우리는 데이비드 O'Brochta 박사와 곤충 유전 기술 조정 연구 네트워크의 모든 구성원들이 유전 기술의 구현에 대해 우리와 다른 사람들에게 제공하는 도움과 훈련을 주셔서 감사합니다. 우리는 특히 컬렉스 배아를 주입하고 부화할 수 있도록 미세 조작 프로토콜을 최적화한 찬나 알루비헤어에게 감사드립니다. 또한 Meuti 연구소에서 일하는 학부 생인 데반테 시몬스와 조셉 우르소(Joseph Urso)와 트랜스제닉 모기를 돌보고 선별하는 데 도움을 준 데반테 시몬스와 조셉 우르소, ITF의 조라 엘름카미(Zora Elmkami)가 모기를 사육하고 주사를 준비하는 데 도움을 준 것에 대해 감사드립니다. 이 작품은 MEM에 제공 OSU에 감염증 연구소에서 학제 간 씨앗 보조금에 의해 지원되었다.

<ol><li>Hamer, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culex+pipiens+%28Diptera%3A+Culicidae%29%3A+a+bridge+vector+of+West+Nile+virus+to+humans%5BTitle%5D)+AND+%22Journal+of+Medical+Entomology%22%5BJournal%5D)">Culex pipiens (Diptera: Culicidae): a bridge vector of West Nile virus to humans.</a> Journal of Medical Entomology. 45 (1), 125-128 (2008).</li>
<li>Bailey, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+St.+Louis+encephalitis+virus+from+overwintering+Culex+pipiens+mosquitoes%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Isolation of St. Louis encephalitis virus from overwintering Culex pipiens mosquitoes.</a> Science. 199 (4335), 1346-1349 (1978).</li>
<li>Sabry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+realistic+index+of+experimental+transmission+efficiency+for+Bancroftian+filariasis%5BTitle%5D)+AND+%22The+Journal+of+Tropical+Medicine+and+Hygiene%22%5BJournal%5D)">A new realistic index of experimental transmission efficiency for Bancroftian filariasis.</a> The Journal of Tropical Medicine and Hygiene. 94 (4), 283-290 (1991).</li>
<li>Cancrini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aedes+albopictus+and+Culex+pipiens+implicated+as+natural+vectors+of+Dirofilaria+repens+in+central+Italy%5BTitle%5D)+AND+%22Journal+of+Medical+Entomology%22%5BJournal%5D)">Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy.</a> Journal of Medical Entomology. 44 (6), 1064-1066 (2007).</li>
<li>Wilton, D. P., Smith, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ovarian+diapause+in+three+geographic+strains+of+Culex+pipiens+%28Diptera%3A+Culicidae%29%5BTitle%5D)+AND+%22Journal+of+Medical+Entomology%22%5BJournal%5D)">Ovarian diapause in three geographic strains of Culex pipiens (Diptera: Culicidae).</a> Journal of Medical Entomology. 22 (5), 524-528 (1985).</li>
<li>Mattingly, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Culex+pipiens+complex%5BTitle%5D)+AND+%22Transactions+of+the+Royal+Entomological+Society+of+London%22%5BJournal%5D)">The Culex pipiens complex.</a> Transactions of the Royal Entomological Society of London. 102 (7), 331-342 (1951).</li>
<li>Eldridge, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+temperature+and+photoperiod+on+blood-feeding+and+ovarian+development+in+mosquitoes+of+the+Culex+pipiens+complex%5BTitle%5D)+AND+%22The+American+Journal+of+Tropical+Medicine+and+Hygiene%22%5BJournal%5D)">The effect of temperature and photoperiod on blood-feeding and ovarian development in mosquitoes of the Culex pipiens complex.</a> The American Journal of Tropical Medicine and Hygiene. 17 (1), 133-140 (1968).</li>
<li>Spielman, A., Wong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+control+of+ovarian+diapause+in+Culex+pipiens%5BTitle%5D)+AND+%22Annals+of+the+Entomological+Society+of+America%22%5BJournal%5D)">Environmental control of ovarian diapause in Culex pipiens.</a> Annals of the Entomological Society of America. 66 (4), 905-907 (1973).</li>
<li>Fritz, M. L., Walker, E. D., Miller, J. R., Severson, D. W., Dworkin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Divergent+host+preferences+of+above-and+below-ground+Culex+pipiens+mosquitoes+and+their+hybrid+offspring%5BTitle%5D)+AND+%22Medical+and+Veterinary+Entomology%22%5BJournal%5D)">Divergent host preferences of above-and below-ground Culex pipiens mosquitoes and their hybrid offspring.</a> Medical and Veterinary Entomology. 29 (2), 115-123 (2015).</li>
<li>Meola, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+temperature+and+humidity+on+embryonic+longevity+in+Aedes+aegypti%5BTitle%5D)+AND+%22Annals+of+the+Entomological+Society+of+America%22%5BJournal%5D)">The influence of temperature and humidity on embryonic longevity in Aedes aegypti.</a> Annals of the Entomological Society of America. 57 (4), 468-472 (1964).</li>
<li>Dong, S., Lin, J., Held, N. L., Clem, R. J., Passarelli, A. L., Franz, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heritable+CRISPR%2FCas9-mediated+genome+editing+in+the+yellow+fever+mosquito%2C+Aedes+aegypti%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Heritable CRISPR/Cas9-mediated genome editing in the yellow fever mosquito, Aedes aegypti.</a> PloS one. 10 (3), (2015).</li>
<li>Kyrou, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+CRISPR-Cas9+gene+drive+targeting+doublesex+causes+complete+population+suppression+in+caged+Anopheles+gambiae+mosquitoes%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.</a> Nature Biotechnology. 36 (11), 1062-1066 (2018).</li>
<li>Liu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Construction+of+an+efficient+genomic+editing+system+with+CRISPR%2FCas9+in+the+vector+mosquito+Aedes+albopictus%5BTitle%5D)+AND+%22Insect+Science%22%5BJournal%5D)">Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus.</a> Insect Science. 26 (6), 1045-1054 (2019).</li>
<li>Itokawa, K., Komagata, O., Kasai, S., Ogawa, K., Tomita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Testing+the+causality+between+CYP9M10+and+pyrethroid+resistance+using+the+TALEN+and+CRISPR%2FCas9+technologies%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies.</a> Scientific Reports. 6, 24652(2016).</li>
<li>Li, M., Li, T., Liu, N., Raban, R. R., Wang, X., Akbari, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+the+generation+of+heritable+germline+mutations+in+the+disease+vector+Culex+quinquefasciatus+using+clustered+regularly+interspaced+short+palindrome+repeats-associated+protein+9%5BTitle%5D)+AND+%22Insect+Molecular+Biology%22%5BJournal%5D)">Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats-associated protein 9.</a> Insect Molecular Biology. 29, 214-220 (2019).</li>
<li>Anderson, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR%2FCas9+gene+editing+in+the+West+Nile+Virus+vector%2C+Culex+quinquefasciatus+Say%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say.</a> PloS one. 14 (11), (2019).</li>
<li>Van der Oost, J., Jore, M. M., Westra, E. R., Lundgren, M., Brouns, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-based+adaptive+and+heritable+immunity+in+prokaryotes%5BTitle%5D)+AND+%22Trends+in+Biochemical+Sciences%22%5BJournal%5D)">CRISPR-based adaptive and heritable immunity in prokaryotes.</a> Trends in Biochemical Sciences. 34 (8), 401-407 (2009).</li>
<li>Heyer, W. D., Ehmsen, K. T., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+homologous+recombination+in+eukaryotes%5BTitle%5D)+AND+%22Annual+Review+of+Genetics%22%5BJournal%5D)">Regulation of homologous recombination in eukaryotes.</a> Annual Review of Genetics. 44, 113-139 (2010).</li>
<li>Monnerat, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anopheles+albitarsis+embryogenesis%3A+morphological+identification+of+major+events%5BTitle%5D)+AND+%22Memorias+do+Instituto+Oswaldo+Cruz%22%5BJournal%5D)">Anopheles albitarsis embryogenesis: morphological identification of major events.</a> Memorias do Instituto Oswaldo Cruz. 97 (4), 589-596 (2002).</li>
<li>Davis, C. W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparative+study+of+larval+embryogenesis+in+the+mosquito+Culex+fatigans+Wiedemann+%28Diptera%3A+Culicidae%29+and+the+sheep-fly+Lucilia+sericata+Meigen+%28Diptera%3A+Calliphoridae%29%5BTitle%5D)+AND+%22Australian+Journal+of+Zoology%22%5BJournal%5D)">A comparative study of larval embryogenesis in the mosquito Culex fatigans Wiedemann (Diptera: Culicidae) and the sheep-fly Lucilia sericata Meigen (Diptera: Calliphoridae).</a> Australian Journal of Zoology. 15 (3), 547-579 (1967).</li>
<li>Port, F., Chen, H. M., Lee, T., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+CRISPR%2FCas+tools+for+efficient+germline+and+somatic+genome+engineering+in+Drosophila%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.</a> Proceedings of the National Academy of Sciences. 111 (29), 2967-2976 (2014).</li>
<li>Kistler, K. E., Vosshall, L. B., Matthews, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+engineering+with+CRISPR-Cas9+in+the+mosquito+Aedes+aegypti%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti.</a> Cell Reports. 11 (1), 51-60 (2015).</li>
<li>Gokcezade, J., Sienski, G., Duchek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+CRISPR%2FCas9+plasmids+for+rapid+and+versatile+genome+editing+in+Drosophila%5BTitle%5D)+AND+%22G3%3A+Genes%2C+Genomes%2C+Genetics%22%5BJournal%5D)">Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila.</a> G3: Genes, Genomes, Genetics. 4 (11), 2279-2282 (2014).</li>
<li>Hwang, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+in+vivo+genome+editing+using+RNA-guided+nucleases%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Efficient in vivo genome editing using RNA-guided nucleases.</a> Nature Biotechnology. 31 (3), 227-229 (2013).</li>
<li>Basu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Silencing+of+end-joining+repair+for+efficient+site-specific+gene+insertion+after+TALEN%2FCRISPR+mutagenesis+in+Aedes+aegypti%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.</a> Proceedings of the National Academy of Sciences. 112 (13), 4038-4043 (2015).</li>
<li>Hammond, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+CRISPR-Cas9+gene+drive+system+targeting+female+reproduction+in+the+malaria+mosquito+vector+Anopheles+gambiae%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.</a> Nature Biotechnology. 34 (1), 78-83 (2016).</li>
<li>Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+homology-directed+human+genome+engineering+by+controlled+timing+of+CRISPR%2FCas9+delivery%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.</a> eLife. 3, 04766(2014).</li>
</ol>

RH는 곤충 유전자 변형 서비스를 제공하는 곤충 변환 시설에서 작동합니다.

이 프로토콜은 Culex 모기의 게놈으로 특정 돌연변이를 소개하는 방법을 제시하고 또한 그밖 모기의 게놈을 편집하기 위하여 이용될 수 있습니다. 이 프로토콜은 주입 물질을 준비하는 방법뿐만 아니라 모기가 계란을 낳도록 유도하는 방법과 그 계란을 준비하고 주입하는 방법에 대한 자세한 비디오 개요를 제공한다는 점에서 중요합니다. 우리는 또한 개별 뗏목에 계란을 낳고 따라서 원하?...

컬렉스 모기는 전 세계의 온대 및 열대 지역에 분포되어 있으며 웨스트 나일 바이러스1,세인트 루이스 뇌염2뿐만 아니라 개 심장벌레3 및 elephantiasis4를일으키는 필리알 선충을 포함한 여러 치명적인 바이러스를 전송합니다. Cx. quinquefasciatus, Cx. pipiens pipiens 및 Cx. pipiens 성추행을포함하는 Culex pipiens 복합체의 구성원은 생물학의 많은 양상에서 눈에 띄는 변화를 보여줍니다. 예를 들어, Cx. quinquefasciatus 및 Cx. pipiens 성추행은 겨울휴면5,6, Cx. pipiens 피피엔스는 짧은 일7,8에대한 응답으로 강력한 계절 반응을 표시하고 diapause를 입력 할 수 없습니다. 또한 Cx. pipiens 성추행은 더 인류애호가경향이 있는 반면 Cx. pipiens와 Cx. quinquefasciatus는 더 많은 동물 애호가6. 그러나 미국과 전 세계 많은 다른 장소에 걸쳐, Cx의 하이브리드로 질병 전염에 강한 영향을 미치는 이 종간 교배. pipiens pipiens 와 Cx. pipiens 성추행은 기회 피더이며 조류와 인간 을 모두 물린것입니다 9,따라서 웨스트 나일 바이러스에 대한 다리 벡터역할을. Culex 모기의 생물학의 이것과 그밖 매혹적인 양상을 공부하는 것은, 부분적으로, Culex 모기는 정지및 건조 저항하는 계란10을 생성하는 Aedes 모기 보다는 실험실에서 서있기 약간 더 어렵기 때문에 및 기능적인 분자 공구가 Culex 종을 위해 잘 개발되지 않기 때문에 방해되었습니다.
CRISPR/Cas9 게놈 편집은남부 집 모기, 컬렉스 퀸케파시아투스14,15,16을포함하여 몇 가지 중요한 모기 종11,12,13의생물학을 평가하는 데 사용되는 강력한 기술이다. 제니퍼 두나와 엠마뉴엘 샤펜티에가 개발한 이 기술은 세균 유래, CRISPR 관련 엔도니클로스(Cas 단백질; Van der Oost 외17)에의한 바이러스에 대한 자연적인 세균 방어를 이용합니다. 동물 배아에 주입할 때, Cas9 단백질은 적당한 가이드 RNA와 조합하여 게놈 내의 이중 좌초 된 휴식을 생성할 수 있습니다. 이것은 가장 빈번하게 게놈의 특정 지구에 엔도누엔리스 활성을 지시하는 가이드 RNA로 복합되는 Cas9 단백질을 사용하여 행해집니다. Cas9 단백질이 사이트별 이중 좌초 휴식을 만든 후, 세포 기계는 두 가지 메커니즘 중 하나를 사용하여 휴식을 복구하려고합니다. 첫 번째는 오류가 발생하기 쉽고 종종 비기능성 단백질을 초래할 수 있는 게놈의 아웃프레임 삽입 및 삭제를 생성하는 비동상종 결합(NHEJ)을 통해 두 끝을 결집시켜 녹아웃 돌연변이를 생성합니다. 또는 셀룰러 기계는 유사한 시퀀스를 찾아 파손을 올바르게 복구하여 동종 학 지향 수리(HDR)를 사용할 수 있습니다. 유사한 서열은 유기체 내의 제2 염색체에 의해 제공될 수 있다(리뷰18참조). 그러나, 수리된 서열이 원래 서열과 정확히 일치하면 Cas9 단백질은 DNA를 다시 절단할 수 있을 것이다. 대안적으로, 연구원은 또한 대체 수리 순서를 가진 표적 서열의 절단 사이트의 양쪽에 동종 서열을 포함하는 기증자 플라스미드를 포함할 수 있습니다 - 종종 형광 마커 단백질, 원래 유전자의 변형 된 버전, 또는 기타 수정 - 게놈에 복사 및 삽입 할 수 있습니다, 또는 "노크-인."
타이밍은 배아를 주입 할 때 중요하며, 특히 CRISPR / Cas9 게놈 편집을 사용하여 곤충의 돌연변이를 만들 때 특히 그렇습니다. 이는 Cas9 단백질과 gRNA가 배아가 동기화 상태에 있을 때만, 세포막이 형성되기 전에, 그리고 다중 핵이 배아 내에서 접근할 수 있을 때만 돌연변이를 생성하는 가장 큰 능력을 갖기 때문이다. 모기의 경우, 핵은 온도19에따라 oviposition 후 주변 ~ 2-4 시간 동안 도달하므로 성공적인 미세 주입이 이 시간 전에 발생해야합니다. 추가적으로, Cas9 단백질은 주입에서 유래된 개별이 세포의 모자이크를 포함할 것이라는 점을, 몇몇은 원하는 돌연변이를 가진, 그리고 그 외는 접근할 수 있는 어떤 핵 DNA든지 자를 것입니다. 이러한 돌연변이가 성공적으로 상속되기 위해서는 Cas9 단백질이 미래의 계란과 정자를 초래할 생식선에 있는 DNA를 잘라야합니다. 생식선에서 돌연변이가 생성되도록 하기 위해 곤충 생식선의 선조인 배아 내극 세포의 위치에 가까운 모든 물질을 주입하는 것이 가장 좋습니다. 극 세포는 Culex 배아20의후방 끝 근처에 위치한다. 배아를 주입하는 것 외에도 원하는 돌연변이를 감지하기 위해 자손을 횡단하고 선별하기 위한 신중한 계획을 개발하는 것이 필수적입니다.
이 프로토콜은 gRNA를 생성하고 주입 믹스를 준비하기 위해 Cas9 단백질로 복합하는 방법뿐만 아니라, 쿨렉스 피피엔스의 여성 모기가 계란을 낳는 방법과 CRISPR / Cas9 매개 게놈 편집을 위해 그 계란을 준비하고 주입하는 방법을 설명합니다. 추가적으로, 우리는 원하는 돌연변이가 얻어졌다는 것을 확인하기 위하여 주입된 태아 및 그들의 자손을 후면, 교차 및 스크린하는 방법을 기술합니다. 이 프로토콜을 사용하여, 우리는 컬렉스 피피엔스의벅아이 균주에서 관심 있는 유전자, 사이클에대한 null 돌연변이를 생성했습니다. 이 균주는 원래 콜럼버스, 오하이오에서 필드 수집 모기에서 2013 년에 설립되었으며 Meuti 실험실에 의해 유지됩니다. 이 프로토콜은 쿨렉스 모기뿐만 아니라 다른 모기 종에서 CRISPR/Cas9 게놈 편집을 요구하는 추가 연구에 사용될 수 있으며, 더 일반적으로 CRISPR/Cas9 게놈 편집을 모든 곤충 종에 사용하는 것과 관련이 있습니다.

<table><tbody><tr><td>Artificial Membrane Feeder</td><td>Hemotek</td><td>SP5W1-3</td><td>Company location: Blackburn, UK</td></tr><tr><td>ATP</td><td>Invitrogen</td><td>18330019</td><td>Company location: Carlsbad, CA, USA</td></tr><tr><td>Borosilicate glass mirocapillary tubes, 1 mm outer diameter</td><td>World Precision Instruments</td><td>1B100-6</td><td>Company Location: Sarasota, FL, USA</td></tr><tr><td>BV10 Needle Beveler</td><td>Sutter Instruments</td><td>BV-10-B</td><td>Company Location: Nobato, CA, USA</td></tr><tr><td>Whatman Circular filter paper (12.5 cm)</td><td>Sigma Aldrich</td><td>WHA1001125</td><td>Company Location: St. Louis, MO, USA</td></tr><tr><td>Conical tube (50 mL)</td><td>Thermo Fisher Scientific</td><td>339652</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Fisherbrand course filter paper with fast flow rate</td><td>Thermo Fisher Scientific</td><td>09-800</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Cover glass (24 x 40 mm)</td><td>Thermo Fisher Scientific</td><td>50-311-20</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Dental dam</td><td>Henry Schein Inc</td><td>1010171</td><td>Company Location: Melville, NY USA</td></tr><tr><td>Scotch double-sided tape</td><td>Thermo Fisher Scientific</td><td>NC0879005</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>FemtoJet 4i microinjector</td><td>Eppendorf</td><td>5252000021</td><td>Company Location: Hamburg, Germany</td></tr><tr><td>Glass vial (2 dram)</td><td>Thermo Fisher Scientific</td><td>033401C</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Halocarbon oil</td><td>Sigma Aldrich</td><td>H8898-50ML</td><td>Company Location: St. Louis, MO, USA</td></tr><tr><td>P-2000 Laser Needle Puller</td><td>Sutter Instruments</td><td>P-2000/G</td><td>Company Location: Nobato, CA, USA</td></tr><tr><td>Parafilm</td><td>Thermo Fisher Scientific</td><td>50-998-944</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>PC-100 Weighted Needle Puller</td><td>Narishige</td><td>PC-100</td><td>This is compatabile with the earlier PC-10 model, which has been discontinued. Company Location: Amityville, NY, USA</td></tr><tr><td>Phire Direct PCR Kit</td><td>Thermo Fisher Scientific</td><td>F140WH</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Kodak Photo-Flo (1%)</td><td>Thermo Fisher Scientific</td><td>50-268-05</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Quartz glass mirocapillary tubes, 1 mm outer diameter</td><td>Capillary Tube Supplies Limited</td><td>QGCT 1.0</td><td>Company Location: Cornwall, UK</td></tr><tr><td>Guide-it&amp;trade; sgRNA Screening Kit</td><td>Takara, Bio USA</td><td>632639</td><td>This kit allows you to determine if gRNAs cut DNA sequences in vitro. Company Location: Mountain View, CA, USA</td></tr><tr><td>Sigmacote</td><td>Sigma Aldrich</td><td>SL2-100ML</td><td>Company Location: St. Louis, MO, USA</td></tr><tr><td>Small petri dishes (35X10 mm)</td><td>Thermo Fisher Scientific</td><td>50-190-0273</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Sodium citrate chicken blood</td><td>Lampire biologicals</td><td>7201406</td><td>Company Location: Everett, PA, USA</td></tr><tr><td>Fisherbrand Square petri dish (10 cm x 10 cm)</td><td>Thermo Fisher Scientific</td><td>FB0875711A</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Tegaderm</td><td>Henry Schein Inc.</td><td>7771180</td><td>Company Location: Melville, NY USA</td></tr><tr><td>Tropical fish food</td><td>Tetramin</td><td>N/A</td><td></td></tr><tr><td>Whatman filter paper</td><td>Thermo Fisher Scientific</td><td>09-927-826</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Whatman filter paper, 4.25 cm</td><td>Sigma Aldrich</td><td>1001-042</td><td>Company Location: St. Louis, MO, USA</td></tr></tbody></table>

preparing and injecting embryos of culex mosquitoes to generate null mutations using crispr/cas9

컬렉스 모기는 웨스트 나일 바이러스와 개 심장벌레와 코끼리와 같은 filarial 선충에 기인한 질병을 포함하여 인간과 동물의 건강에 부정적인 영향을 미치는 몇몇 질병의 중요한 벡터입니다. 최근에는 제네라 아노필스와 에데스에속하는 모기를 포함하여 여러 곤충 종의 갓 배치된 배아에 가이드 RNA(gRNA)로 복합된 Cas9 단백질을 주입하여 현장 지향 돌연변이를 유도하는 데 사용되어 왔다. 컬렉스 모기를 조작하고 주입하는 것은 이 모기가 다른 모기 종처럼 뗏목에서 알을 똑바로 세우기 때문에 약간 더 어렵습니다. 여기에서 우리는 gRNA를 디자인하는 방법, Cas9 단백질로 그(것)들을 복잡하게 하고, Culex pipiens의 여성 모기를 계란을 낳기 위하여 유도하고, Cas9/gRNA를 가진 미세 주입을 위한 새로 놓인 태아를 준비하고 주입하는 방법을 기술합니다. 우리는 또한 원하는 돌연변이를 위해 주입된 모기를 후방및 스크린하는 방법을 설명합니다. 대표적인 결과는 이 기술이 Culex 모기의 게놈에 있는 사이트 지시돌연변이를 유도하기 위하여 이용될 수 있고, 약간의 수정으로, 그밖 모기 종에 있는 널 돌연변이를 생성하는 것을 이용될 수 있다는 것을 보여줍니다.

설명된 프로토콜을 사용하여, 우리는 성공적으로 Cx. pipiens의배아를 주입할 수 있었고, 주입된 태아 중 생존율이 높은 것을 관찰하였다(~55%, 도 1). 이전 시험은 달걀 여포의 전방이 의학 드레싱 스트립에 부착되었기 때문에 생존의 비율이 낮았으며, 모기 유충이 초리온에서 탈출하고 성공적으로 물로 수영하는 것을 막았습니다. 전방 끝이 의료 드레싱의 스트립을 넘어 ...

Watch this Scientific Journal Video about Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9 at JoVE.com

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9는 비모델 유기체에서 유전자 기능을 특성화하는 데 점점 더 사용됩니다. 이 프로토콜은 주입 믹스 준비에서 모기 배아를 획득하고 주입하는 것뿐만 아니라 주입 된 모기와 원하는 돌연변이에 대한 자손을 후면, 교차 및 선별하는 방법에 이르기까지 Culex pipiens의녹아웃 라인을 생성하는 방법을 설명합니다.

CRISPR/Cas9를 사용하여 Null 돌연변이를 생성하기 위해 컬렉스 모기의 배아를 준비하고 주입합니다.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

preparing-injecting-embryos-culex-mosquitoes-to-generate-null

Research

JoVE Journal

Biology

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

6.9K 조회수.  The Ohio State University. CRISPR/Cas9는 비모델 유기체에서 유전자 기능을 특성화하는 데 점점 더 사용됩니다. 이 프로토콜은 주입 믹스 준비에서 모기 배아를 획득하고 주입하는 것뿐만 아니라 주입 된 모기와 원하는 돌연변이에 대한 자손을 후면, 교차 및 선별하는 방법에 이르기까지 Culex pipiens의녹아웃 라인을 생성하는 방법을 설명합니다.

비디오: Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

의 사출. stephensi의 태아는 말라리아 방지 모기를 생성하는

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

연구

생물학

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

트렌스 제닉 모기를 구하는 A. aegypti의 태아의 Microinjection

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

Xenopus 에 Leapfrogging CRISPR/Cas9-기반에 대 한 원시 생식 세포 이식

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

유전학

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

컬렉스 퀸케파시아투스의 효율적인 부위 별 돌연변이 발생을 위한 배아 미세 주입 기술

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

샌드 플라이 (플레보토무스 파파타시) CRISPR/Cas9 돌연변이 발생을 위한 배아 미세 주입

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. 
High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

모기 Aedes aegypti에서 고해상도 용융 분석을 사용하여 CRISPR/ Cas9 돌연변이 발생 에 따른 인델 검출

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

수확 및 준비 Drosophila</emElectrophysiological 녹화 및 기타 수속을위한> 태아

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.

Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology.

CRISPR-Cas9을 이용한 황열병 모기 이집트숲모기(Mosquito Aedes aegypti )의 게놈 편집

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering ...

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

CRISPR/Cas9 게놈 편집 헬리코파 아르미게라 의 배아 미세 주입 및 녹아웃 돌연변이 식별 (휘브너)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

행동분석학

Mosquito Embryo Microinjection: A Technique to Deliver Exogenous DNA into Embryo Yolk of Anopheles Gambiae to Generate Germline Mutations

Use a micro-loader tip to fill the needles with two microliters of DNA mixture. Insert the needle into the needle-holder, and connect the automated pressure pump tubing. Align the needle so that it makes an angle of 15 degrees with the slide&#39;s plane.
Open the needle tip by carefully touching the first egg. Then, insert the needle&#8217;s tip to about 10 micrometers in the posterior pole. A successful injection will lead to a small movement of the cytoplasm within the egg.
Use the microscope co-axial stage controls to move to the next egg to continue injection. To ensure that the needle tip remains open and has not been clogged, press the &#39;Inject&#39; button before entering another embryo, and visualize the small droplet at the opening of the needle. If the needle gets clogged, press the &#39;Clear&#39; button to clear the needle, and repeat the droplet visualization test.
Adjust the pressure as needed, if the needle tip opening gets slightly bigger to ensure that the droplet size stays small. Inject about 40 to 50 eggs with one needle. After injections are complete, rinse the eggs off into a glass container lined with filter paper and filled with 50 milliliters of deionized water.