Biz aslında başvuru 11 sayfa 130 çıktı mikroenjeksiyon pipeti üretim rakamı yayınlamak için izin Humana Basın teşekkür ederim; videografisi için Larry Dlugas ve Steven Wong, Eğitim İletişim OHSU Bölümü, video tasarım ve düzenleme için Larry Dlugas; Adam M. Ç &#39;Quinn, Kıdemli Tasarımcısı, teknik şematik sağlamak için özel yatay laminer akış kaput ve Les Goldsmith tasarımı için Trion / Envirco; Victor Monterroso, OG, Yüksek Lisans, Doktora ve Tom Chatkupt, DVM, Karşılaştırmalı Tıp OHSU Bölümü, rehberlik için bizim ile hayvan bakım protokolü, cerrahi teknikler ve profilaktik analjezi rejimi; Marcel Perret-Gentil, DVM, MS, veteriner dikiş teknikleri üzerine sadaka paylaşmak için; bizim Adobe Premiere Pro video mikroskopi bilgisayar iş istasyonu tasarımı için Edward Porsov, MS, ve Leah Beyaz ve LNS Jonas Hinckley (Portland, OR) Captioning. Bu çalışma Sağırlık ve Othe Ulusal Enstitüsü bağışları ile desteklenmiştirr İletişim Bozuklukları: DC R01 008.595 ve DC R01 008.595-04S2 (JB) ve P30 DC005983 (Oregon İşitme Araştırma Merkezi Çekirdek Grant, Peter Gillespie, Proje Yürütücüsü).

1. Ventral Laparotomi <ol><li> Sodyum pentobarbital anestezik solüsyonu (gram vücut ağırlığı başına 7.5 uL) intraperitoneal enjeksiyonu ile; olan embriyoların embriyonik günde 11.5 (vajinal fiş algılandığında gün öğleden embriyonik gelişimin günlük 0.5 E11.5) altındadır baraj uyutmak. Anestezik solüsyonu Çalışma; mutlak etanol, 100 uL, 65 mg / mL sulu magnezyum sülfat (uterin tonu modüle) arasında 320 uL, 50 mg / mL sodyum pentobarbital çözeltisinin 180 uL ve propilen glikol 400 uL (sahip araç karışabilir sulu ve organik bileşenleri). </li><li> Ağrılı uyaranlara testleri yapılarak anestezi bütünlüğü değerlendirin: pençe sıkmak; kuyruk tutam, yanak ve burun kılı dokunma ve göz kırpma yanıtı. Kornealara steril oftalmik merhem uygulayın. </li><li> Suprapubik bölgeden makası ile göğüs kafesi ve ince bir bıçak (Oster # 40 bıçak) için karın kürk Tıraş. % 70 etanol ile karın,% 10 povidon iyot (Betadine), bir dezenfektend% 70 etanol, sırayla. Yer steril bir örtü üzerine fare karın tarafı aşağı ve sonra 2-5 dakika için bir ısıtma yastığı veya sıcak plaka (37 ° C) ayarlanır. </li><li> Top uçlu makasla ventral orta hat karın cildi sarın. 10-14 mm kesi uzatın. Linea alba, karın duvarının ventral orta hat boyunca bir avasküler, beyaz bağ dokusu bandı tanımlayın. Top uçlu makas ile linea alba kesilirken ve kesi 10-14 mm uzatmak. Hemen önceden ısıtılmış (37 ° C) Laktatlı Ringer solüsyonu ile karın sulanması. </li><li> Implantasyon sitelerde aşırı basınç uygulamadan halka forseps ile rahmin iki boynuzu yansıtmak. Ön ısıtması, Laktatlı Ringer solüsyonu ile dış kaynaklardan laparotomi sulayın. </li></ol> 2. Transuterine Mikroenjeksiyon <ol><li> 12-16 μ: aşağıdaki özelliklere sahip bir kalın duvarlı, borosilikat cam kapiller mikroenjeksiyon pipeti fabricatem dış çapı ve 20 derece eğimli. ; Basıncı = 200; hız = 46;; = 0 çekin zamanı = 100) ısı = rampa testi artı 3 ünite: küçük kutu filamanlı bir Sutter P-97 pipet çektirmenin, aşağıdaki programı kullanın. Sutter BV-10 beveler ile, geniş çaplı pipetler için 104C (altın) aşındırıcı disk kullanın. Kalsiyum fosfat 3-4 mikrogram / uL de plazmid Pastör pipetiyle ifade (pH 7,2-7,4) tamponlu tuz. 30 saniye boyunca yavaşça konsantre DNA, vortex ile kristal hızlı yeşil ekleyin ve 10 saniye boyunca 10.000 g dönerler. Görsel enjeksiyonunun etkinliğini izlemek için gerekli hızlı yeşil minimum tutar ampirik olarak belirlenir. DNA / hızlı yeşil çözümü ile eğimli pipet dolgu. Basınçlı enjektör (Picospritzer kaynak gazı olarak&gt;% 99 saf azot kullanarak) pipet sahibine yüklü mikroenjeksiyon pipeti bağlayın. </li><li> Düşük yoğunluklu, kendi implantasyon site içinde embriyo görselleştirmek için halojen ışık ile rahim Transillüminasyonda. Bea tanımlayınting kalp, beyin veziküller, bacak tomurcukları, arka beyin arasında doğmakta olan 4. ventrikül ve göz. Ön ısıtması ile her 2 dakika, hidrasyon korumak için Ringer çözeltisi laktatlı rahim sulayın. Embriyo yönlendirme ve anatomik yukarıda belirtildiği tanımlamak için rahim üzerinde hafif basınç uygulayın. </li><li> Orient embriyo anterior ve posterior dalları kanadını otocyst bulunduğu mezenkimal toprakları birincil baş damarı tespit etmek. Uygun otocyst rahim transillüminasyon tarafından görülemez. Otocyst damarın ana gövde ile birlikte, bir Amerikan futbol sahası üzerinde dikey veya goalposts şekli oluşturan, primer baş venin anterior ve posterior dalları arasındaki ortasında bulunur. Otocyst sabitlenmesini ortasındadır. </li><li> Otocyst ve olası konumu ile uyumlu bir yörünge içinde rahim yoluyla enjeksiyon pipeti yerleştirin. Uter geçtikten sonra bir kez mikroenjektör darbeliBizim izleyici boya ve pipet yaklaşık konumunu görselleştirmek için. Mikrometre kontrol altında pipet Advance ve derinlik değerlendirmek için tekrar darbe. Ayrıca sürekli yanal baş mezenşim ve nabız içine pipet ilerlemek. Başarılı otocyst hedefleme dorsale endolenfatik kanal ve tarak Vestibül kabuk şekli konik şekli ortaya çıkaracaktır. Rahim üzerinde baskı bırakın, tek hareketle embriyo / uterustan pipet çıkarın ve hemen ısıtması ile rahim sulanması, Ringer çözeltisi laktatlı. </li></ol> 3. Vivo olarak Elektroporasyon <ol><li> Laktatlı Ringer solüsyonu ile rahim sulayın. Taze uygulanmış laktatlı Ringer çözeltisi elektriksel çift kürek tarzı elektrotlara rahim gereklidir. Laktatlı Ringer ile elektrot tungsten yüzeyleri nemlendirin. Elektrotlar yolunda Merkezi enjekte otocyst. Yavaşça elektroporasyon pa rahim sıkıştırmakddles. Katod enjekte otocyst ve anot uninjected otocyst bitişik uterus duvarı ile temas halinde olduğu lateral uterus duvar ile temas halindedir. Elektroporatörün üzerine ayak pedalı anahtarı ile bir kare dalga darbe tetikler. Elektroporasyon parametreler şunlardır: puls başına 43 volt ve 950 msn interpulse gecikme az 5, 50 ms darbe. Puls teslim hemen sonra rahim sulanması. 60-100 mAmps Otik epitel ataları transferinde yeterlidir: doku teslim mevcut kaydedin. Enjekte edilir ve baraj başına 4-6, E11.5 embriyolar electroporate. </li><li> Olan iç kulaklarında olan bu embriyoların 4. ventriküle sulu floresan dekstran (Alexa Fluor 488 plazmid ifade bir yeşil floresan proteini kodlayan eğer plazmid ifade bir kırmızı floresan proteini veya Alex Fluor 594 kodlar varsa) ikinci bir bağımsız transuterine mikroenjeksiyon gerçekleştirin doğru enjekte edilir ve puls başına akımının en az 60 mAmps de olduelektroporasyon sırasında yürekli. Floresan dekstran ardbeyin doğumda tespit edilebilir ve iç kulak (3.5) embriyogenez sırasında manipüle edildi yavrularının seçimi sağlayacaktır. </li><li> Laktatlı Ringer ile laparotomi sulayın. Karın boşluğuna laparotomi yeniden takın. Ön ısıtması, Laktatlı Ringer çözeltisi 2-4 ml ile rahim boşluğuna boşaltır ve taşma steril örtüyü üzerine kesi dışarı akmasını sağlar. Kuru, steril malzeme ile dökümlülüğü değiştirin. Olmayan bir kesici iğne ve bir 6-0 emilebilir sütür ile karın duvarı dikin. Biz karın duvarı hem de deri için, her dikiş kilitleme, çalışan bir dikiş tercih. </li><li> Barajlar &#39;kürk kurulayın ve deri altı enjeksiyon yoluyla bir non-steroid anti-inflamatuar gibi Meloksikam gibi yönetmek. Steril bir örtü üzerinde önceden ısıtılmış kurtarma kafesine baraj dönün. Monitör ve kanlı akıntı için barajlar &#39;solunumda, yara yeri açıklığı ve vajina kaydedin. Kanama ra olduğunuyeniden ama mevcut ve hız kesmeden eğer o anestezi altında iken, baraj euthanize. O bilinç ve ambulasyon girişimleri kavuşur zaman unutmayın. O yeme ve içme belirtileri gösterir ve yuva inşa etmeye başlamıştır fare koloniye baraj dönün. Genellikle, bu 12 saat içinde gerçekleşir. </li><li> Doğumda (postnatal gün 0), flaş bir GFP veya Texas Red filtresi ile stereofluorescence mikroskop kullanarak floresan dekstran algılamak için çöp her yavru ile arka beyin bölgesi uygun şekilde ayarlayın. Sadece emziren baraj ardbeyin etiketleme gösterilecek bu yavrulardan dönün. </li></ol> 4. Temsilcisi Sonuçlar <img src="/files/ftp_upload/3653/3653fig1.jpg" alt="Şekil 1" /> Şekil 1.. Gelişmekte cochlea&#39;ya Elektroporasyon aracılı gen transferi. E11.5 otocyst (darbe tra bir ifade plazmid kodlama gelişmiş yeşil floresan protein (EGFP) ve electroporated ile enjekte edildiparametreler: Beş, 50 msn / nabız ve 950 msn interpulse gecikme) az 43 volt bakliyat. Otocyst koklea ortasında açmak yoluyla tabanından EGFP ifade gösteren E11.5 enjekte edildi ve electroporated yavru postnatal günde 6 (P6) A) bir temsilci iç kulak. Koklea lateral duvarında orta dönüş ve apeks sadece çıkarıldı. Apeks doğuran E11.5 ataları transfekte değildi. B) koklea İmmünoboyama Tüm mount (A) saç hücre belirteci ile, miyozin 7a (Myo7a), bu EGFP ifade Corti organı yörüngesini takip eder ve fena halde saç hücresi taşıyan duyusal epitel yerleştiğini gösterir. Otocyst E11.5 enjekte edildi ve electroporated bir E18.5 embriyo C) bir temsilci iç kulak. Lazer konfokal projeksiyon Myo7a pozitif duyusal kıl hücrelerinin EGFP ifade gösterir. EGFP expressio belirten Corti E18.5 organ koklear duyu epiteli D) Lazer konfokal projeksiyonn iç saç hücrelerinde (IHC), dıştaki saç hücreleri (OHC), iç falankslarında hücreleri (IPC), ayak hücreleri (PC), ve Deiters &#39;hücreleri (dc). (B) &#39;de ölçekli çubuğu (A) için de geçerlidir.

<ol>
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Çıkar çatışması ilan etti.

Gelişmekte olan fare iç kulağa gen transferi: Fare iç kulak postimplantation gelişim 12,13 ilk haftasında kulak taslakları gelişir. Embriyonik günde 9.5 (E9.5) tarafından, taslakları invajine ve otocyst 2 adı verilen bir sıvı dolu vesikül dönüştü. Vezikül içinde kulak öncüleri olgun iç kulak içinde duyusal ve duyunun hücreleri gibi vestibüler ve işitsel epitelde mekanik hassas kıl hücreleri innerve nöronlar doğurur. Geç evrelerinde olarak, iç kulak...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Mikro Sterilizasyon Kutusu </td><td> ROBOZ </td><td> RS-9900a </td><td> 8X8.5X1.25 inç </td></tr><tr><td> Bilye uçlu makas </td><td> Güzel Bilim Araçları </td><td> 14109-09 </td><td></td></tr><tr><td> Halka forseps </td><td> Güzel Bilim Araçları </td><td> 11106-09 </td><td> 4.8mm ID/6mm OD </td></tr><tr><td> Adson Doku Forseps </td><td> Güzel Bilim Araçları </td><td> 11027-12 </td><td></td></tr><tr><td> İğne sürücüsü </td><td> Güzel Bilim Araçları </td><td> 12502-12 </td><td></td></tr><tr><td> Alerji Şırınga Tepsi </td><td> Becton Dickison </td><td> 305536 </td><td></td></tr><tr><td> Sütür 6-0 </td><td> Syneture </td><td> GL-889 </td><td> 0.7 metrik gastrointestinal sütür </td></tr><tr><td> Ringer USP </td><td> Baxter </td><td> 2B2323 </td><td></td></tr><tr><td> Hızlı yeşil </td><td> Sigma Aldrich </td><td> F7258 </td><td></td></tr><tr><td> Borosilikat cam kılcal </td><td> Harvard Apparatus </td><td> 30-0053 </td><td></td></tr><tr><td> Nembutal Sodyum Çözüm </td><td> OVATION Pharmaceuticals Inc </td><td> NDC 67386-501-52 </td><td></td></tr><tr><td> MgSO4.7H2O </td><td> Fisher Scientific </td><td> M63-500 </td><td></td></tr><tr><td> Propilen glikol </td><td> Fisher Scientific </td><td> P355-1 </td><td></td></tr><tr><td> Etanol </td><td> Sigma Aldrich </td><td> E7023-500 </td><td></td></tr><tr><td> Meloksikam </td><td> Boehringer Ingeheim </td><td> ADA 141-219 </td><td></td></tr><tr><td> Mikropipet Çektirme </td><td> Sutter Aletleri </td><td> P-97 </td><td> FB255B kutusu filament; Sutter araçlardan Pipet Yemek danışın </td></tr><tr><td> Mikroelektrod Beveler </td><td> Sutter Aletleri </td><td> BV-10 </td><td> Büyük pipetler için 104C beveling diski; teorisi beveling için kullanım kılavuzuna başvurun </td></tr><tr><td> Mikropipet tutucu </td><td> Warner Aletleri </td><td> MP-S15T </td><td> Picospritzer tüp için 1,5 mm dış çaplı, pipet ve kadın basınç portu için. </td></tr><tr><td> Cımbız tarzı elektrot </td><td> Protech International Inc </td><td> CUY650P5 </td><td> 5 mm dış çapı </td></tr><tr><td> Kare Dalga Elektroporatör </td><td> Protech International Inc </td><td> CUY21EDIT </td><td> Footpedal önerilen </td></tr><tr><td> PICOSPRITZER III </td><td> Parker Hannifin </td><td> 051-0500-900 </td><td> Footpedal önerilen </td></tr><tr><td> Manuel Kontrol Mikromanipülatör </td><td> Harvard Apparatus </td><td> 640056 </td><td></td></tr><tr><td> Yatay laminer akış temiz tezgah </td><td> Envirco </td><td></td><td> LF 630-10.554 için özel modifikasyonlar. Kaput şematik için ek bilgilere bakın. </td></tr><tr><td> Leica stereofluorescence Lumencor SOLA Hafif bir motor ile mikroskobuyla diseksiyon </td><td> Bartels ve Stout ve Lumencor </td><td> Lumencor SOLA Hafif bir motor ile MZ10F </td><td> MZ10F odaklanmak ve SOLA ışık motor tetiklemek için Footpedals tavsiye edilir </td></tr><tr><td> Alexa Fluor 594 Dextran </td><td> Invitrogen </td><td> D22913 </td><td> 10mg/ml, sulu </td></tr><tr><td> Alexa Fluor 488 Dextran </td><td> Invitrogen </td><td> D22910 </td><td> 10mg/ml, sulu </td></tr><tr><td> Enviro-dri </td><td> Çoban Özel Papers </td><td></td><td> <a href="http://www.ssponline.com" target="_blank">www.ssponline.com</a> </td></tr></tbody></table>

gene transfer to the developing mouse inner ear by in vivo electroporation

; Kesecik ve kesecik içinde makulaları ve kıvrılmış kokleadaki Corti organı semisirküler kanalların ampullae 3 krista: memelilerin iç kulağının 6 farklı duyusal epitel vardır. Krista ve makulaları Corti organı işitsel saç hücreleri 1 işitme için birincil dönüştürücüler ise mekanik uyarıcılar, denge, özel anlamda hizmet etmek için transdüksiyonu vestibüler saç hücreleri içerir. Semisirküler kanallar ve koklea bu duyusal epitel ve morfonogenezi Hücre kaderi şartname fare gebeliğin ikinci haftasında gerçekleşecek ve büyük ölçüde doğumdan 2,3 önce tamamlanır. Fare iç kulak gelişim çalışmaları rutin hücresel ve / veya morfolojik fenotipleri 4,5 moleküler temelini anlamak için farklı embriyonik ya da doğum sonrası aşamalarında transgenik embriyolar hasat tarafından yürütülmektedir. Biz utero gelişmekte fare iç kulak veren gen transferi hipotez &lt;/ Em&gt; kazanç ve kayıp fonksiyon-çalışmaları bağlamında memelilerin iç kulağının gelişimi 6 altında yatan genetik mekanizmalar sorgulama için geleneksel fare transgenesis için ücretsiz bir yaklaşımı temsil ediyor. Ventral laparatomi) 1, 2) transuterine mikroenjeksiyon; ve in vivo elektroporasyon 3): Gelişmekte olan fare iç kulağa gen misexpression çalışmalar yapmak, deneysel paradigma burada gösterdiği üç genel adım içine giderir. Ventral laparotomi implante embriyo 7 deneysel erişmek için rahim dışsallaştırılmasını izin veren bir fare hayatta bir cerrahi tekniktir. Transuterine mikroenjeksiyon otik vezikül veya otocyst lümenine plazmid ifade tanıtmak Eğimli, cam kılcal mikropipetler kullanılmasıdır. Vivo elektroporasyon progenitör hücreler 8-10 içine plazmid ifade sürmek için kare dalga, akım darbeleri uygulamasıdır. 11 her adımla ilgili ayrıntılı notlar yer. Fare deneysel embriyolojik teknikleri nesir öğrenmek ve hareketsiz görüntüleri tek başına zor olabilir. Bu çalışmada, biz gen transferi prosedürü 3 adım göstermektedir. En önemlisi, biz nasıl tam olarak göstermek için dijital video mikroskopi dağıtmak: 1) anne karnında embriyo yönünü belirlemek; 2) otocyst için enjeksiyon hedefleme için embriyo edinenlerin; 3) embriyonik gün 11.5 olarak otocyst içine izleyici boya solüsyonu ile karıştırılarak DNA microinject ve 12.5; doğumda doğum sonrası seçim için ve 5) etiket electroporated embriyolar; 4) enjekte otocyst electroporate. Ortak metodolojik hataları önlemek için nasıl tartışmak;; otocyst mistargeting en sık nedenler resimsel bir kılavuz; başarıyla transfekte iç kulaklarında temsilcisi örnekler sağlamak ve utero g bir yazmak için mevcut esaslarıene transferi hayvan bakım protokolü.

Watch this Scientific Journal Video about Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation at JoVE.com

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

Fare iç kulak olan gelişim programı gebelikte özenli bir taslakları kaynaklı duyu organıdır. Biz bir tanımlamak<em&gt; Utero</emÜç adımdan oluşan&gt; gen transferi tekniği: fare ventral laparatomi, transuterine mikroenjeksiyon ve<em&gt; In vivo</em&gt; Elektroporasyon. Biz kritik deney embriyolojik tekniklerini göstermek için dijital video mikroskobu kullanın.

Tarafından geliştirilmesi Fare İç Kulak için Gen Transferi<em&gt; In Vivo</em&gt; Elektroporasyon

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 ...

gene-transfer-to-developing-mouse-inner-ear-vivo

Research

JoVE Journal

Neuroscience

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

13.9K Görüntüleme.  Oregon Health & Science University. Fare iç kulak olan gelişim programı gebelikte özenli bir taslakları kaynaklı duyu organıdır. Biz bir tanımlamak Utero gen transferi tekniği: fare ventral laparatomi, transuterine mikroenjeksiyon ve In vivo Elektroporasyon. Biz kritik deney embriyolojik tekniklerini göstermek için dijital video mikroskobu kullanın.

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

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Non-ventriküler Plazmid Enjeksiyon ve Elektroporasyon Postnatal Rat Beyin Gen Teslimat

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Nörobilim

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.

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

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

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

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

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

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Aktivitesi miktarının Cis</emEksplant Elektroporasyon Fare Retina> Düzenleme Elemanları

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

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.

Gelişmekte olan Fare Retina in vivo Elektroporasyon olarak

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Fare Utero Elektroporasyon: Kontrollü Spatiotemporal Gen Transfeksiyon

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Yalıtımlı LacZ ifade Sitometrisi kullanarak Fare İç Kulak Dokular Hücreler

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Gelişmekte Neuroepithelium Hücre Davranış Yüksek çözünürlüklü Canlı Görüntüleme

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

İç Kulak Saç Hücreleri Patch Clamp Kayıtlar İzole Zebrafish gelen

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

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.

Fare Genetik Manipülasyon yoluyla Hipotalamus geliştirilmesi<em&gt; Utero olarak</em&gt; Elektroporasyon

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

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Yuvarlak Pencere Membran aracılığıyla Fare İç Kulak için virally ve Gen Teslimat Cerrahi Yöntemi

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...