저자 dysfusion cDNA, Dys 항체 및 W 1118 파리, 스티븐 크루스 감사하고 싶습니다.

1. 배아 컬렉션 <ol><li> 25 새장을 설정 ° 2-4일 된 w 1118 flies.Grape 주스 플레이트를 사용하는 C는 수령 전에 1-2일 기간 동안 계란 컬렉션을 동기화하기 위해 하루 동안 매 시간마다 변경됩니다 </li><li> 25 1 시간에 대한 배아를 수집 ° C </li><li> 면도날로 중간에 가볍게 절단 포도 주스 한천의 사각형 조각을 잘라 한천의 라인을두고 </li><li> 포도 쥬스 한천의 조각에 포도 주스 플레이트에서 배아를 전송하는 금속 프로브를 사용하여 한천의 중간에있는 라인에 45도 각도에서 배아의 긴 축을 가진 직선에서 그들을 라인 업. 약 50 배아의 직선을하십시오. 라인에서 멀리 가리키는 후부 엔드와 모든 배아 지점 같은 방향을 확인하십시오. </li><li> 이중 스틱 테이프의 사각형 조각을 잘라 및 18x18mm 커버 슬립에 놓으십시오. </li><li> 커버 슬립을 더블 스틱 테이프와 배아를 들고 </li><li> , 유리 슬라이드에 커버 슬립을 플레이스 약 10 분 공중에 배아를 건조. </li><li> 700 할로 카본 오일의 얇은 층과 배아를 맡아, 지금은 주사를위한 준비. </li></ol> 2. 니들을 풀링 유리 모세관 튜브를 당겨 microinjection의 바늘을 만듭니다. 우리는 세계 정밀 악기 (모델 PUL - 1)에서 바늘 풀러를 사용합니다. 바늘 풀러 프로그램 사양은 다음과 같습니다 열 : 1, 지연 4. 3. 니들 필링 Eppendorf Microloader 팁 장착되어 Eppendorf p20 피펫을 사용하여 백업 부하 바늘은 일반적으로 1.5-2 μL dsRNA를 로드할 수 4. 니들 브레이크 <ol><li> 바늘을 깨는 전에 슬라이드로 coverslip을 넣으십시오. </li><li> 슬라이드 중간에 기름 한 방울과 슬라이드를 준비 </li><li> 공기 압력을 조정 Picospritzer III picopump 켭니다 </li><li> 샵 포인트를 작성, 표지 용지의 가장자리에 대한 가득한 바늘을 휴식. </li><li> 팁이 깨진 경우, Picospritzer III picopump 기슭 페달에 단계는, 일부 dsRNA는 바늘 끝 밖으로 누출됩니다. </li><li> coverslip으로 슬라이드를 꺼내주세요. </li><li> 전에 준비한 슬라이드의 중간에 오일 드롭에서 바늘을 삽입합니다. </li><li> 당신은 발 페달을 밟아 때 100 - 200pl dsRNA를 얻을 수있는 Picospritzer III picopump에 대한 설정을 조정합니다. dsRNA는 작은 비말로 볼 수 있습니다. </li></ol> 5. 주사 <ol><li> 동일한 초점 비행기로 최초의 배반엽 배아의 후부 부분에 바늘 끝을 준비하고 30~50% 달걀 길이 주위 센터에서 배아를 주입. </li><li> 당신이 Picospritzer III picopump 기슭 페달 밟아 후, dsRNA의 작은 금액은 사출 사이트의 작은 점으로 표시됩니다. </li></ol> 6. Phenotypic 분석 <ol><li> 태아가 원하는 배아 단계로 개발할 때까지 주입 후 18 덮여 촉촉한 챔버 (플라스틱 페트리 접시) ° C에 슬라이드를 놓으십시오. </li><li> 이중 양면 테이프에서 배아를 제거하고 커버 용지의 가장자리로 밀어 프로브를 사용합니다. 헵탄를 사용하여 아래에 나일론 메쉬와 컬렉션 약병에 슬라이드에서 배아를 와시, PBT로 3 회 씻는다. Dechorionate 5 분 50 % 표백제로 배아하고 PBT로 3 회 씻는다. 수집 약병에서 나일론 메쉬의 한 부분으로 배아를 전송하고 배아를 공개 정착액 솔루션 (5ml 헵탄, 4.5ml PBS, 0.5ml 37 % 포름 알데히드)에 나일론 메쉬를 찍어. 30 분 대한 고정력있는 솔루션에 배아를 수정. eppendorf 튜브에 배아를 전송하고, 표준 프로토콜을 사용하여 배아를 immunostain, 메탄올과 배아를 Devitellinize. </li><li> 배아는 해당 애벌레 단계로 개발할 수 있습니다. </li><li> 중간에 할로 카본 700 기름 한 방울과 슬라이드를 준비 </li><li> 슬라이드 하나의 유충을 전송, 상단에 커버 슬립을하다 </li><li> 명시야 현미경으로 애벌레를 관찰. </li></ol> 7. 대표 결과 : dsRNA의 예는 Fig1에 표시됩니다 Drosophlia의 배아에서 dysfusion (dys) 유전자를 노크. GFP - dsRNA 주입 태아는 부정적인 통제하고 있습니다. Dys는 tracheal 융합 세포의 표현 bHLH PAS의 전사 인자이다. GFP - dsRNA 주입 배아가 정상 tracheal 융합을 표시하고, Dys 단백질이 융합 세포 (그림 1A)에 속해 있습니다. 한편, dys - dsRNA는 배아가 실패 지점 융합을 보여 주입하고, Dys 단백질이 융합 세포 (그림 1B)에 존재하지 않습니다. dsRNA는 배아가 애벌레 단계로 개발 주입하면 dys - dsRNA 주입 애벌레는 GFP - dsRNA 주입 대조군 (그림 1C)에 비해 실패 지점 퓨전 (Fig.1D)을 보여주었다. 이러한 결과는 효과가 Drosophila의 배아에 주입하여 dsRNA 내생 dys 유전자 표현의 다운 노크했다 <img src="/files/ftp_upload/2477/2477fig1.jpg" alt="그림 1" /> 그림 1. dys - RNAi에 의해 dys 기능의 제거 tracheal 퓨전 D를 보여efects. 배반엽의 배아 (W 1118)는 GFP - dsRNA (대조군) 또는 dys - dsRNA 중 하나와 함께 주입하고 tracheal 결함에 대한 assayed했다. (A) 단계 16 배아는 GFP - dsRNA로 주입과 α - Dys 및 MAb 2A12 그 얼룩이 tracheal 루멘 물들다. 눈에 띄게 융합있는 측면 트렁크는, 표시됩니다. 화살표는 측면 트렁크 Dys - 긍정적인 융합 세포를 가리 킵니다. (B) 16 단계 배아는 dys - dsRNA로 주입과 α - Dys과 MAb 2A12 물들다. 배아는 측면 트렁크 퓨전 (별표 제어 배아의 측면 - 트렁크 융합의 정상 위치를 나타냅니다)의 부족을 보여주었다. 없음 Dys 양성 세포는 dys - RNA 효과적으로 Dys 단백질을 폐지한다는 의미 관찰되지 않았습니다. GFP - dsRNA 또는 dys - dsRNA 중 하나와 함께 (C D)을 제 2 instar 애벌레는 tracheal 결함에 대한 밝은 - 필드 현미경으로 조사되었다. (C) 퓨전의 사이트는 GFP - dsRNA 주입 제어 애벌레의 별표로 표시됩니다. (D) 측면 트렁크 dys - RNA 주입 애벌레의 퓨즈에 실패, (*)는 측면 트렁크 융합의 정상 위치를 나타냅니다.

<ol>
	<li>St Johnston, D., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+pattern+and+polarity+in+the+Drosophila+embryo.">The origin of pattern and polarity in the Drosophila embryo.</a> Cell. 68, 201-219 (1992).</li><li>Venken, K. J., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+technologies+for+gene+manipulation+in+Drosophila+melanogaster.">Emerging technologies for gene manipulation in Drosophila melanogaster.</a> Nat. Rev. Genet. 6, 167-178 (2005).</li><li>Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Kennerdell, J. R., Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+dsRNA-mediated+genetic+interference+to+demonstrate+that+frizzled+and+frizzled+2+act+in+the+wingless+pathway.">Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.</a> Cell. 95, 1017-1026 (1998).</li><li>Misquitta, L., Paterson, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+gene+function+in+Drosophila+by+RNA+interference+(RNA-i):+a+role+for+nautilus+in+embryonic+somatic+muscle+formation.">Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 1451-1456 (1999).</li><li>Horowitz, A., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+morphogenesis.">Branching morphogenesis.</a> Circ. Res. 103, 784-795 (2008).</li><li>Hogan, B. L., Kolodziej, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis:+molecular+mechanisms+of+tubulogenesis.">Organogenesis: molecular mechanisms of tubulogenesis.</a> Nat. Rev. Genet. 3, 513-523 (2002).</li><li>Ghabrial, A., Luschnig, S., Metzstein, M. M., Krasnow, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+morphogenesis+of+the+Drosophila+tracheal+system.">Branching morphogenesis of the Drosophila tracheal system.</a> Annu. Rev. Cell Dev. Biol. 19, 623-647 (2003).</li><li>Jiang, L., Crews, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfusion+transcriptional+control+of+Drosophila+tracheal+migration,+adhesion,+and+fusion.">Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion.</a> Mol. Cell. Biol. 26, 6547-6556 (2006).</li><li>Jiang, L., Crews, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+dysfusion+basic+helix-loop-helix+(bHLH)-PAS+gene+controls+tracheal+fusion+and+levels+of+the+trachealess+bHLH-PAS+protein.">The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein.</a> Mol. Cell. Biol. 23, 5625-5637 (2003).</li></ol>

관심 없음 충돌 선언하지 않습니다.

dsRNA 주입 방식 현재는 여기에 Drosophila tracheal 개발 유전자 기능의 매우 민감하고 신속한 분석이 가능합니다. 이 방법은 잠재적으로 다른 조직 및 장기 개발을위한 유전자 기능을 분석하기 위해 적용할 수 있습니다.

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		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Halocarbon oil 700</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H8898</td>
<td>&nbsp;</td>
<tr>
<td>Picospritzer III picopump</td>
<td>&nbsp;</td>
<td>Parker Precision Fluidics</td>
<td>051-0500-900</td>
<td>&nbsp;</td>
<tr>
<td>Micro-pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>21170M</td>
<td>&nbsp;</td>
<tr>
<td>Microloaders</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>930001007</td>
<td>&nbsp;</td>		</tbody>
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rnai interference by dsrna injection into drosophila embryos

유전자 검사는 복잡한 생물 학적 과정을 한 통찰력을 확보하고 사용할 수있는 가장 강력한 방법 중 하나입니다. 수년 동안 유전자 조작을위한 많은 개선 및 도구 Drosophila 2 사용할 수 있습니다. 곧 두 번 좌초 RNA가 분해 RNA 간섭 (RNAi)가 Drosophila 장기 개발에 유전자 기능을 분석하는 강력한 반대로 유전자 접근 방법을 제공하기 위해 표시되었습니다 Caenorhabditis 엘레간스에서 개별 유전자의 활동을하는 데 사용할 수있는 프리와 멜로 3 초기 발견 이후 4, 5. 폐, 신장, 간, 그리고 혈관 계통을 포함한 많은 장기는, 분기 관 네트워크로 구성하는 중요 교통 유체 또는 가스 6, 7. Drosophila tracheal 형성의 분석은 다른 관형 기관 8 morphogenesis을 연구하는 훌륭한 모델 시스템을 제공합니다. 버클리 Drosophila 게놈 프로젝트는 tracheal 시스템에 표현됩니다 유전자의 수백을 보여주었다. 튜브 형성의 분자 및 세포 메커니즘을 연구하기 위해 도전 tracheal 개발에서 이러한 유전자의 역할을 이해하는 것입니다. 여기, 우리는 최저의 개별 유전자 발현에 Drosophila의 배아에 주입 dsRNA에 대한 자세한 방법을 설명했다. 우리는 성공적으로 dsRNA 주입에 의해 내생 dysfusion (dys) 유전자 발현을 무너 뜨 렸어. Dys는 tracheal 융합 세포의 표현 bHLH - PAS 단백질이며, 그것은 tracheal 지점 융합 9, 10 필요합니다. dys - RNAi 완전히 dys 표현을 제거하고 tracheal 퓨전 결함의 결과. 이것은 비교적 간단한 방법은 Drosophila의 tissure 및 장기 개발을위한 requried 유전자를 식별할 수있는 도구를 제공합니다.

Watch this Scientific Journal Video about RNAi Interference by dsRNA Injection into Drosophila Embryos at JoVE.com

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA 간섭은 유전자의 기능을 분석하는 매우 효과적인 입증되었습니다<em&gt; Drosophila</em&gt; tracheal 개발. 최저의 유전자 발현에 플라이 배아에 dsRNA를 주입하기 위해 티베트 연구실에서 사용하는 자세한 프로토콜은 그림입니다. 이 기술의 조직 및 장기 개발에 필요한 심사 유전자의 가능성을 가지고<em&gt; Drosophila</em&gt;.

dsRNA 주입하여 RNAi 간섭에 Drosophila 태아

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

rnai-interference-by-dsrna-injection-into-drosophila-embryos

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

RNAi Interference by dsRNA Injection into Drosophila Embryos

16.1K 조회수.  Oakland University. RNA 간섭은 유전자의 기능을 분석하는 매우 효과적인 입증되었습니다 Drosophila tracheal 개발. 최저의 유전자 발현에 플라이 배아에 dsRNA를 주입하기 위해 티베트 연구실에서 사용하는 자세한 프로토콜은 그림입니다. 이 기술의 조직 및 장기 개발에 필요한 심사 유전자의 가능성을 가지고 Drosophila.

비디오: RNAi Interference by dsRNA Injection into Drosophila Embryos

Injection of dsRNA into Female A. aegypti Mosquitos

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus.  The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

여성 A. aegypti 모기에의 dsRNA의 분사

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

연구

생물학

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 ...

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

E16 랫 태아의 Utero의 심실 주입 및 Electroporation에

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

라이브 해부 Drosophila 태아 : 항체 스테 이닝에 의해 돌연변이 컬렉션을 검사 늘씬한 방법

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

Drosophila의 배아들 중에서 - 생체내의 원심 분리

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNAi Effectors의 세균성 배송 : Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

성인 Zebrafish에 Intraperitoneal 사출

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

의 입식 이미징 Drosophila 태아

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

에서 기본 셀 문화 Drosophila Gastrula의 태아

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.