우리는 결실 토론 및 기술 지원을위한 실험실의 구성원 감사합니다. 이 비디오 프레 젠 테이션은 연구 베티 Pathobiology학과에 대한 준회원 딘, 베티 건강 과학 센터, 오클라호마 주립 대학에 의해 지원되었다. 연구는 정부의 드 Ciencia 전자 Innovación, 스페인 (프로젝트 BFU2008-01244/BMC), JF에 CSIC 교내 프로젝트 PA1002451에 의해 후원 되었음 : 월터 R. Sitlington는 KMK, CVHS 2,009 RAC 부여 OAES 음식 동물 연구를위한 의자를 둘러 싸인 동물 건강 자금과 USDA, 국가 연구 이니셔티브 경쟁 그랜트 번호 2007-04613.

1. dsRNA의 생성. <ol><li> dsRNA의 시험 관내 전사 및 합성 (예를 들어, Dermacentor에 대한 oligonucleotide primers subolesin에게 D8AAT75를 사용 variabilis에 대한 T7 프로 모터 시퀀스를 포함하는 oligonucleotide primers를 합성 : 5&#39; - TAATACGACTCACTATAGGGTACTGACTGGGATCCCCTGCACAGT - 3 &#39;와 D8DVT73 : 5&#39; - TAATACGACTCACTATAGGGTACTCGAGCTTGGTGGAAAGGACG - 3&#39;). </li><li> RT - PCR은 각 oligonucleotide 뇌관과 틱 총 RNA 중 1-10 NG 10 pmol를 사용하여 대상 유전자를 증폭. </li><li> PCR 제품을 정화. </li><li> 정화 PCR 제품의 8 μL를 사용하여 dsRNA를 합성. </li><li> 분석법에 의해 dsRNA를 계량. </li></ol> 2. dsRNA와 진드기의 사출. 2.1. 주사 진드기의 준비. <ol><li> 첫째, 50 ML 일회용 원심 분리기 튜브의 각 솔루션에서 그들을 흔들어 진드기를 유지하기 위해 튜브 톱을 통해 고급 메쉬 와이어 화면을 통해 솔루션을 decanting하여 솔루션의 일련의 진드기 씻으십시오. 세탁 진드기에 대한 솔루션의 순서는 수돗물, 3 % 과산화수소, 증류수, 70 % 에탄올과 증류수로 두 번 더 씻는다 두 씻는다입니다. </li><li> 종이 타올에있는 진드기를 얼룩 건조. </li><li> 실험에 따라, 20 50 그룹으로 진드기를 계산, 실험 그룹의 번호로 단단히 장착되어 뚜껑과 라벨을 가진 1.25 온스 플라스틱 컵의 각 그룹에서 진드기를 놓습니다. </li></ol> 2.2. 사출 팀 틱. RNAi 팀은 세 사람으로 구성되어 있습니다 : (1) 붉은 치과 왁스의 시트에 부착된 이중 끈적끈적한 테이프의 각 체크를 위치 한 사람이, 후에 진드기를 모니터링 진드기 (3) 한 사람을 주사 (2) 한 사람 사출 그들을 활성화하는 진드기에 CO 2를 통기하여 실험 그룹의 번호로 표시 컵에 사는 진드기로 계산됩니다. 모든 팀 구성원은 일회용 장갑을 착용해야합니다. 2.3. 주사 진드기의 배치. <ol><li> 더몬트 미세 집게를 사용하여 체크를 캡처하여 붉은 치과 왁스의 3 &quot;X 6&quot;시트에 부착된 스티커를 두 번 테이프에 복부 쪽을 놓으십시오. 진드기는 밀접한 5 진드기의 그룹에서 함께 위치하고 있습니다. </li><li> 모든 5 진드기의 mouthparts 더들을 구속하기 위해 이상의 마스킹 테이프의 작은 스트립을 플레이스하지만 사출 과정이 체크 주입기 (그림 1)에 의해 관찰 수 있도록 노출 신체의 대부분을 떠나있는 동안. </li></ol> 2.4. 진드기의 사출. <ol><li> 진드기는 외골격의 복부 표면의 오른쪽 하단 사분면에 주입됩니다. </li><li> 첫째, 피어스 ½ &quot;, 29 게이지 바늘 (그림 2A)가 장착되어 Monoject의 인슐린 주사기를 사용하여 외골격에 구멍. </li><li> 45 ° beveled 포인트와 1 인치 33 게이지 바늘로 맞춤 제작 해밀턴 주사기를 사용하여 - (μL 당 5 X 10 11 분자 5 × 10 10) (그림 2B) dsRNA 솔루션의 0.2-0.5 μL 즉시 개나 주사 . 바늘은 dsRNA의 배치 및 유지를 확보하기 위해 틱 캐비티에 잘 배치해야합니다. 어떤 액체가 주입 사이트 (그림 2C)에서 탈출 가능성이 높습니다. 관리는 이상 hemolymph의 손실을 일으킬 것입니다 진드기를 주입하고 진드기의 죽음을 일으킬 수하지 않도록해야합니다. </li><li> 각 실험 그룹의 주사를 완료한 후 해밀턴 주사기를 청소합니다. 또 다른 실험 그룹에 대한 사용하기 전에. 폐기물 컨테이너에 추방, 15 번 반복 후 3 % 과산화수소를 포함하는 비커에서 먼저 주사기를 작성합니다. 폐기물 컨테이너에 추방, 15 번 반복 후 멸균 물을 들어있는 비커에서 주사기를 작성합니다. 벤트 경우, 플런저가 원활하게 이동하고 진드기의 주입에 필요한 부드러운 터치에 응답하지 않습니다, 때문에 해밀턴 주사기의 플런저를 구부 않도록주의를 가져가라. </li></ol> 2.5. 주사 후 진드기의 치료. <ol><li> 좋은 포셉 즉시 더블 끈적끈적한 테이프에서 주입 체크를 들고 플라스틱 복구 컨테이너 (약 6 &quot;X 6&quot;과 진드기의 탈출을 방지하기 위해 마스킹 테이프와 함께 반지를 낀)에 놓으십시오. 진드기는 주사 후 간략하게 운영 중지 상태가됩니다하지만 곧 접시 주위에 크롤 링을 시작합니다. </li><li> 입김을 즉시 진드기를 활성화에 도움이되는 복구 컨테이너에서 그들을 배치 후 진드기에 2 CO. 진드기가 크롤 링하고 활성화되면, 사출 상처가 빠르게 치유하고 그들이 가장 가능성이 살아남을 것입니다. </li><li> 각 실험 그룹의 숫자에 따라 진드기를 카운트하고 단단하게 장착되어 뚜껑이있는 표시 플라스틱 컵에서 그들을 놓으십시오. 대체 진드기는 언제는 다음 실험 그룹을 주입하기 전에 죽었를 대체하는 주입해야합니다. </li></ol> 2.6. 개최 틱. <ol><li> 습도 챔버에있는 진드기 (12hr 밝은 장소 : 22-25 12 시간 어두운 photoperiod을 ° C 및 95 %의 상대 Humidity) 및 1 일 개최. </li><li> 틱 수유 세포에 넣어 거려, 실험 그룹 당 하나는 양에 접착시키고 uninjected 남성 또는 여성 진드기 (주입 아니었 중에서 성별)의 동일한 번호와 피드 수 있습니다. 충만 피드 여성 진드기는 10 먹이의 일 때 제어 여성 후 양에서 제거됩니다 이들은 호스트 수집 무게 아르를 떨어뜨리고있다. </li><li> 종이팩에 진드기를 삽입하고, 산란의 완성까지 습도 챔버하다. 가중치 그룹의 모든 진드기에 의해 생산되는 계란 질량에 의해 산란을 평가해보십시오. </li></ol> 2.7. RNAi 후 틱 표현형 분석. <ol><li> , 틱 중량, 산란 및 달걀 다산 살아남은 진드기의 수를 결정하여 수유 후 표현형 틱 평가. 그러나, 다른 분석은 연구의 대상 유전자와 목적에 따라 수행할 수 있습니다. </li></ol> 3. RT - PCR로 유전자 입을를 확인 분석. <ol><li> 수유 후 제어 주입 및 dsRNA - 주입 그룹에서 개별 진드기의 침샘과 배짱을 해부하다. </li><li> 개별 조직 샘플에서 총 RNA를 추출합니다. </li><li> 실시간 RT - PCR에 의해 개별 조직의 목표 유전자 성적표를 분석하고 틱 16에 대한 RNA 수준을 정상화는 genNorm 방법 (생물 래드 iQ5 일반용, 버전 2.0에 의해 구현으로 ddCT 방법)을 사용하여 rRNA. </li><li> 한 amplicon이 형성되도록하는 반응의 끝에 그 해리 곡선을 실행하여 모든 시료에 대해 동일한 온도 범위에서 일관되게 amplicons의 변성. </li><li> mRNA 수준 (표준 CT 값) 제어 주입 및 dsRNA - 주입 진드기는 학생`S T 테스트 (P는 = 0.05)를 사용하여 사이를 비교합니다. </li></ol> 4. 대표 결과 : 프로토콜은 여기에 (표 1) 다양한 ixodid 틱 수종에 RNAi을 위해 저희 실험실에서 사용되었습니다 설명했다. 진드기에 주입 dsRNA의 금액은 체크의 크기에 따라 다릅니다; 큰 틱 종 더 큰 볼륨을 수용할 수 있습니다. 부정적인 제어 진드기는 관련이없는 dsRNA로 주입해야합니다. 이러한 subolesin 14-19,22-25,27-32,34 및 베타 굴지 20,21 등 여러 dsRNAs는 긍정적인 컨트롤로 사용할 수 있습니다. 그것이 혼합 dsRNA 솔루션을 피하기 위해 트리 트먼트를 사이에 주사기를 씻어하는 것이 중요합니다. 프로토콜이 제대로되면 5 % 미만의 사망률은 24 시간 후 주사 절차에서 얻을 수 있습니다. 진드기의 유전자 최저 후 전형적인 표현형 보호하는 항원을 체크를 위해 화면을 위해 dsRNA의 수영장과 함께 주입 진드기의 패널​​로 그림 3에 표시됩니다. <table border="1"><tbody><tr><td> 종 틱 </td><td> dsRNA는 주입 </td><td> 참조 </td></tr><tr><td> Ixodes scapularis의 </td><td> cDNA 도서관, subolesin, 굴지, nucleotidase, NF - KB, akirin </td><td> 21, 22, 29, 30 </td></tr><tr><td> Dermacentor variabilis </td><td> subolesin, GST, ubiquitin, vATPase, selenoproteins M과 W2a, 조혈 줄기 / 전구 세포 굴지 Proteasome 26S subunit, ferritin1, varisin, akirin, 단백질과 같은 </td><td> 15, 19, 22, 24, 26, 30-32 </td></tr><tr><td> Dermacentor marginatus </td><td> subolesin </td><td> 22 </td></tr><tr><td> Amblyomma americanum </td><td> cDNA 도서관, subolesin, akirin </td><td> 17, 22, 30 </td></tr><tr><td> Amblyomma hebraeum </td><td> subolesin, voraxin </td><td> 28 </td></tr><tr><td> Rhipicephalus의 sanguineus </td><td> Rs86, subolesin </td><td> 22, 23 </td></tr><tr><td> Rhipicephalus microplus </td><td> GST, ubiquitin, selenoprotein, Bm86, Bm91, subolesin, GI, GIII, EF1a, flagelliform 실크 단백질, 폰 Willebrand 인자 </td><td> 16, 18, 25, 27 </td></tr><tr><td> Rhipicephalus의 annulatus </td><td> ubiquitin, subolesin, EF1a, GIII </td><td> 16 </td></tr></tbody></table> 표 1. RNAi 프로토콜이 사용되었습니다있는 수종으로 톡톡 두드려. <img src="/files/ftp_upload/2474/2474fig1.jpg" alt="그림 1" /> 그림 1. 빨간 치과 왁스의 시트을 준수 더블 스티커 테이프에 진드기의 배치, 최대 복부 측면. 진드기는 마스킹 테이프의 작은 스트립이 주입기는 주입 중에 진드기의 사체를 관찰 수 있도록하면서 추가로 진드기를 확보하기 위해 mouthparts 위에 배치되는 후, 5 그룹에 배치됩니다. <img src="/files/ftp_upload/2474/2474fig2.jpg" alt="그림 2" /> 그림 2. 분사 절차는 인슐린 싸이로 틱 외골격의 (A) 오른쪽 아래 사분면 피어싱을 포함ringe는 주사 사이트를 만들기 위해 29 게이지 바늘로 장착되어, (C) 대부분은 / 틱 hemolymph의 일부 누수가 발생합니다 33 게이지 바늘로 해밀턴 주사기를 사용하여이 사이트에서 dsRNA의 (B) 즉시 분사 유체. <img src="/files/ftp_upload/2474/2474fig3.jpg" alt="그림 3" /> 그림 3. RNAi가 Amblyomma americanum에서 진드기 항원에 대한 보호 화면으로 사용된있는 체크 여섯 그룹의 패널. 긍정적인 subolesin RNAi 제어 및 부정 무관한 dsRNA 제어와 비교하면 진드기의 phenotypic 변화를 볼 수 있습니다. 본 실험에서는 각 그룹의 틱 사망률, 무게, 그리고 산란에 대한 RNAi의 효과는 통계적으로 분석했다.

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Biol. 12, 299-305 (2003).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+for+the+study+and+genetic+manipulation+of+ticks.">RNA interference for the study and genetic manipulation of ticks.</a> Trends Parasitol. 23, 427-433 (2007).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+studies+of+tick+cells+in+response+to+infection+with+the+cattle+pathogen,+Anaplasma+marginale.">Functional genomic studies of tick cells in response to infection with the cattle pathogen, Anaplasma marginale.</a> Genomics. 90, 712-722 (2007).</li><li>Almaz&#228;N, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+Rhipicephalus+(Boophilus)+microplus+candidate+protective+antigens+for+the+control+of+cattle+tick+infestations.">Identification and characterization of Rhipicephalus (Boophilus) microplus candidate protective antigens for the control of cattle tick infestations.</a> Parasitol. Res. 106, 471-479 (2010).</li><li>de la Fuente, J. <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+protective+antigens+by+RNA+interference+for+control+of+the+lone+star+tick,+Amblyomma+americanum.">Identification of protective antigens by RNA interference for control of the lone star tick, Amblyomma americanum.</a> Vaccine. 28, 1786-1795 (2010).</li><li>Zivkovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+genes+in+salivary+glands+of+male+Rhipicephalus+(Boophilus)+microplus+in+response+to+infection+with+Anaplasma+marginale.">Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus) microplus in response to infection with Anaplasma marginale.</a> BMC Genomics. 11, 186-186 (2010).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+tick+infections+with+Anaplasma+marginale+and+A.+phagocytophilum+by+targeting+the+tick+protective+antigen+subolesin.">Reduction of tick infections with Anaplasma marginale and A. phagocytophilum by targeting the tick protective antigen subolesin.</a> Parasitol. Res. 100, 85-91 (2006).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Ixodes+scapularis+anticoagulation+by+using+RNA+interference.">Disruption of Ixodes scapularis anticoagulation by using RNA interference.</a> Proc. Natl. Acad. Sci U.S.A. 101, 1141-1146 (2004).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screening+in+ticks+for+identification+of+protective+antigens.">RNA interference screening in ticks for identification of protective antigens.</a> Parasitol. Res. 96, 137-141 (2005).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tick+protective+antigen,+4D8,+is+a+conserved+protein+involved+in+modulation+of+tick+blood+ingestion+and+reproduction.">The tick protective antigen, 4D8, is a conserved protein involved in modulation of tick blood ingestion and reproduction.</a> Vaccine. 24, 4082-4095 (2006).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+effect+of+silencing+the+expression+of+tick+protective+antigens+4D8+and+Rs86+in+Rhipicephalus+sanguineus+by+RNA+interference.">Synergistic effect of silencing the expression of tick protective antigens 4D8 and Rs86 in Rhipicephalus sanguineus by RNA interference.</a> Parasitol. Res. 99, 108-113 (2006).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocidal+control+of+ticks+by+silencing+of+a+single+gene+by+RNA+interference.">Autocidal control of ticks by silencing of a single gene by RNA interference.</a> Biochem. Biophys. Res. Commun. 344, 332-338 (2006).</li><li>Nijhof, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bm86,+Bm91+and+subolesin,+in+the+silencing+of+the+tick+protective+antigens.">Bm86, Bm91 and subolesin, in the silencing of the tick protective antigens.</a> Int. J. Parasitol. 37, 653-662 (2007).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+the+defensin,+varisin,+in+male+Dermacentor+variabilis+by+RNA+interference+results+in+reduced+Anaplasma+marginale+infections.">Silencing of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections.</a> Exp. Appl. Acarol. 46, 17-28 (2008).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+the+role+of+tick+subolesin+in+gene+expression.">Evidence of the role of tick subolesin in gene expression.</a> BMC Genomics. 9, 372-372 (2008).</li><li>Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+RNA+interference+of+the+subolesin+and+voraxin+genes+in+male+Amblyomma+hebraeum+(Acari:+Ixodidae)+on+female+engorgement+and+oviposition.">The impact of RNA interference of the subolesin and voraxin genes in male Amblyomma hebraeum (Acari: Ixodidae) on female engorgement and oviposition.</a> Exp. Appl. Acarol. 47, 71-86 (2009).</li><li>Galindo, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+subolesin+is+an+ortholog+of+the+akirins+described+in+insects+and+vertebrates.">Tick subolesin is an ortholog of the akirins described in insects and vertebrates.</a> Dev. Comp. Immunol. 33, 612-617 (2009).</li><li>Canales, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+and+immunogenicity+of+the+mosquito+ortholog+of+the+tick+protective+antigen,+subolesin.">Conservation and immunogenicity of the mosquito ortholog of the tick protective antigen, subolesin.</a> Parasitol. Res. 105, 97-111 (2009).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+genes+involved+in+Anaplasma+marginale-tick+interactions+affects+the+pathogen+developmental+cycle+in+Dermacentor+variabilis.">Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis.</a> BMC Dev. Biol. 9, 42-42 (2009).</li><li>Zivkovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subolesin+expression+in+response+to+pathogen+infection+in+ticks.">Subolesin expression in response to pathogen infection in ticks.</a> BMC Immunol. 11, 7-7 (2010).</li><li>Karim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+tool:+gene+silencing+in+Ixodes+scapularis+eggs+and+nymphs+by+electroporated+dsRNA.">Functional genomics tool: gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA.</a> BMC Biotechnol. 10, 1-1 (2010).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transovarial+silencing+of+the+subolesin+gene+in+three-host+ixodid+tick+species+after+injection+of+replete+females+with+subolesin+dsRNA.">Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA.</a> Parasitol. Res. 100, 1411-1415 (2007).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+tick-pathogen+interface+for+novel+control+strategies.">Targeting the tick-pathogen interface for novel control strategies.</a> Front. Biosci. 13, 6947-6956 (2008).</li><li>Kurscheid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+tick+RNAi+pathway+by+comparative+genomics+and+reverse+genetics+screen+of+targets+with+known+loss-of-function+phenotypes+in+Drosophila.">Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila.</a> BMC Mol. Biol. 10, 26-26 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

다른 방법은 진드기 14 33 RNAi에 대해 설명하고 있지만, dsRNA의 주입은 가장 널리 unfed (표 1)과 먹이 진드기 16,25,34 모두에서 사용됩니다 여기에 설명되어 있습니다. RNAi는 틱 유전자 기능 연구, 경마 병원체 인터페이스의 특성 및 심사와 진드기 항원 보호 14,35의 특성화를위한 유용한 도구로 표시되었습니다. 특히, RNAi가 나 35 기능 분석을위한 가장 중요한 도구가되고 있습니다. Methodologically, RNAi 가능성이 높습 개인의 다수의 유전자 최저를 허용할 수 있습니다보다 효율적인 방법으로 진화합니다. 진드기의 dsRNA 유도된 RNAi의 메커니즘은 더 나은 이해하고이 수종 35,36에서이 유전자 접근 방식의 활용에 기여하기 위해 정제해야합니다. 진드기의 RNAi의 오프 대상 효과의 정도도 충분히 14,27 연설을해야 할 중요한 질문이다. 마지막으로, RNAi는 대부분 틱 유전자 규정 및 시스템 생물학의 연구에 종합적인 기여를 제공하고 경마 병원체 인터페이스와 틱 infestations과 틱 통한 병원균의 전송을 제어하는​​ 백신의 개발에 영향을 미칠 수 있습니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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>Access RT-PCR system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A1250</td>
<td>&nbsp;</td>
<tr>
<td>Purelink PCR purification kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K3100-02</td>
<td>&nbsp;</td>
<tr>
<td>Megascript RNAi kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1626M</td>
<td>&nbsp;</td>
<tr>
<td>Red dental wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72674</td>
<td>&nbsp;</td>
<tr>
<td>Plastic cups, 1.25 oz and lids</td>
<td>&nbsp;</td>
<td>Solo Cup Company, Urbana Ill.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe</td>
<td>&nbsp;</td>
<td>Monoject</td>
<td>&nbsp;</td>
<td>Fitted with a &frac12;&#x201D;, 29 gauge needle</td>
</tr>
<tr>
<td>Hamilton syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>701SN,33/.375&#x201D;/45DGR</td>
<td>Custom made</td>
</tr>
<tr>
<td>TriReagent</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>93289</td>
<td>&nbsp;</td>
<tr>
<td>iScript One-Step RT-PCR Kit with SYBR Green</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>170-8892</td>
<td>&nbsp;</td>
<tr>
<td>Real-time PCR detection system</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>Several</td>
<td>Please refer to <a href="http://www.bio-rad.com/">http://www.bio-rad.com/</a></td>
</tr>		</tbody>
		</table>
		</div>

rna interference in ticks

진드기는 야생 국내 동물과 인간의 의무 hematophagous ectoparasites하고 있으며, 인간의 질병 1과 가장 중요한 벡터의 벡터는 가축 업계 세계 2에 영향을 미치는 것으로 모기의 두번째 세계로 간주됩니다. 진드기는 하위 클래스 Acari, 주문 Parasitiformes, 아목 Ixodida 분류하고 북극에서 열대 지역 3 전세계 배포됩니다. 틱 infestations을 제어하기위한 노력에도 불구하고, 이러한 ectoparasites는 4,5 인간과 동물의 건강에 심각한 문제가 남아 있습니다. RNA 간섭 (RNAi) 6 신진 대사 경로에 유전자 기능 또는 효과를 결정하기 위해 유전자 발현의 중단과 관련된 핵산 기반 반대 유전자 접근 방식이다. 작은 방해 RNAS (siRNAs)는 dsRNA 트리거 7-9과 같은 순서를 포함하는 세포질 mRNAs의 강력한 일련의 특정 저하를 두 번 좌초 RNA (dsRNA)과 결과에 의해 시작되는 RNAi 경로의 효과기 분자 수 있습니다. dsRNA에 의해 시작 포스트 transcriptional 유전자 입을 메커니즘은 모든 eukaryotes에서 발견되었습니다 것은 지금까지 공부하고, RNAi는 급속하게 기능 게놈 연구와 다른 응용 프로그램 10 도구로 생물의 다양한 개발되었습니다. RNAi는 유전자 조작에 대한 대체 방법을 사용할 수 없거나 신뢰할 수 5,11 아르 진드기 및 다른 유기체에서 가장 널리 사용되는 유전자 입을 기술되었다. 진드기의 유전적 특성은 RNAi 12,13의 최근 응용까지 제한되었습니다. RNAi를 사용할 것으로 짧은 시간에, 그것은 틱 유전자 기능을 연구를위한 유용한 도구로, 경마 병원체 인터페이스의 특성 및 심사와 진드기 항원 보호 14 특성 것으로 판명되었습니다. 여기에, unfed 거려에 dsRNA의 주입을 통해 RNAi에 대한 방법을 설명합니다. 그것은이 실험 방법에서 얻은 지식은 기본적인 생물 학적 시스템의 이해와 틱 infestations을 제어하고 체크 통한 병원균 15-19의 전송을 방지하기 위해 백신의 발전에 현저하게 기여할 가능성이 높습니다.

Watch this Scientific Journal Video about RNA Interference in Ticks at JoVE.com

RNA Interference in Ticks

unfed 거려에 dsRNA의 주입에 의한 RNA 간섭 (RNAi)의 방법을 설명합니다. RNAi는 유전자 조작의 다른 방법의 사용이 제한되었습니다 진드기에서 가장 널리 사용되는 유전자 입을 기술입니다.

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 ...

Research

JoVE Journal

Immunology and Infection

18.0K 조회수.  Oklahoma State University. unfed 거려에 dsRNA의 주입에 의한 RNA 간섭 (RNAi)의 방법을 설명합니다. RNAi는 유전자 조작의 다른 방법의 사용이 제한되었습니다 진드기에서 가장 널리 사용되는 유전자 입을 기술입니다.

비디오: RNA Interference in Ticks

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

siRNA 전달을위한 셀 타입의 특정 안티 HIV gp120 aptamers의 개발

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

연구

면역학 및 감염병학

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 ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

라임 병 연구의 변속기 및 Xenodiagnosis에 대한 동물에 진드기의 먹이

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

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 ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

뉴 클레오 캡시드 소둔 매개 전기 영동 (NAME) 분석은 HIV-1 뉴 클레오 캡시드 억제제의 신속한 식별을 할 수 있습니다

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

유도 발현 시스템을 적용하면 세포 내 신호와 세균 독성 요인의 간섭을 연구하기 위해

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA 정제는 세포 내에서 재배<em&gt; 리스테리아</em&gt; 식세포 세포에

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

차세대 일렉트로 차 인간 Th17 세포 작은 RNA 형질

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

코딩 및 비 코딩 RNA 클래스의 식별 표시 돼지에서 전 혈

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

이중 가닥 RNA와 패턴 인식 수용 체 부정적인 감 RNA 바이러스 감염에의 confocal 영상

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...