私たちは、デビッド・オブロフタ博士と昆虫遺伝技術調整研究ネットワークのすべてのメンバーに、彼らが遺伝技術の実施に関して私たちや他の人々に提供する助けと訓練に感謝します。我々は特に 、Culex 胚を注入し、孵化できるようにマイクロマニピュレーションプロトコルを最適化してくれたチャンナ・アルヴィヘアに感謝する。また、ムーティラボで働く学部生のデバンテ・シモンズとジョセフ・ウルソは、トランスジェニック蚊の世話とスクリーニングを支援し、ITFのゾラ・エルムカに対して、蚊の注射の飼育と準備を支援してくれたことに感謝します。この研究は、MEMに提供されたOSUの感染症研究所からの学際的種子助成金によって支えられた。

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<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>
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</ol>

RHは昆虫の遺伝子組み換えでサービスを提供する昆虫変換施設で働いています。

このプロトコルは 、Culex 蚊のゲノムに特定の突然変異を導入する方法を提示し、他の蚊のゲノムを編集するためにも使用することができます。このプロトコルは、注射材料を準備する方法だけでなく、蚊に卵を産む方法の詳細なビデオ概要、ならびにそれらの卵を準備して注入する方法の詳細なビデオ概要を提供するという点で重要です。また、メスの Cx.ピピエン の生物学を?...

Culex蚊は、世界の温帯および熱帯地域全体に分布し、西ナイルウイルス1、セントルイス脳炎2だけでなく、イヌ心虫3および象の毛虫症を引き起こすフィラリア線虫を含むいくつかの致命的なウイルスを伝播する。Cx.クインケファシアトゥス、Cx.ピピエンスピピエンス、Cx.ピピエンス痴漢を含むCulex pipiens複合体のメンバーは、彼らの生物学の多くの側面で顕著なバリエーションを示しています。例えば、Cx.quinquefasciatusおよびCx.ピピエンス痴漢は越冬休眠5、6、Cx.ピピエンスピピエンは堅牢な季節応答を表示し、短い日7、8に応答してジアポーズに入ることができない。さらに、Cx.ピピエンス痴漢はより人類愛的である傾向があり、Cx.ピピエンとCx.キンケファシアトゥスはより動物性の6です。しかし、米国および世界の他の多くの場所で、これらの種は、Cx.ピピエンスピピエンとCx.ピピエンス痴漢のハイブリッドとして病気の伝染に強い影響を与える交雑種は日和見フィーダーであり、鳥と人間の両方を噛み、それによって西ナイルウイルスの橋梁ベクトルとして機能します。Culex蚊の生物学のこれらの側面やその他の魅力的な側面を研究することは、Culex蚊が静止と乾燥耐性卵10を生成するAedes蚊よりも実験室で飼育するのがわずかに難しく、機能的な分子ツールがCulex種のために十分に開発されていないため、部分的に妨げられております。
CRISPR/Cas9ゲノム編集は、南部の家の蚊、カレックスキンケファシアトゥス14、15、16を含むいくつかの重要な蚊種11、12、13の生物学を評価するために使用されている強力な技術です。ジェニファー・ダウドナとエマニュエル・シャルパンティエによって開発されたこの技術は、細菌由来のCRISPR関連エンドヌクレアーゼ(Casタンパク質、ファン・デル・ウーストらによるレビューを参照)によってウイルスに対する自然な細菌防御を利用しています。動物の胚に注入されると、Cas9タンパク質と適切なガイドRNAを組み合わせて、ゲノム内で二本鎖破断を生じさせることができる。これは、エンドヌクレアーゼ活性をゲノムの特定の領域に導くガイドRNAと複合体化されたCas9タンパク質を使用することによって最も頻繁に行われます。Cas9タンパク質が部位特異的な二本鎖破断を作成した後、細胞機械は2つのメカニズムのいずれかを使用して破断を修復しようとします。1つ目は、非相同の端結合(NHEJ)を通じて2つの端を結び付ける必要があり、これはエラーが起こりやすいため、ゲノムにアウトフレーム挿入および欠失を生じさせ、非機能的なタンパク質を引き起こし、ノックアウト突然変異を生じさせる。あるいは、細胞機械は、正しく破断を修復するために類似した配列を見つけることによって相同性指向の修復(HDR)を使用するかもしれません。同様の配列は、生物内の第2染色体によって提供され得る(レビュー18を参照)。しかし、修復された配列が元の配列と正確に一致する場合、Cas9タンパク質は再びDNAを切断することができます。あるいは、標的配列の切断部位の両側に相同配列を含むドナープラスミドを、代替修復配列(蛍光マーカータンパク質、元遺伝子の改変版、またはその他の改変)を含み、ゲノムにコピーして挿入したり、「ノックイン」したりすることもできます。
胚を注入する際にはタイミングが重要であり、CRISPR/Cas9ゲノム編集を使用して昆虫の突然変異を作り出す場合は特にそうです。これは、Cas9タンパク質とgRNAは、胚が間合状態にある場合、細胞膜が形成される前、および胚内で複数の核がアクセス可能である場合にのみ突然変異を生成する最大の能力を有するためである。蚊の場合、原子核は、温度19に応じて、卵子から〜2〜4時間後に周囲に到達するため、この時間までにマイクロインジェクションが成功する必要があります。さらに、Cas9タンパク質は、注射に起因する個体が細胞のモザイクを含み、所望の突然変異を有する個体が含まれるなど、アクセス可能な核DNAを切断する。これらの突然変異を正常に継承するためには、Cas9タンパク質は、将来の卵子と精子を生み出す生殖細胞系列に存在するDNAを切断する必要があります。生殖細胞系列に突然変異が発生することを確実にするためには、昆虫生殖細胞系の前駆体である胚内の極細胞の位置に近い全ての物質を注入するのが最善である。極細胞は 、Culex 胚20の後端付近に位置する。胚を注入することに加えて、所望の突然変異を検出するためには、子孫の交育とスクリーニングのための慎重な計画を策定することが不可欠である。
このプロトコルは、gRNAを生成し、注射ミックスを準備するためにCas9タンパク質でそれらを複雑にする方法だけでなく、卵を産むためにCulexピピエンのメスの蚊を誘導する方法とCRISPR / Cas9媒介ゲノム編集のためにそれらの卵を準備し、注入する方法を説明します。さらに、注射された胚の後部、横断、スクリーニングの方法とその子孫を説明し、所望の突然変異が得られたことを確認する。このプロトコルを使用して、私たちは、カレックスピピエンのバックアイ株で、関心のある遺伝子、サイクル、ヌル突然変異を生成しました。この株はもともとオハイオ州コロンバスのフィールド収集蚊から2013年に設立され、Meutiラボによって維持されています。このプロトコルは、Culex蚊および他の蚊種におけるCRISPR/Cas9ゲノム編集を必要とする追加の研究に使用することができ、より一般的には、任意の昆虫種にCRISPR / Cas9ゲノム編集を採用することに関連しています。

<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

Culex蚊は、西ナイルウイルスやイヌの心臓虫やゾウガシスなどのフィラリア線虫によって引き起こされる病気を含む人間と動物の健康に悪影響を及ぼすいくつかの疾患の主要なベクターです。最近では、CRISPR/Cas9ゲノム編集は、アノフェレス属に属する蚊を含むいくつかの昆虫種の新たに敷設された胚にガイドRNA(gRNA)と複合体化されたCas9タンパク質を注入することによって、部位指向の突然変異を誘導するために使用されている。Culex蚊の操作と注入は、これらの蚊が他の種の蚊のように個別に卵をいかだに置くのではなく、いかだで直立させるので、少し難しいです。ここでは、gRNAを設計し、Cas9タンパク質とそれらを複雑にする方法、卵を産むためにCulexピピエンの雌の蚊を誘導する方法、およびCas9 / gRNAを用いたマイクロインジェクションのために新しく産卵した胚を調製して注入する方法について説明します。また、必要な突然変異のために注入された蚊を飼育およびスクリーニングする方法についても説明する。代表的な結果は、この技術がCulex蚊のゲノムに部位指向突然変異を誘導するために使用され、わずかな改変を伴って、他の蚊種でもヌル突然変異体を生成するために使用できることを示している。

記載されたプロトコルを用いて、Cx.pipiensの胚を正常に注入することができ、注入された胚の間で高い生存率を観察した(〜55%、図1)。以前の試験では、卵胞の前部が医療ドレッシングストリップに取り付けられ、蚊の幼虫が絨毛膜から脱出するのを防ぎ、水に泳ぐことに成功したため、生存率が低かった。前端が医療用ドレッシングのストリップを超えて延びるこ...

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/Cas9は、非モデル生物の遺伝子機能を特徴付けるためにますます使用されています。このプロトコルは、注射ミックスの調製から蚊の胚の取得と注入に、および必要な突然変異のために注入された蚊とその子孫を飼育、横断、およびスクリーニングする方法に 、Culex pipiensのノックアウトラインを生成する方法を記述します。

CRISPR/Cas9を使用してヌル突然変異を生成するための Culex 蚊の胚の調製と注入

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 ビュー.  The Ohio State University. CRISPR/Cas9は、非モデル生物の遺伝子機能を特徴付けるためにますます使用されています。このプロトコルは、注射ミックスの調製から蚊の胚の取得と注入に、および必要な突然変異のために注入された蚊とその子孫を飼育、横断、およびスクリーニングする方法に 、Culex pipiensのノックアウトラインを生成する方法を記述します。

ビデオ: 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.

の注入。ハマダラカの胚は、マラリア耐性の蚊を生成する

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

生物学

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.

トランスジェニック蚊を取得するA.ネッタイシマカの胚のマイクロインジェクション

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.

アフリカツメガエルのリープフロッギング CRISPR/Cas9 ベースの始原生殖細胞の移植

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 ...

遺伝学

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.

キュレックス・キンケファシアトゥスにおける効率的な部位特異的変異誘発のための胚微小注入技術

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.

CRISPR/Cas9突然変異誘発のためのサンドフライ(フレボトムスパパタシ)胚微小注射

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.

モスキートAedes aegyptiにおける高解像度メルト分析を用いたCRISPR/Cas9突然変異誘発に続くインデル検出

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.

収穫と準備ショウジョウバエ</em電気生理学的記録とその他の手続のための>胚

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-Cas9を用いた黄熱病ネ ッタイシマカ のゲノム編集

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ゲノム編集 ヘリコベルパ・アルミゲラ (Hübner)の胚マイクロインジェクションとノックアウト変異体の同定

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

行動学

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.

CRISPR/Cas9を使用してヌル突然変異を生成するための Culex 蚊の胚の調製と注入

要約

さらに動画を探す

の注入。ハマダラカの胚は、マラリア耐性の蚊を生成する

トランスジェニック蚊を取得するA.ネッタイシマカの胚のマイクロインジェクション

アフリカツメガエルのリープフロッギング CRISPR/Cas9 ベースの始原生殖細胞の移植

キュレックス・キンケファシアトゥスにおける効率的な部位特異的変異誘発のための胚微小注入技術

CRISPR/Cas9突然変異誘発のためのサンドフライ(フレボトムスパパタシ)胚微小注射

モスキートAedes aegyptiにおける高解像度メルト分析を用いたCRISPR/Cas9突然変異誘発に続くインデル検出

収穫と準備ショウジョウバエ胚

CRISPR-Cas9を用いた黄熱病ネッタイシマカのゲノム編集

CRISPR/Cas9ゲノム編集ヘリコベルパ・アルミゲラ (Hübner)の胚マイクロインジェクションとノックアウト変異体の同定

蚊胚マイクロインジェクション:ハマダラカの胚卵黄に外因性DNAを送達して生殖細胞変異を生成する技術

CRISPR/Cas9を使用してヌル突然変異を生成するための Culex 蚊の胚の調製と注入

要約

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の注入。ハマダラカの胚は、マラリア耐性の蚊を生成する

トランスジェニック蚊を取得するA.ネッタイシマカの胚のマイクロインジェクション

アフリカツメガエルのリープフロッギング CRISPR/Cas9 ベースの始原生殖細胞の移植

キュレックス・キンケファシアトゥスにおける効率的な部位特異的変異誘発のための胚微小注入技術

CRISPR/Cas9突然変異誘発のためのサンドフライ(フレボトムスパパタシ)胚微小注射

モスキートAedes aegyptiにおける高解像度メルト分析を用いたCRISPR/Cas9突然変異誘発に続くインデル検出

収穫と準備ショウジョウバエ胚

CRISPR-Cas9を用いた黄熱病ネ ッタイシマカ のゲノム編集

CRISPR/Cas9ゲノム編集 ヘリコベルパ・アルミゲラ (Hübner)の胚マイクロインジェクションとノックアウト変異体の同定

蚊胚マイクロインジェクション:ハマダラカの胚卵黄に外因性DNAを送達して生殖細胞変異を生成する技術

CRISPR-Cas9を用いた黄熱病ネッタイシマカのゲノム編集

CRISPR/Cas9ゲノム編集ヘリコベルパ・アルミゲラ (Hübner)の胚マイクロインジェクションとノックアウト変異体の同定