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.

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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.

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			<td height="38" style="height:38px;width:216px;">Artificial Membrane Feeder</td>
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			<td style="width:119px;">SP5W1-3</td>
			<td style="width:167px;">Company location: Blackburn, UK</td>
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			<td height="38" style="height:38px;width:216px;">ATP</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:119px;">18330019</td>
			<td style="width:167px;">Company location: Carlsbad, CA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Borosilicate glass mirocapillary tubes, 1 mm outer diameter</td>
			<td style="width:121px;">World Precision Instruments</td>
			<td style="width:119px;">1B100-6</td>
			<td style="width:167px;">Company Location: Sarasota, FL, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">BV10 Needle Beveler</td>
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			<td style="width:167px;">Company Location: Nobato, CA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Whatman Circular filter paper (12.5 cm)</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">WHA1001125</td>
			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
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			<td height="38" style="height:38px;width:216px;">Conical tube (50 mL)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">339652</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Cover glass (24 x 40 mm)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
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			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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			<td height="38" style="height:38px;width:216px;">Dental dam</td>
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			<td style="width:121px;">Thermo Fisher Scientific</td>
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			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">FemtoJet 4i microinjector</td>
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			<td style="width:167px;">Company Location: Hamburg, Germany</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Glass vial (2 dram)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">033401C</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Halocarbon oil</td>
			<td style="width:121px;">Sigma Aldrich</td>
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			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">P-2000 Laser Needle Puller</td>
			<td style="width:121px;">Sutter Instruments</td>
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			<td style="width:167px;">Company Location: Nobato, CA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Parafilm</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-998-944</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">PC-100 Weighted Needle Puller</td>
			<td style="width:121px;">Narishige</td>
			<td style="width:119px;">PC-100</td>
			<td style="width:167px;">This is compatabile with the earlier PC-10 model, which has been discontinued. Company Location: Amityville, NY, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Phire Direct PCR Kit</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F140WH</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Kodak Photo-Flo (1%)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-268-05</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Quartz glass mirocapillary tubes, 1 mm outer diameter</td>
			<td style="width:121px;">Capillary Tube Supplies Limited</td>
			<td style="width:119px;">QGCT 1.0</td>
			<td style="width:167px;">Company Location: Cornwall, UK</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Guide-it&#8482; sgRNA Screening Kit</td>
			<td style="width:121px;">Takara, Bio USA</td>
			<td style="width:119px;">632639</td>
			<td style="width:167px;">This kit allows you to determine if gRNAs cut DNA sequences in vitro. Company Location: Mountain View, CA, USA</td>
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			<td height="38" style="height:38px;width:216px;">Sigmacote</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">SL2-100ML</td>
			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Small petri dishes (35X10 mm)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-190-0273</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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			<td height="38" style="height:38px;width:216px;">Sodium citrate chicken blood</td>
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			<td style="width:167px;">Company Location: Everett, PA, USA</td>
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			<td height="38" style="height:38px;width:216px;">Fisherbrand Square petri dish (10 cm x 10 cm)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
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			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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			<td height="38" style="height:38px;width:216px;">Tegaderm</td>
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			<td style="width:167px;">Company Location: Melville, NY USA</td>
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			<td height="38" style="height:38px;width:216px;">Tropical fish food</td>
			<td style="width:121px;">Tetramin</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Whatman filter paper</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">09-927-826</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Whatman filter paper, 4.25 cm</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">1001-042</td>
			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
		</tr>
	</tbody>
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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

6.7K 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 ...

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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 ...

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 ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

CRISPR / Cas9를 통해 포유 동물 세포주의 게놈에 삭제 세대

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

불꽃을 침묵 : 약한 전기 물고기에서 CRISPR / Cas9 게놈 편집

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

CRISPR/Cas9를 사용하여 1차 인간 B 세포의 게놈 공학

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

TGF-β 매개 내피- 중간엽 전환(EndMT)과 CRISPR/Cas9 유전자 편집을 사용하여 EndMT 이펙터의 기능평가

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no prevention. Control of mosquito vectors can rely on chemicals applied to the immature and adult stages, which can contribute to the mortality of non-targets and more importantly, lead to insecticide resistance in the vector. The sterile insect technique (SIT) is a method of controlling populations of pests through the release of sterilized adult males that mate with wild females to produce non-viable offspring. This paper describes the process of producing sterile males for use in an operational SIT program for the control of Aedes aegypti mosquitoes. Outlined here are the steps used in the program including rearing and maintaining a colony, separating male and female pupae, irradiating and marking adult males, and shipping Aedes aegypti males to the release site. Also discussed are procedural caveats, program limitations, and future objectives.

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The sterile insect technique (SIT) is used to control specific, medically important mosquito populations that may be resistant to chemical controls. Here, we describe a method of mass rearing and preparation of sterile male mosquitoes for release in an operational SIT program targeting the Aedes aegypti mosquito.

운영 멸균 곤충 기술 프로그램에서 방출하기 위해 조사되고 표시된 수컷 Aedes aegypti 모기 준비

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

CRISPR/Cas9 기술을 이용한 철새 메뚜기의 동형접합 돌연변이 구축

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.