저자는 SCE micromicropipette 팁의 전자 현미경 이미지에 대한 미숙 신경 세포와 데릭 던필드의 시간 경과 영화 캡처 성장을위한 Sharmin Hossain 감사합니다.

장비 설정 : <ol><li> 이 기법에 필요한 전기 장비 자극기, 오실로 스코프 및 micropipette에 삽입하는 은색 철사가 장착되어 micropipette 홀더입니다. 우리는 일반적으로 액슨 악기 Axoporator 800A 자극기를 사용하지만 같은 그라스 악기와 같은 다른 일반적인 stimulators는 SD9 stimulators는 단일 셀 electroporation에도 적합합니다. 자극기는 내부 지휘 솔루션을 포함하고있는 유리 micropipette 안에 배치하는 실버 와이어 전극과 인터페이스 headstage에 연결되어 있습니다. 자극기에서 다른 연결은 또한 올챙이 외부 접지로부터 입력을받는 오실로 스코프 입력에 연결되어 있습니다. 올챙이 외부 접지는 단순히 electroporation 동안 올챙이와 electrotonic 접촉에 남아 은색 와이어입니다. micropipette이 올챙이와 접촉에 배치되면 회로가 완료됩니다. </li><li> 이 책략 공간을 제공하고 micropipette 대해서 30-45 도의 각도에서 가져온 수 있습니다 때문에 단세포 electroporation은 잘 작동 거리 (최소 5cm)와 현미경이 필요합니다. </li></ol> micropipettes의 제조 : 적절한 micropipettes의 작성이 프로토콜에서 중요한 단계입니다. 어려움은 조직을 펑쳐링 때 쉽게 부숴지지 하나 좁은 팁 (직경 1μm 이하)를 균형에있다. 우리는 또한 큰 10도 정도의 테이퍼 각도와 도움말을 바람직하다는 사실을 발견했습니다. <ol><li> 1μm 이하의 팁 직경, 그리고 이상 10도의 팁 테이퍼 각도로 뽑아 유리 micropipette를 준비합니다. <ol><li> 0.75mm의 1.5mm와 내경의 외경과 내부 필라멘트와 Borosilicate 유리가 잘 이러한 목적에 가장 적합합니다. <ol><li> 비교적 두꺼운 유리 팁에 강성의 학위를 제공합니다. </li><li> 내부 필라멘트는 micropipette 팁쪽으로 채워진 액체를 그리기에 중요합니다. </li></ol></li></ol></li><li> 적절한 도움말 제작은 종종 관찰 결과에 따라 몇 가지 수정을 요구할 것입니다. 다른 조직은 약간 다른 모양 팁 직경과 팁을 요구할 수 있습니다. </li></ol> 단일 셀 electroporation 프로토콜 : 신규 사용자를 위해, 그것은 유전자 코드 형광 단백질과는 달리, 그들은 즉시 epifluorescence 아래에서 볼 수있다, 때문에 형광 dextran의 염료와 함께 시작하는 것이 좋습니다. 형광 염료의 사용은 사용자가 직접 조직 내에서 micropipette 팁의 위치를​​ 볼 수있게하고,시 electroporation은 장비가 제대로 micropipette 팁 적절한 여부를 설정하고되었는지 여부에 대해서 즉각적인 피드백을 제공합니다. <ol><li> 중 로딩 주사기를 사용하거나 백업 작성하여 electroporated하는 화합물의 솔루션 micropipette를 입력합니다. <ol><li> 플라스미드 DNA를 사용하는 경우, 물 속에 준비, 샘플은 내독소 - 무료 확인하고, 1-2μg/ml 사이의 농도에서. </li><li> 형광 dextran의 염료의 농도는 경험적으로 결정하여야한다. 우리는 종종 약 300μM로 물에 희석 형광 염료를 사용합니다. </li><li> 일반적으로, 0.5 - 1μl의 볼륨이 충분합니다. </li><li> 이전 로딩하기 위해 간략하게 괴롭히는가 발생할 수 있습니다 micropipette로 그려진 미립자 파편의 양을 줄이기 위해 고속 솔루션을 원심 분리기. </li><li> 팁 내에 공기 방울은 종종 현재의 흐름에 영향을하고 적극적으로 headstage에 장착하기 전에 micropipette를 flicking하여 dislodged해야합니다. </li></ol></li><li> 홀더에 micropipette를 삽입하거나 headstage 3 축 micromanipulator에 장착. 그 은색 와이어 전극이 micropipette에 삽입하고, 내부 솔루션 electrotonic 연락입니다 확인하십시오. </li><li> 올챙이 양육 매체 (스타 인 버그의 솔루션, 산도 7.4)에 준비가 0.02 % MS - 222 (3 - 아미노 벤 초산 에틸 에스테르)의 침수에 의해 tadpoles를 마취. <ol><li> 우리는 일반적으로 실험에서 무대 44-48 흰둥이 Xenopus laevis tadpoles를 사용합니다. </li><li> Tadpoles는 약 5 분 후에 일반적으로 완전히 anesthetized 있습니다. </li><li> 여러 tadpoles 기다리는 시간을 줄이기 위해 동시에 anesthetized 수 있습니다, 그러나 1 시간 이상의 기간 동안 MS - 222에 노출하지 마십시오. </li></ol></li><li> 플라스틱 전송 micropipette를 사용하여 electroporation 챔버에 하나 anesthetized 올챙이을 전송합니다. <ol><li> electroporation 챔버 쉽게 작은 Sylgard ® 실리콘 블록의 올챙이 모양의 구멍을 조각하고, 그것은 챔버에있는 올챙이와 electrotonic 연락을 것입니다 이러한 캐비티에 은색 접지 와이어를 삽입하여 내부에서 만들 수 있습니다. </li></ol></li><li> 를 사용하여 위치 올챙이 등의 측면 페인트를 moistened. </li><li> 그것은 피부와 접촉에 대한 관심의 영역에 직접 인접한 때까지 micropipette를 낮춥니다. <ol><li> 밀도가 높은 포장 세포 기관과 지역을 타겟팅하면 크게 성공 electroporation의 기회를 증가할 것이다.우리는 일반적으로 올챙이 광학 tectum를 타겟팅할 수 있습니다. </li><li> 30-45 도의 각도에서 micropipette 가져와. 험한 각도가 자주 하나 전망을 방해하면서 얕은 궤도는 피부 찔린 더 어렵게합니다. </li></ol></li><li> 지속적인 micropipette의 저하 처음 피부 보조개 다음 기본 조직으로 통과됩니다. <ol><li> 피상적인 세포는 생체내 이미징에 대한 선호하고 있으며, micropipette 팁은 피부의 dimpling에 따라 천천히 저하되어함으로써 타겟팅할 수 있습니다. </li></ol></li><li> 하나는 지속되면 조직 내에 위치 micropipette 저항을 모니터링해야합니다. Axoporator 800A (액슨 인 스트 루먼트), 약 10 40MΩ의 resistances를 사용하는 경우 최적입니다. 저항의 팁 직경은 단일 셀 electroporation에 적합한지 여부의 좋은 지표입니다. <ol><li> 하이 micropipette의 resistances은 (&gt; 100MΩ) 너무 좁고 조언들로 가득 찼어 팁 또는 micropipettes 나타내는 있습니다. </li><li> 낮은 micropipette의 resistances (&lt;10MΩ)는 종종 깨진 팁의 결과가 너무 커서 팁 직경을 나타내는 있습니다. </li><li> 직접 저항 측정을 제공하지 않습니다 자극기를 사용하는 경우, micropipette 저항은 간접적으로 오실로 스코프 (아래 설명)에 의해 측정 전압 펄스의 진폭을 관찰하여 측정하실 수 있습니다. </li></ol></li><li> 전압 열차를 적용합니다. <ol><li> 플라스미드 DNA를, 펄스는 500ms의 펄스 트레인 기간과 300Hz에서 제공 기간이 1ms 부정 사각형 웨이브 펄스의 이루어져 가장 효과적으로 발견 열차. <ol><li> 펄스 전압은 오실로 스코프에서 측정된 펄스의 진폭이 0.75과 1.5μA 사이는 것을 경험적으로 그러한 결정됩니다. </li></ol></li><li> 형광 염료 dextran의 경우, 300μs 기간의 긍정적인 사각형 웨이브 펄스는 10ms의 펄스 트레인 기간과 300Hz의 주파수에서 전달됩니다. <ol><li> DNA에 대한 프로토콜과 마찬가지로 펄스 전압은 오실로 스코프에서 측정된 펄스의 진폭이 0.75과 1.5μA 사이는 것을 경험적으로 그러한 결정됩니다. </li><li> 자극 매개 변수 추가 조정은 epifluorescence 아래 electroporation의 결과를 관찰을 바탕으로 만들 수 있습니다. <ol><li> electroporated 세포가 희미 나타나면 세포가 밝은 나타날 때까지, 펄스 매개 변수는 점차 증가한다. </li><li> 세포의 클러스터는 레이블이있는 경우 하나의 세포가 표시되기 전까지는 반면에, 펄스 매개 변수는 감소한다. </li></ol></li></ol></li></ol></li><li> micropipette을 접어야 조직 내의 다른 사이트에 다시 삽입하고, 전압 기차를 적용할 수 있습니다. <ol><li> 수율을 개선하기 위해, 우리는 일반적으로 해당 레이블이 붙은 세포가 중복되지 않도록 충분한 간격 올챙이 광학 tectum 각 반구에서 여러 사이트를 electroporate. </li></ol></li><li> 스타 인 버그의 양육 솔루션 컨테이너에 electroporated 올챙이을 전송합니다. <ol><li> Tadpoles는 빠른 속도로 몇 분 이내에 마취에서 회복됩니다. </li></ol></li><li> 동일한 micropipette는 수많은 tadpoles 사용할 수 있습니다. 끊임없이 오실로 스코프에서 발견된 펄스의 진폭을 관찰하는 것이 중요합니다. <ol><li> 펄스 진폭이 크게 펄스 강도를 증가 후 낮은 남아있다면 이런 현상이 가능성이 한 번에 많은 tadpoles를 electroporating 때 종종 볼 수 막힌되는 팁의 결과입니다. <ol><li> 사소한 팁 방해할을 이동시키다하는 방법은 조직에 micropipette 삽입 후 하나의 긍정적인 펄스를 적용하는 것입니다. </li><li> 덩어리가 dislodged, 또는 자주 반복을 막히​​는 수없는 경우, micropipette를 교체하십시오. </li></ol></li><li> 펄스 진폭이 크게 펄스 매개 변수를 감소 이후 매우 큰 남아있을 경우, 이것은 끝 고장 및 교체해야하는 기호입니다. </li></ol></li></ol> 성공적으로 electroporated 세포에 대한 검사 : <ol><li> electroporation 후, tadpoles는 똑바로 epifluorescence 현미경을 사용하여 레이블이 세포에 대한 검사를하고 있습니다. </li><li> 형광 염료의 경우, tadpoles는 빠르면 30 분 이후 electroporation 후 심사 수 있습니다. <ol><li> 우리는 더 이상 간격이 낮은 배경 형광 수준과 연관된 것을, 그러나 발견했다. </li></ol></li><li> 유전자 인코딩 형광 단백질의 경우, 심사는 일반적으로 최소 12 시간 이후의 electroporation을 실시합니다. <ol><li> 형광 단백질을 표현하는 세포는 시간을 더 밝게하려면 것입니다 따라서 희미한 전지 12~24시간의 추가 간격 후 다시 심사 수 있습니다. </li></ol></li><li> Sylgard ® 챔버에 배치, 위에서 설명한 및 coverslipped로 Tadpoles는 anesthetized 있습니다. 개인 tadpoles는 다음 epifluorescence에 따라 심사됩니다. <ol><li> epifluorescence 빛에 과도하게 노출이 표시된 세포를 죽일 수 있습니다 phototoxicity 발생됩니다. 따라서 세포가 형광 불빛에 노출되는 시간을 최소화합니다. </li></ol></li><li> 스타 인 버그의 양육 솔루션 컨테이너에 tadpoles를 반환합니다. </li></ol>

<ol>
	<li>Bestman, J. E., Ewald, R. C., Chiu, S. L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+single-cell+electroporation+for+transfer+of+DNA+and+macromolecules.">In vivo single-cell electroporation for transfer of DNA and macromolecules.</a> Nature Protocols. 1, 1267-1272 (2006).</li><li>Dunfield, D., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+electroporation.">Single cell electroporation.</a> Encyclopedia of Neuroscience (4th Ed.). , .</li><li>Haas, K., Jensen, K., Sin, W. C., Foa, L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+in+Xenopus+tadpoles+in+vivo--from+single+cells+to+the+entire+brain.">Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain.</a> Differentiation. 70, 148-154 (2002).</li><li>Haas, K., Sin, W. C., Javaherian, A., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29, 583-591 (2001).</li></ol>

단일 셀 electroporation (SCE)는 유전자 기능을 결정하고 타겟 유전자 조작을 수행하기위한 강력한 도구입니다. 흰둥이 올챙이의 투명성과 두뇌의 접근이 이상적으로 살아있는 유기체 내에서 성장과 세포의 연결 이벤트를 시각화에 적합한이 모델 시스템을합니다. SCE는 단일 신경 세포의 성장의 시각화를 허용하고, 그렇지 않으면 변형 두뇌 속에 휴대 자율 조​​작을 수행할 수 있습니다. 이 동영상이 문서 Xenopus laev...

single cell electroporation in vivo within the intact developing brain

단일 셀 electroporation (SCE)는 생체내 준비를 포함한 그대로 조직 내의 개별 세포로 DNA 또는 기타 macromolecules의 전달을 허용하는 전문 기술입니다. 이 기법의 독특한 장점은 주변 조직이 변형 떠나는 동안 실험 조작은이를 통해 글로벌 트리 트먼트로 인한 사람의 휴대 자율 효과를 구별, 각각의 세포에서 수행할 수 있습니다됩니다. 생체내 이미징 기술의 고급과 결합하면 형광 마커 SCE는 세포 형태, 세포 성장, 그리고 초에서 일까지 timescales 이상의 세포 사건의 직접적인 시각화 수 있습니다. 이 기법은 생체내 및 전직 생체내 준비의 다양한 사용되지만, 우리는 Xenopus laevis의 tadpoles에 사용하기 위해이 기술을 최적화합니다. 이 동영상이 문서에서 우리는 세부 흰둥이 Xenopus 올챙이의 손상 뇌 내의 뉴런에 형광 염료 또는 플라스미드 DNA의 SCE에 대한 절차를. 우리는 또한 수율을 최적화하기위한 방법을 논의하고, 찬란 SCE에 의해 분류 뉴런의 사는 두 개의 광자 형광 이미징의 예를 보여줍니다.

Watch this Scientific Journal Video about Single Cell Electroporation in vivo within the Intact Developing Brain at JoVE.com

Single Cell Electroporation in vivo within the Intact Developing Brain

단일 셀 electroporation (SCE)는 생체내 준비를 포함한 그대로 조직 내의 개별 세포로 DNA 또는 기타 macromolecules의 전달을 허용하는 전문 기술입니다. Xenopus laevis 올챙이의 손상 뇌 내의 뉴런에 형광 염료 또는 플라스미드 DNA의 SCE에 대해 우리가 세부 절차를.

완전한 개발 두뇌 속에 생체내에서 단일 세포 Electroporation

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

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Biology

13.2K 조회수.  University of British Columbia - UBC. 단일 셀 electroporation (SCE)는 생체내 준비를 포함한 그대로 조직 내의 개별 세포로 DNA 또는 기타 macromolecules의 전달을 허용하는 전문 기술입니다. Xenopus laevis 올챙이의 손상 뇌 내의 뉴런에 형광 염료 또는 플라스미드 DNA의 SCE에 대해 우리가 세부 절차를.

비디오: Single Cell Electroporation in vivo within the Intact Developing Brain

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Mycobacteria의 Electroporation

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

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생물학

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

마우스 망막 신경절 세포의 유전자 발현에 대한 utero 및 전직 생체내의 Electroporation에

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

사용 포유류 골격 근육의 유전자 Transfection VIVO에서 Electroporation

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

마우스 콘 Photoreceptors에서 단일 셀 석션 레코딩

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

생성중 성인 Zebrafish 테일 핀으로 Morpholinos의 생체내 Electroporation에

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

단일 미토콘드리아 과산화물 온전한 마음에 점멸 또는 공 촛점 이미징<em&gt; 생체</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

칼슘의 평가는 그대로 골격 근육 섬유에서 불꽃

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

마우스 고환의 생체 내 미세 주입 및 Electroporation에에서

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...