ES의 연구실에서 작업은 스위스 국립 과학 재단 (National Science Foundation)에서 지원됩니다. 우리는 촬영과 도움 박사 비트 Kunz 감사드립니다.

<ol>
	<li>Ch&#233;dotal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+tales+of+the+midline.">Further tales of the midline.</a> Current Opinion in Neurobiology. 21, 68-75 (2011).</li><li>Avraham, O., Hadas, Y., Vald, L., Zisman, S., Schejter, A., Visel, A., Klar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+axonal+guidance+and+sorting+in+dorsal+interneurons+by+the+Lim-HD+proteins+Lhx9+and+Lhx1.">Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.</a> Neural Development. 4, 21 (2009).</li><li>Wilson, N. H., Stoeckli, E. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88, 49-92 (1951).</li><li>Boulland, J., Halasi, G., Kasumacic, N., Glover, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+Human+Stem+Cells+into+the+Chicken+Embryo.">Xenotransplantation of Human Stem Cells into the Chicken Embryo.</a> J. Vis. Exp. (41), e2071 (2010).</li><li>Kim, B. G., Dai, H. -. N., McAtee, M., Vicini, S., Bregman, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+of+dendritic+spines+with+the+carbocyanine+dye+DiI+for+confocal+microscopic+imaging+in+lightly+fixed+cortical+slices.">Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices.</a> Journal of Neuroscience Methods. 162, 237-243 (2007).</li><li>Murphy, M. C., Fox, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterograde+tracing+method+using+DiI+to+label+vagal+innervation+of+the+embryonic+and+early+postnatal+mouse+gastrointestinal+tract.">Anterograde tracing method using DiI to label vagal innervation of the embryonic and early postnatal mouse gastrointestinal tract.</a> Journal of Neuroscience Methods. 163, 213-225 (2007).</li><li>Helms, A. W., Abney, A. L., Ben-Arie, N., Zoghbi, H. Y., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+and+multiple+enhancers+control+Math1+expression+in+the+developing+nervous+system.">Autoregulation and multiple enhancers control Math1 expression in the developing nervous system.</a> Development. 127, 1185-1196 (2000).</li><li>Li, X., Lufkin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+recombinase+expression+in+the+floorplate,+notochord+and+gut+epithelium+in+transgenic+embryos+driven+by+the+Hoxa-1+enhancer+III.">Cre recombinase expression in the floorplate, notochord and gut epithelium in transgenic embryos driven by the Hoxa-1 enhancer III.</a> Genesis. 26, 121-122 (2000).</li><li>Mauti, O., Baeriswyl, T., Stoeckli, E. T. G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+by+Injection+and+Electroporation+of+dsRNA+in+Avian+Embryos.">Silencing by Injection and Electroporation of dsRNA in Avian Embryos.</a> Cold Spring Harbor Protocols. , (2008).</li><li>Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+using+in+ovo+electroporation+to+analyze+gene+function.">A primer on using in ovo electroporation to analyze gene function.</a> Developmental Dynamics. 229, 433-439 (2004).</li><li>Cullen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+and+confirming+the+specificity+of+RNAi+experiments.">Enhancing and confirming the specificity of RNAi experiments.</a> Nature Methods. 3, 677-681 (2006).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 간단한, 벡터 기반의 인공 miRNA 표현 전략은 닭 신경 튜브에 최저 내생 유전자 발현에 사용할 수 있습니다. 이 기능 도구는 복잡한 발달 경로의 해설을 촉진하기 위해 여러 유전자 입을, 시간적 제어 및 세포 유형 특이성을 제공합니다. plasmids은 commissural 뉴런에 또는 중간 타겟, floorplate 3 최저 별개의 유전자하는 데 사용할 수 있기 때문에 특히, 우리는 commissural 축삭지도에서 이러한 plasmids의 유틸리티를 증명하고있다. miRNA - 표현의 plasmids (및 따라서, 침묵 세포)에서 생성 된 형광 단백질 표현의 강도와 위치는 다음 매개 변수에 따라 달라집니다 : <ol><li> 증폭기는 사용 : <ol class="lalpha"><li> β-고를업자는 ubiquitously 활성화 </li><li> Math1 증강은 dI1 뉴런 12 활성 </li><li&gt;을 Hoxa1 증강 III는 바닥 판 13 활성 </li></ol></li><li> 플라스미드의 농도. 플라스미드 농도가 너무 높은 경우 너무 작은 플라스미드는 형광 단백질의 표현을 줄이고 추적되는 것을 electroporated 세포를 방지 할 수 있지만, 증강 활동 및 원치 않는 세포의 후속 표현의 &#39;leakiness&#39;을 일으킬 수 있습니다. RNAi의 plasmids의 높은 농도는 오프 대상 효과를 줄이고 내생 miRNA의 biogenesis 경로의 포화로 인해 발생하는 비 특정 독성의 위험을 최소화하기 위해 피해야한다. 우리는 사용 <ol class="lalpha"><li> β-고를업자 : 0.25 μg / μl </li><li> Math1 증강 : 0.7 μg / μl </li><li> Hoxa1 증강 III : 0.5-1.0 μg / μl </li></ol></li><li> electroporation의 정확성. 비켜에서 닭 배아를 취급하는 것은 교육을 통해 취득해야 매뉴얼 기술이 필요합니다. 우리와 다른이전에이 절차 14,15의 문제 해결 팁을 발표했다. </li></ol> 적절한 후보가 발견되기 전에 몇 가지 miRNAs는 검사 할 필요가 있습니다. 이 과정은 관찰 된 표현형, 음성 컨트롤 (스크램블 및 / 또는 관련이없는 miRNAs)과 구조 16을 구성을 확인하기 위해 여러 독립 miRNAs의 식별을 포함해야합니다. 오픈 도서 준비가 commissural 뉴런의 axonal 예측의 구조 정보를 시각화하기 위해 적절한 위치로 최적 고정 DiI으로 주입해야합니다. 높은 배경은 오픈 책이나 DiI의 spillage의 오랜 고정로 인해 발생할 수 있습니다. 너무 dorsally에 위치한 셀에 DiI의 주입은 일반적으로 바닥 판에 대한 분류 axonal 계획의 부족을 야기 할 것이다. 너무 ventrally에 위치한 세포는 중간 선에서 많은 ipsilateral와 contralateral 계획을 가지게 될 것이며, 따라서 피해야한다. beginne에 대한RS, 우리는 DiI의 정확한 재현 순위를 보장하기 위해 wildtype 배아에서 촬영 오픈 도서에 연습하는 것이 좋습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 0.5 mm 유리 모세관 </td><td> 세계 정밀 계측기 </td><td> 1B120F-4 </td></tr><tr><td> 유리 바늘 풀러 </td><td> Narishige </td><td> PC-10 </td></tr><tr><td> Electroporator </td><td> BTX </td><td> ECM 830 </td></tr><tr><td> Sylgard 실리콘 엘라스토머 </td><td> 세계 정밀 계측기 </td><td> SYLG184 </td></tr><tr><td> 텅스텐 와이어, 0.075 mm </td><td> 세계 정밀 계측기 </td><td> TGW0325 </td></tr><tr><td> 곤충 핀, 0.20 mm </td><td> 정밀 과학 도구 </td><td> 26002-20 </td></tr><tr><td> 곤충 핀, 0.10 mm </td><td> 정밀 과학 도구 </td><td> 26002-10 </td></tr><tr><td> 봄 가위 </td><td> 로 정밀 과학OLS </td><td> 15003-08 </td></tr><tr><td> 뒤몽 # 5 포셉 </td><td> 정밀 과학 도구 </td><td> 11252-20 </td></tr><tr><td> 뒤몽 # 55 포셉 </td><td> 정밀 과학 도구 </td><td> 11255-20 </td></tr><tr><td> 빠른 DiI </td><td> 분자 프로브 </td><td> D-7756 </td></tr><tr><td> 형광 현미경 </td><td> 하늘 </td><td> SZX12, BX51 </td></tr></tbody></table>

in ovo electroporation of mirna-based plasmids in the developing neural tube and assessment of phenotypes by dii injection in open-book preparations

Commissural dI1 뉴런은 광범위하게 개발 1,2 동안 축삭 안내를 근간 메커니즘을 명료하게하다하는 연구되었습니다. 이 뉴런은 등쪽 척추에 위치하고 있으며, 틀에 박힌 궤도를 따라 자신의 axons을 보내 수 있습니다. Commissural axons는 처음으로 다음 floorplate에서 ventrally 프로젝트. 중간 선을 넘어 한 후에는 다음과 axons은 길이 방향으로 뇌에 대한 날카로운 뱃 부리 장식이있는 회전과 프로젝트를합니다. 다음 단계에 각각 매력적이고 혐오스러운지도 단서의 조율 된 활동에 의해 조절된다. 이러한 신호의 올바른 해석은 demarcated 경로를 따라 axons의지도에 매우 중요합니다. 따라서, commissural 축삭 안내에 특정 분자의 생리 기여 이상적인 생활 배아의 맥락에서 조사하고 있습니다. 따라서, 생체 내 유전자 최저는 정확하게 신중하게 여러 재생할 수 있습니다 유전자의 축삭 유도 활동을 구분하기 위해 통제되어야합니다개발하는 동안 역할. 여기, 우리는 셀 타입 별, 추적 방식으로 닭 신경 튜브의 분해 유전자 발현하는 방법을 설명합니다. 우리는 3 셀 타입 별 발기인 / 형광 단백질 마커의 표현을 유도 강화를 품고 miR30-RNAi의 성적표 4 (cDNA 인코딩 형광 단백질의 3&#39;-UTR 내에있는 ()에 의해 직접 다음 소설 플라스미드 벡터를 사용하여 그림 1). 개발 신경 튜브에 electroporated 때,이 벡터는 유전자 발현의 효율적인 downregulation을 유도하고 최저 3 체험 세포의 추적 직접 사용 밝은 형광 마커 단백질을 표현한다. electroporation하기 전에 다른 RNAi 벡터를 혼합하면 척추의 독립적 인 지역에서 두 개 이상의 유전자의 동시 분해 할 수 있습니다. 이 빠른 방식, S에서 개발되는 동안 검사되는 복잡한 세포와​​ 분자 상호 작용을 허용, imple 정확하고 저렴. 오픈 도서 준비 5 commissural 축삭의 탄도의 DiI 추적과 함께,이 방법은 commissural 축삭의 성장과지도의 세포와 분자 메커니즘의 생체 연구를위한 유용한 도구입니다. 원칙적으로 모든 발기인 / 인핸서는 잠재적으로 개발 6시 유전자 기능의 생체 연구에 대한 기술이 널리 적용하고, 사용할 수 있습니다. 이 동영상을 먼저 처리하는 방법을 보여줍니다 및 창 계란, 신경 튜브에 DNA의 plasmids의 주입과 electroporation 절차. commissural 축삭 안내를 조사하려면, 척추은 오픈 북 준비로 배아에서 제거 고정하고, 축삭 경로를 추적 할 수 있도록 DiI으로 주입된다. 척추 코드는 coverslips 사이에 장착 공 촛점 현미경을 사용하여 시각화합니다.

Watch this Scientific Journal Video about In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations at JoVE.c

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

신경 튜브의 유전자 발현은 세포 유형의 특정, 추적 방식으로 downregulated 할 수있는이 방법이 설명되어 있습니다. 우리는 방법을 보여줍니다<em&gt; 비켜에서</emspatiotemporally 제어 RNA 간섭을 이끌어내는 microRNA 기반 plasmids의&gt; electroporation은 개발 신경 튜브에 commissural 축삭 안내를 조사하는 데 사용할 수 있습니다.

오픈 책 준비에 DiI 주입하여 개발 신경 튜브와 Phenotypes의 평가에 miRNA 기반 Plasmids의 비켜 Electroporation에

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

in-ovo-electroporation-mirna-based-plasmids-developing-neural-tube

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

16.4K 조회수.  University of Zurich. 신경 튜브의 유전자 발현은 세포 유형의 특정, 추적 방식으로 downregulated 할 수있는이 방법이 설명되어 있습니다. 우리는 방법을 보여줍니다 비켜에서 electroporation은 개발 신경 튜브에 commissural 축삭 안내를 조사하는 데 사용할 수 있습니다.

비디오: In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

배아 척수에서 뉴런의 Commissural 해부와 문화

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

연구

신경과학

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

행동 내분비학 및 실험 경제학을 결합 : 테스토 스 테론과 사회 의사 결정

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

개발 Zebrafish Forebrain에 Clonally 관련 신경 전구 세포의 시간 경과 라이브 영상

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

개발 마우스 망막의 생체내 Electroporation에

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

에 의해 개발 마우스 내이 (内耳)에 진 전송<em&gt; VIVO에서</em&gt; Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

의 배아와 성인 신경 줄기 세포의 확장<em&gt; 자궁에</em&gt; Electroporation 또는 바이러스 성 Stereotaxic 주입

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

메뚜기에 신경 활동을 특성화하기 위해 다중 단위 기록 방법 (<em&gt; Schistocerca 아메리카나</em&gt;) 강한 회로

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

마우스의 유전자 조작을 통해 시상 하부 개발<em&gt; 자궁 내</em&gt; 일렉트로

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

본래 Vomeronasal 기관 및 액세서리 후각 망울의 생체 준비

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...