저자 electroporation 챔버의 섹션에 대해 설명 건설과 그녀의 도움 카렌 로렌스 감사하고 싶습니다.

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-Seq+identification+of+weakly+conserved+heart+enhancers.">ChIP-Seq identification of weakly conserved heart enhancers.</a> Nat Genet. 42, 806-810 (2010).</li><li>Visel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+accurately+predicts+tissue-specific+activity+of+enhancers.">ChIP-seq accurately predicts tissue-specific activity of enhancers.</a> Nature. 457, 854-858 (2009).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proc Natl Acad Sci U S A. 101, 16-22 (2004).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+transgenes+introduced+by+in+vivo+electroporation.">Controlled expression of transgenes introduced by in vivo electroporation.</a> Proc Natl Acad Sci U S A. 104, 1027-1032 (2007).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cereb Cortex. 19, 120-125 (2009).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gene+function+in+the+retina.">Analysis of gene function in the retina.</a> Methods Mol Biol. 423, 259-278 (2008).</li><li>Lee, J., Myers, C. A., Williams, N., Abdelaziz, M., Corbo, 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=Quantitative+fine-tuning+of+photoreceptor+cis-regulatory+elements+through+affinity+modulation+of+transcription+factor+binding+sites.">Quantitative fine-tuning of photoreceptor cis-regulatory elements through affinity modulation of transcription factor binding sites.</a> Gene Ther. 17, 1390-1399 (2010).</li><li>Carter-Dawson, L. D., LaVail, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rods+and+cones+in+the+mouse+retina.+II.+Autoradiographic+analysis+of+cell+generation+using+tritiated+thymidine.">Rods and cones in the mouse retina. II. Autoradiographic analysis of cell generation using tritiated thymidine.</a> J Comp Neurol. 188, 263-272 (1979).</li><li>Young, 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=Cell+differentiation+in+the+retina+of+the+mouse.">Cell differentiation in the retina of the mouse.</a> Anat Rec. 212, 199-205 (1985).</li><li>Corbo, 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=CRX+ChIP-seq+reveals+the+cis-regulatory+architecture+of+mouse+photoreceptors.">CRX ChIP-seq reveals the cis-regulatory architecture of mouse photoreceptors.</a> Genome Res. 20, 1512-1525 (2010).</li><li>Kim, D. S., Matsuda, T., Cepko, C. 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관심 없음 충돌 선언하지 않습니다.

Explant의 electroporation은 개발 마우스 망막에서 CIS - 규제 활동을 quantifying의 간단한 수단입니다. 마우스 transgenesis 통해 CIS - 규제 분석에 비해, electroporation은 신생아 마우스 새끼, DNA, 해부 악기, 그리고 electroporation / 조직 문화 장비를 필요로하는 많은 저렴합니다. 그것은 훨씬 적은 시간 또한 많이 소요 : 한 실험 영상 및 데이터 분석을위한 실험의 끝에 유일한 준비 시간이 몇 시간에 대한 팔일의 문화 시대, 그리고 몇 시간이 필요합니다. 실제 망막 조직이 활용되기 때문에 Explant의 electroporation은 또한 세포 문화 기반 CIS - 규제 분석 우수합니다. 망막은 explant에서 매우 일반적으로 개발 문화는 photoreceptors은 외부 세그먼트를 정교하지 있지만, 세 개의 서로 다른 세포 레이어를 (외부 핵 계층 내부 핵 층과 신경절 세포 층) 양식. 추가 장점은 explant의 electroporation 높은 재현성입니다. 심지어 다른 일에 다른 망막에 electroporated 동일한 구조, 동일한 상대 표현 수준에서 일반적으로 발생합니다. 또한, electroporated plasmids이 핵에 episomally 유지 생각하고 염색체에 포함되지 않습니다 때문에, 그들은 유전자 변형 생쥐에서 수행 CIS - 규제 분석을 현혹하는 동일한 통합 사이트 효과를 적용하지 않습니다. Explant의 electroporation 몇 가지 제한이 없습니다. 첫째, 세포주기에 여전히에만 세포가 효율적으로 electroporation 15 일까지 transduced 수 있습니다. P0에서 막대 및 기타 나중에 태어난 망막 세포 유형 (바이폴라 세포, amacrine 세포, M ller의 glia)이 방법에 의해 대상의 주요 세포 집단입니다. P0 electroporation에 의해 원뿔 photoreceptors의 Electroporation은 16을보고하지만 효율이 낮은 것으로 나타납니다되었습니다. 두 번째 제한은 망막의 진보보기 흉한 것 2 주 결과 너머 그 explant 문화이므로 권장하지 않습니다. 제 9에서 설명한대로 발기인의 부량 늦게 timepoints에 필요한 경우 단, 생체내의 electroporation 9가 원하는 timepoint에서 망막 절개로 뒤를 수행할 수 있습니다 해부하는 망막의 평면 장착하고, 부량. 세 번째 제한은이 분석은 적당히 높은 처리량있다는 것입니다. 한 실험에 구조의 수백을 테스트할 수 세포 배양 기반 assays과는 달리,이 프로토콜에 설명된 기술은 구성 당 하나의 전체를 마우스 망막의 최소 필요합니다. 따라서, 전용 핀셋으로 구성이 합리적 하루에 electroporated 수 있습니다. 현재 접근법을 사용 발기인 활동의 부량과 관련하여 한 추가 주의해야 할 점은 매우 강력한 발기인을 시금 때 특히, GFP의 채널로 DsRed 형광의 &#39;출혈을 통한&#39;에 대한 가능성이있다는 것입니다. 그 이유는의 방출 스펙트럼 그 GFP의 일부 중복을 DsRed 것입니다. 이 문제를 회피하기 위해 최적화된 방출 필터는 DsRed와 GFP 사이의 스펙트럼 중복을 최소화 것을 사용해야합니다. 이러한 최적화된 필터 설정을 사용할 수없는 경우, 다른 잠재적인 솔루션은 GFP 대신 푸른 이동 형광 단백질 (예, BFP 또는 CFP)를 사용하는 것입니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> Electroporation 요리, Microslide 453 </td><td> BTX 하버드 장치 </td><td> 45-0105 </td><td> 수정에 대한 프로토콜 섹션 1을 참조 </td></tr><tr><td> 100 % 실리콘 고무 수족관 시멘트 </td><td> 퍼펙토 제조 </td><td></td><td></td></tr><tr><td> 플라스틱 microtube 걸이 </td><td> 피셔 사이 언티픽 </td><td> 05-541 </td><td> 전용 랙 손잡이가 사용됩니다 </td></tr><tr><td> Dremel 도구 </td><td></td><td></td><td> 플라스틱 튜브 대뿐이야 핸들을 절단 </td></tr><tr><td> DMEM </td><td> Gibco / Invitrogen </td><td> 11,965 </td><td></td></tr><tr><td> F12 </td><td> Gibco / Invitrogen </td><td> 11,765 </td><td></td></tr><tr><td> L-Glu/pen/strep </td><td> Gibco / Invitrogen </td><td> 10378-016 </td><td> 100X 농도 </td></tr><tr><td> 인슐린 </td><td> 시그마 - 알드리치 </td><td> I - 6634 </td><td> 1000X 주식, 5mM HCL 및 필터 - 소독의 5mg/ml에 resuspend </td></tr><tr><td> FBS </td><td> Gibco / Invitrogen </td><td> 26140-079 </td><td></td></tr><tr><td> ECM 830 스퀘어 - 웨이브 electroporator </td><td> BTX 하버드 장치 </td><td></td><td></td></tr><tr><td> Nuclepore 필터 </td><td> 워트 먼지 </td><td> 110606 </td><td> 25mm, 0.2μm </td></tr><tr><td> 조직 문화의 인큐베이터 </td><td></td><td></td><td> 37 ° C 5 % CO 2 </td></tr><tr><td> 유리 coverslips # 1.5 </td><td> 피셔 사이 언티픽 </td><td> 12 - 544E </td><td> 두께 0.16mm </td></tr><tr><td> 카메라가 장착된 형광등 복합 현미경 </td><td></td><td></td><td> 카메라 단색 (하마 마츠에 의해 예, 오르카 - ER 카메라)되어야합니다 </td></tr><tr><td> EGFP / 복합 현미경에 대한 DsRed 필터 세트 </td><td> 채도 기술 주식 회사 </td><td> 86,007 </td><td> 빨간색과 초록색 chanenels 사이에이 필터 세트 최소화 bleedthrough </td></tr><tr><td> ImageJ 소프트웨어 </td><td> NIH </td><td></td><td> <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> </td></tr></tbody></table>

quantifying the activity of cis-regulatory elements in the mouse retina by explant electroporation

세포 유전자 네트워크 내에서 전사 요소 짧은 CIS - 규제 요소 (CRES)에 바인딩하여 spatiotemporal 패턴과 목표 유전자의 표현 수준을 제어 (~ 300-600 BP) 상류 거짓말을 할 수 게놈의 DNA 뻗어, 하류, 또는 시간 그들이 제어 유전자의 introns. 크르 (즉, 강화 / 발기인)은 일반적으로 1-3 transcriptional activators 및 repressors 모두에 대해 여러 개의 클러스터 바인딩 사이트로 이루어져 있습니다. 그들은 transcriptional 입력 논리적으로 통합이 spatiotemporally 정확하고 양적 정확한 발기인 활동의 형태로 하나의 출력을 제공 역할을합니다. 최신 CIS 규제 포유류의 대부분의 연구 4-5 CRES의 확장기 기능을 시금의 수단으로 마우스 transgenesis에 의존합니다. 이 기술은 주로 비 양적, 삽입 사이트 효과를 계정에 시간이 걸리는 비용과입니다. 한편, 포유 동물 CRE 기능에 대한 정량 assays는 조직 문화 시스템 (예를 들어, 듀얼 루시페라제 assays)에서 개발하지만, 이러한 결과의 생체내 관련성에서 종종 확실치되었습니다. Electroporation은 포유류의 조직 생활에서 CIS - 규제 활동 spatiotemporal와 양적 평가 모두를 허용한다는 점에서 전통적인 마우스 transgenesis에 훌륭한 대안을 제공합니다. 이 기술은 특히 대뇌 피질과 망막 6-8에서, 중추 신경 시스템에서 CIS 규제의 분석에 특히 유용했습니다. 생체내 및 전직 생체내 마우스 모두에서 망막 electroporation은, 개발 광범위하게 쓰다 및 Cepko 6-7,9에 의해 설명하고있는 동안, 우리는 최근 electroporated 마우스 망막 10 photoreceptor 특정 CRES의 활동을 계량하는 간단한 방법을 개발했습니다. electroporation에 의해 망막에 도입 DNA의 양이 실험에서 실험에 따라 다를 수 있습니다 감안할 때, 그것은 모든 실험에서 공동 electroporated &#39;로딩 제어&#39;를 포함하는 것이 필요합니다. 이러한 측면에서 기술 교양 세포에서 발기인 활동을 계량하는 데 사용되는 듀얼 루시페라제 분석과 매우 비슷합니다. photoreceptor CIS - 규제 활동을 시금 때, electroporation은 일반적으로 최고 막대 생산 11-12의 시간 신생아 생쥐 (출생 후의 일 0, P0)에서 수행됩니다. 일단 망막 세포 유형 사후 mitotic되고, electroporation들은 훨씬 더 효율적입니다. 신생아 마우스 및 막대는 성인 마우스 망막에있는 세포의 이상이 70 %를 구성한다는 사실에 막대의 출생의 높은 비율을 감안할 때, P0에서 electroporated 아르 세포의 대부분은 막대합니다. 이러한 이유로, 막대의 photoreceptors은 electroporation을 통해 공부하는 가장 쉬운 망막 세포 유형입니다. 우리가 여기서 설명하는 기술은 photoreceptor 크르의 활동을 quantifying 주로 유용합니다.

Watch this Scientific Journal Video about Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation at JoVE.com

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

이 프로토콜은 explant의 electroporation을 통해 마우스 망막 생활 CIS - 규제 요소의 활동 (즉, 확장기 / 발기인)를 수치 간단하고 저렴한 방법을 설명합니다. DNA의 준비, 망막 절개, electroporation, 망막 explant의 문화, 포스트 고정 분석과 부량이 설명되어 있습니다.

의 활동을 Quantifying CIS</emExplant Electroporation하여 마우스 망막에서> - 규제 요소

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

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Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

14.4K 조회수.  Washington University School of Medicine. 이 프로토콜은 explant의 electroporation을 통해 마우스 망막 생활 CIS - 규제 요소의 활동 (즉, 확장기 / 발기인)를 수치 간단하고 저렴한 방법을 설명합니다. DNA의 준비, 망막 절개, electroporation, 망막 explant의 문화, 포스트 고정 분석과 부량이 설명되어 있습니다.

비디오: Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

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 ...

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신경과학

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 ...

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle (reviewed in 1). The rhombic lip can be subdivided into the upper rhombic lip (URL) which encompasses rhombomere 1 (r1) and generates neurons of the cerebellum and the lower rhombic lip (LRL) which gives rise to diverse neuronal brainstem lineages 2-4. LRL derivatives include the auditory neurons of the cochlear nuclei and those of the precerebellar nuclei that are involved in regulating balance and motor control 5-8. Neurogenesis from the LRL occurs over a large temporal window that encompasses embryonic days (E) 9.5-16.55, 9. Different neuronal lineages emerge from the LRL as postmitotic cells (or are born) during distinct developmental days during this neurogenic window. 
Electroporation of gene expression constructs can be used to manipulate gene expression in LRL progenitors and can potentially change the fate of the neurons produced from this region 10-12. Altering gene expression of LRL progenitors in the mouse via in utero electroporation has been highly successful for manipulating lineages born on embryonic day E12.5 or later 10, 12-14. In utero electroporations prior to E12.5 have been unsuccessful primarily due to the lethality associated with puncturing the fourth ventricle roofplate, a necessary step in delivering exogenous DNA that is electroporated into the LRL. However, many LRL derived lineages arise from the LRL earlier than E12.5 9. These earlier born lineages include the neurons that comprise the lateral reticular, external cuneate, and inferior olivary nuclei of the precerebellar system which function to connect inputs from the spinal cord and cortex to the cerebellum 5. In order to manipulate expression in the LRL of embryos younger than E12.5, we developed an in vitro system in which embryos are placed into culture following electroporation.
This study presents an efficient and effective method for manipulating the gene expression of LRL progenitors at E11.5. Embryos electroporated with green fluorescent protein (GFP) driven from the broadly active CAG promoter reproducibly expressed GFP after 24 hours of culture. A critical aspect of this assay is that gene expression is only altered because of the expression of the exogenous gene and not because of secondary effects that result from the electroporation and culturing techniques. It was determined that the endogenous gene expression patterns remain undisturbed in electroporated and cultured embryos. This assay can be utilized to alter the fate of cells emerging from the LRL of embryos younger than E12.5 through the introduction of plasmids for overexpression or knock down (through RNAi) of different pro-neural transcription factors.

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

This study describes the development of an in vitro electroporation technique that allows for the manipulation of gene expression in the lower rhombic lip of midgestation embryos.

Midgestation 마우스 태아의 아래쪽 사방 정계 립의 시험 관내 Electroporation에

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

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 ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

마우스 망막을 사용하여 중추 신경계의 혈관 재생의 평가

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

마우스 뇌의 다양한 지역에서 텔로 머라 제 활동 : 비 방사성 텔로 머라 제는 증폭 프로토콜을 반복 (TRAP) 분석

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

이미징 CA<sup&gt; 2 +</sup마우스 망막의 광 수용체 콘 축삭 터미널에서&gt; 역학

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Electroporation에 의해 쥐 좌골 신경의 슈반 세포로 생체 내 유전자 전달에

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

성인 마우스 지 루트 신경 절의 Electroporation Vivo에서 조사 포유류 축 삭 재생:

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

의 활동을 Quantifying CIS - 규제 요소