (μL / 1μg) 및 5 μL (μL / 1μg)의 경우, EC 버퍼의 94 μL를 추가합니다. </li><li> 1 μL pBFP (μL / 1μg)와 10 μL (μL / 1μg)의 경우, EC 버퍼 89 μL를 추가합니다. </li></ol></li><li> 그런 다음 천천히 사용되는 DNA 1 μg 당 향상제를 추가합니다. <ol class="lalpha"><li> 1 μL 플라스미드 DNA의 경우, 8 μL 향상제를 추가합니다. </li><li> 1 μL pGFP 들어, 8 μL 향상제를 추가합니다. </li><li> 1 μL pBFP 들어, 8 μL 향상제를 추가합니다. </li><li> 1 μL pBFP 1 μL의 경우, 16 μL 향상제를 추가합니다. </li><li> 1 μL pBFP 5 μL의 경우, 48 μL 향상제를 추가합니다. </li><li> 1 μL pBFP 10 μL 들어, 88 μL 향상제를 추가합니다. </li></ol></li><li> 1 초과 솔루션의 모든 튜브의 맨 아래에 있는지 확인을 위해 소용돌이 DNA / 버퍼 / 확장기는 혼합물. 그렇다면 5 분 실온에서 품어. </li><li> 10 초 1 μg 플라스미드 DNA와 와동 당 Effectene 시약을 추가합니다. 솔루션의 모든 10 분 상온에서 부화 후 튜브의 하단에하고 있는지 확인합니다. <ol class="lalpha"><li> 1 μL 플라스미드 DNA를 10 μL Effectene를 (표 I) 추가합니다. </li><li> 1 μL pGFP 들어, 10 μL Effectene를 (표 II) 추가합니다. </li><li> 1 μL pBFP 들어, 10 μL Effectene를 (표 II) 추가합니다. </li><li> 1 μL pBFP 1 μL의 경우, 20 μL Effectene (표 II)를 추가합니다. </li><li> 1 μL pBFP 5 μL의 경우, 60 μL Effectene를 (표 II) 추가합니다. </li><li> 1 μL pBFP 10 μL의 경우, 110 μL Effectene를 (표 II) 추가합니다. </li></ol></li><li> DNA / 버퍼 / 확장기 / Effectene 혼합물에 dropwise 예열 미디어를 추가하여 1000 μL의 최종 볼륨 각 튜브를 가져와. 미디어가 너무 신속하게 추가하면 DNA 수있다는이 시점에서 침전물. 부드럽게 3-4 회를 아래와 pipetting하여 섞는다. <ol class="lalpha"><li> 1 μL 플라스미드 DNA 들어, 882 μL 미디어 (표 I) 추가합니다. </li><li> 1 μL pGFP 들어, 882 μL의 미디어 (표 II)를 추가합니다. </li><li> 1 μL pBFP 들어, 882 μL의 미디어 (표 II)를 추가합니다. </li><li> 1 μL pBFP 1 μL 들어, 864 μL 미디어 (표 II)를 추가합니다. </li><li> 1 μL pBFP 5 μL 들어, 792 μL 미디어 (표 II)를 추가합니다. </li><li> 1 μL pBFP 10 μL 들어, 702 μL 미디어 (표 II)를 추가합니다. </li></ol></li><li> 다음 천천히 잘 각 한 번에 한 방울에 해당하는 DNA / 확장기 / Effectene 혼합 1 ML를 추가합니다. </li><li> 소용돌이는 플레이트 부드럽게 세포 transfection 혼합물의 균일한 분배를 보장합니다. Incubat정상적인 성장 조건에서 전자 세포. </li><li> 어느날 transfection 후 부드럽게 미디어를 대기음 천천히 신선한 예열 미디어의 상당 볼륨 바꿉니다. 이렇게하면 우물의 하단에있는 세포 레이어를 방해하지 않는 것이 중요합니다. </li><li> transfection 후 이틀 transfection 효율 (그림 2)를 확인하기 위해 형광 현미경 pGFP와 transfected 긍정적인 제어 세포를 검사합니다. </li><li> SUA5B 전지는 매우 빠르게 성장하고 우거져 될 것입니다. 나흘 기존의 미디어를 aspirating 갓 예열 미디어 천천히 상응하는 볼륨을 추가하여 transfection 얇은 이러한 세포 후에. </li><li> 우물에 새로운 미디어를 추가하면 최대 pipeting하여 각 우물의 바닥에서 모든 세포를 분리하고 아래로, 세포의 혼합물의 절반 볼륨 (1 ML)를 버리고 2 최종 볼륨을 가지고 신선한 예열 미디어 1 ML을 추가 ML. </li><li> 필요한 얇은, 그들은 건강하고 자란되지 않도록 매일 세포를 확인하십시오. </li><li> 세포 분석을 수집하기 전에 5~7일 게시물 transfection을위한 정상적인 조건에서 성장을 허용합니다. </li><li> 5~7일 게시물 transfection에서 모든 우물에서 이전 미디어를 aspirating 각 잘하는 살균 방 온도 PBS 1 ML을 추가하여 세포를 수집합니다. </li><li> 그리고 아래로 pipetting하여 우물의 바닥에서 세포를 분리 5 ML 스냅 캡 튜브에 세포 혼합물을 전송하고 즉시 유동세포계측법를 통해 세포를 분석할 수 있습니다. </li></ol> 대표 결과 <img src="/files/ftp_upload/2355/2355fig1.jpg" alt="그림 1" /> 그림 1. Untransfected 건강한 A. stephensi MSQ43 (A)와 A. 약 80 %에 합류 gambiae SUA5B (B) 세포. <img src="/files/ftp_upload/2355/2355fig2.jpg" alt="그림 2" /> 그림 2. A. stephensi MSQ43 (A)와 A. gambiae SUA5B (B) 세포가 플라스미드을 표현하는 긍정적인 제어와 GFP transfected. <img src="/files/ftp_upload/2355/2355fig3.jpg" alt="그림 3" /> 그림 3. BFP 특정 잠금 핵산 - 수정 oligonucleotides (LNA 기능)과 단일 염기 다음 transfection의 돌연변이 유발을 통해 GFP에 BFP의 전환을 확인 유동세포계측법 데이터입니다. SUA5B 세포는 대조군으로 (pBFP) 플라스미드를 표현 BFP와 긍정적인 제어로 플라스미드를 표현 GFP (pGFP)와 transfected 또는 pBFP 및 BFP 특정 LNA 기능의 증가 농도로했다. 왼쪽에서 오른쪽으로 표시 유동세포계측법 데이터 (A) 게이팅 전략 : 라이브 세포, propidium 요오드화물의 (PI) 부정적인 세포와 transfection 다음 GFP 양성 세포 5 일간의 기대 대 측면 산란. (B) GFP 양성 세포의 비율의 그래프는 pBFP와 LNA 기능의 증가와 함께 농도 (A) 다음과 transfection에 표시됩니다. <img src="/files/ftp_upload/2355/2355table1.jpg" alt="표 1" /> 표 I. Transfection의 조건. <img src="/files/ftp_upload/2355/2355table2.jpg" alt="표 2" /> 표 II. 그림 1에서 사용되는 Transfection 조건.

<ol>
	<li>Corby-Harris, V. D. A., Watkins de Jong, L., Pakpour, N., Antonova, Y., Ziegler, R., Ramberg, F., Lewis, E. E., Brown, J. M., Luckhart, S. L., Riehle, 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=A+novel+strategy+for+controlling+malaria+transmission+in+the+mosquito+Anopheles+stephensi.">A novel strategy for controlling malaria transmission in the mosquito Anopheles stephensi.</a> PLOS Pathogens. , (2010).</li><li>Braasch, D. A., Corey, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locked+nucleic+acid+(LNA):+fine-tuning+the+recognition+of+DNA+and+RNA.">Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA.</a> Chem Biol. 8, 1-7 (2001).</li><li>Andrieu-Soler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transmission+of+targeted+gene+modification+using+single-stranded+oligonucleotides+with+flanking+LNAs.">Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.</a> Nucleic Acids Res. 33, 3733-3742 (2005).</li><li>Parekh-Olmedo, H., Drury, M., Kmiec, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+nucleotide+exchange+in+Saccharomyces+cerevisiae+directed+by+short+oligonucleotides+containing+locked+nucleic+acids.">Targeted nucleotide exchange in Saccharomyces cerevisiae directed by short oligonucleotides containing locked nucleic acids.</a> Chem Biol. 9, 1073-1084 (2002).</li><li>Rhee, W. J., Santangelo, P. J., Jo, H., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+accessibility+and+signal+specificity+in+live-cell+detection+of+BMP-4+mRNA+using+molecular+beacons.">Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons.</a> Nucleic Acids Res. 36, e30-e30 (2008).</li><li>Marras, S. A., Kramer, F. R., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+SNPs+with+molecular+beacons.">Genotyping SNPs with molecular beacons.</a> Methods Mol Biol. 212, 111-128 (2003).</li><li>Tyagi, S., Bratu, D. P., Kramer, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+molecular+beacons+for+allele+discrimination.">Multicolor molecular beacons for allele discrimination.</a> Nat Biotechnol. 16, 49-53 (1998).</li><li>Blandin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+genetic+basis+of+resistance+to+malaria+parasites+in+Anopheles+gambiae.">Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae.</a> Science. 326, 147-150 (2009).</li><li>Niare, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+loci+affecting+resistance+to+human+malaria+parasites+in+a+West+African+mosquito+vector+population.">Genetic loci affecting resistance to human malaria parasites in a West African mosquito vector population.</a> Science. 298, 213-216 (2002).</li><li>Riehle, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+malaria+infection+in+Anopheles+gambiae+is+regulated+by+a+single+genomic+control+region.">Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region.</a> Science. 312, 577-579 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기 A.로 LNA 기능의 transfection 다음 episomal 유전자 대상에서 단일 염기의 목표 전환에 대한 체외 방법 및 증거를 제공 stephensi 및 A. gambiae 세포. LNA - ON - 중재 유전자 변환은 사이트 특정 DNA 수리 대응의 유도가 필요합니다, 우리의 데이터는 모기 세포가 특정 사이트 돌연변이 유발이 메커니즘을 지원할 수 있음을 나타냅니다. 방법 여기서 제시가 episomal 대상의 변환...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Effectene transfection reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
<td>&nbsp;</td>
<tr>
<td>GFP control plasmid (pcDNA6.2-GW/EmGFP-miR-neg)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K4935-00</td>
<td>&nbsp;</td>
<tr>
<td>BFP plasmid (pcDNA5/FRT/TO)</td>
<td>&nbsp;</td>
<td>Gift from Dr. Concordet3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LNA-ONs </td>
<td>&nbsp;</td>
<td>Gift from Dr. Concordet3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MEM, Earle's w/ glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11095</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum (FCS)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Schneider&#x2019;s Drosophila medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11730</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140</td>
<td>&nbsp;</td>
<tr>
<td>10% D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5767</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin antibiotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transfection and mutagenesis of target genes in mosquito cells by locked nucleic acid-modified oligonucleotides

Plasmodium의 기생충, 말라리아의 원인이되는 대리인은 만 250 새로운 감염이 매년 발생하는 감염 아노 펠 레스 모기의 물린 통해 전송됩니다. 연구의 수십에도 불구하고, 말라리아에 대한 백신도 새로운 제어 전략의 필요성을 강조 아직 없습니다. 한 혁신적인 접근은 효과적으로 말라리아 기생충 전송을 제어하는​​ 유전자 조작 모기를 사용하는 것입니다. 대상 돌연변이 유발을 통해 모기의 세포 신호 전달 경로의 고의적인 변경은, 기생충 개발 1 조절 발견되었습니다. 이러한 연구에서, 우리는 변화를위한 잠재적인 유전자 목표를 식별하기 시작할 수 있습니다. 타겟 돌연변이 유발은 전통적으로 대상 유전자와 대형 DNA 분자 사이의 동종 재조합에 의존하고 있습니다. 그러나, 안정적으로 변형 세포 라인의 생성 등 복잡한 DNA 분자의 건설 및 사용은 비용, 시간 소요 종종 비효율적이다. 따라서, 고정 핵산 - 수정 oligonucleotides (LNA 기능)을 사용하여 전략 episomal 및 염색체 DNA의 유전자 타겟 (2 검토)에 인공 단일 염기 대체를 소개하는 유용한 대안을 제공합니다. LNA - ON - 중재 대상으로 돌연변이 유발은 효모와 생쥐 3,4 모두 교양 세포에 대한 관심의 유전자에 변이 점을 소개하기 위해 사용되었습니다. 우리는 LNA 기능이 아노 펠 레스 gambiae과 아노 펠 레스 stephensi 세포 모두에서 녹색 형광 단백질 (GFP) 표현에 파란색 형광 단백질 (BFP) 표현에서 스위치로 이어지는 transfected episomal 대상에서 하나의 염기 변화를 소개하는 데 사용할 수있는 것을 여기에 표시 . 이 변환은 모기 세포 대상 유전자의 돌연변이 유발 효과가 LNA 기능에 의해 중재이 기술은 체외에서 생체내의 염색체 돌연변이 유발 타겟에 적용 될 수있는 제안이 될 수 있다고 처음으로 보여줍니다.

Watch this Scientific Journal Video about Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides at JoVE.com

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Oligonucleotides은 특히 모두 transfected 대상 유전자의 단일 염기 대체 사이트로 사용할 수 있습니다<em&gt; 아노 펠 레스 gambiae</em&gt;과<em&gt; 아노 펠 레스 stephensi</em&gt; 세포.

옥내 핵산 - 수정 Oligonucleotides에 의해 Transfection 및 모기 세포 대상 유전자의 돌연변이 유발

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

transfection-mutagenesis-target-genes-mosquito-cells-locked-nucleic

Research

JoVE Journal

Immunology and Infection

10.3K 조회수.  University of California, Davis. Oligonucleotides은 특히 모두 transfected 대상 유전자의 단일 염기 대체 사이트로 사용할 수 있습니다 아노 펠 레스 gambiae과 아노 펠 레스 stephensi 세포.

비디오: Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Kaposi의 육종 - 관련 Herpesvirus (KSHV)에 의해 유도된 유전자를 식별 Dysregulated

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

연구

면역학 및 감염병학

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

항원 특정 VIVO에서</emCFSE 라벨 대상 세포를 사용하여> 죽이는 분석

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

세포 성숙없이 돌기 세포의 효율적인 Transfection을 위해 최적화된 프로토콜

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

에 셀 운명을 따라<em&gt; E. 대장균</em&gt; 파지 람다에 의해 감염 후

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

특정 유전자의 Overexpression 또는 최저 후 라모스의 B 세포에서 체세포 Hypermutation을 평가

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

옥내 핵산 흐름 Cytometry - 형광 현장에서 하이브 리다이 제이션 (LNA 흐름 - 물고기) : 세균의 소형 RNA 감지하는 방법

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

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.

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 ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

에 알긴산 생산과 관련된 새로운 유전자의 식별<em&gt; 녹농균</em&gt; 사용 미니<em&gt; himar1</em&gt; 마리너 트랜스포존 매개 돌연변이 유발

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.