Name: hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice
Uploaded: 2016-07-09T13:00:00.000+00:00
Duration: 7 min 30 s
Description: Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

NIH R01MH084894 Bu çalışmanın finansmanı katıldı. Biz Dr teşekkür etmek istiyorum. farelerin bağış ve bu çalışmanın çekimleri sırasında cerrahi ve davranış tesislerin kullanımı için Mount Sinai Tıp Okulu Yasmin Hurd ve Scott Russo.

NOT: Hayvan ıslahı ve bakımları için tüm prosedürler Sina Dağı&#39;nda Tıp Icahn Okulu Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) yönetmeliğine göre yapılmıştır. prosedür boyunca steril eldiven kullandığınızdan emin olun. 1. İlaç ve Virüs Hazırlık <ol><li> İlaç Hazırlama <ol><li> 12.9 ml% 0.9 tuzlu su çözeltisi içinde 100 mg / ml Ketamin 1.35 ml ve 20 mg / ml ksilazin 0.75 ml eritilmesi ile 15.0 mi, ketamin / ksilazin anestezik hazırlayın. Iyice çözüm karıştırmak. </li></ol></li><li> Virüs hazırlanması <ol><li> MGlu2 Clone ve mGlu2ΔTM4N standart protokoller daha önce 6 açıklanan aşağıdaki bir bicistronic herpes simpleks virüsü (HSV) vektörü içine oluşturur. Daha önce tarif edildiği gibi 6,13,14 viral partikülleri paketleyin. Kalıntıların Değişiklik Ala-677 4.40, mGlu2 Ala-681 4.44 ve Ala685 4.48 Ser686 4.40, Ph içinE690 4.44 ve Gly-694 4.48 mGlu3 (HA-mGlu2ΔTM4N) &#39;de daha önce 6 tarif edilmiştir. Not: Bu, daha önce kimerik yapısı, HA-mGlu2ΔTM4N sağlam G-protein-bağlı 6 Sinyal plazma membranında eksprese edildiği gösterilmiştir. </li><li> viral vektörlerin saklayın -80 ° C kullanılmadığı zaman. Daha sonra çözülme viral buz üzerinde vektör ve 10 ul kısma bölmeyin. cerrahi işlem için, buz üzerinde tutmak. </li></ol></li></ol> 2. Cerrahi <ol><li> cerrahi Hazırlık <ol><li> (1.1.1 ayrıntılar için bkz) fare tartılır ve ketamin / ksilizin kokteyl uygun dozda ile fare enjekte. </li><li> Düzgün anestezi olmadığını görmek bir tepki ortaya çıkarmak için yapamaz fare düzgün anestezi ise, ağrı yanıt ayak ve kuyruk sıkmak için fareyi kontrol edin. </li><li> makası kullanarak burun ucu kafatası tabanından fare kafasını tıraş. Fare af oftalmik jel uygulayınfare körlüğü önlemek için terward. </li><li> stereotaksik çerçeve üzerine her şırınga yükleyin. onlar 10 derece uzakta normal böylece daha sonra stereotaksik çerçeve her kolun dik kısmını yatırın. iğneler birbirine bakacak şekilde, kollar eğik olduğundan emin olun. </li><li> % 70 etanol ile iğne doldurarak her bir şırınga temizleyin. Şırınga temiz olduğundan emin olmak için iğne en az üç kez doldurun. </li><li> İğne temizlendikten sonra, çift distile H2O ile iğne doldurarak iğne floş Bir kez kızardı, çift distile H2O 1.3 ul her iğne doldurun Çift distile H2O 0.3 ul serbest bırakmak için şırınga pistonu Büküm İğnenin ucunda su boncuklar, dikkatlice su uzakta silin. hiçbir şey şırınga çıkarsa, aşağı tamamen pistonu itin ve şırınga temizlik tekrarlayın. </li><li> Su ile doldurduktan sonra, daha sonra şırınga ile dolum şırınga yukarı çekinHavanın 0.5 ul. </li><li> Hava ve su şırınga kez, dikkatle virüs çözümü 1.3 ul şırınga doldurun. Bu noktada şırınga toplam hacim 2.8 ul olduğundan emin olun. Yine virüsün 0.3 ul serbest bırakmak için şırınga ucu bükün. İğnenin ucundaki sıvı boncuklar, dikkatle sıvıyı uzak silin. hiçbir şey şırınga çıkarsa, aşağı tamamen pistonu itin ve şırınga temizlik tekrarlayın. </li></ol></li><li> cerrahlık <ol><li> Kafatası seviyesi ve düz olacak şekilde stereotaksik çerçeve ayarlamak için emin olun, stereotaksik çerçeve için fareyi takın. maruz kafa derisi povodine-iyot uygulayın. Bir neşter kullanılarak, maruz traş alanı içinde kafatası orta hat boyunca sagittal bir kesi yapmak. Sonra kafatası maruz kalır emin olmak için kesi yerinde cilde büret kelepçeleri takın. </li><li> Ve dikişler maruz periost uzak çözmek için H 2 O 2 kullanınkafatası. Şimdi Bregma ve dikişler görünür olduğundan, kafatası seviyesi olduğundan emin olmak için stereotaksik çerçeve ayarlamak için emin olun. </li><li> bregma ile şırınga iğne uçları hizalayın ve bregma koordinatlarını kaydetmek. iğneler takılı olacak koordinatlarını hesaplamak. <ol><li> Rostral-Cauldal (RC) düzlem için, kaydedilen RC Bregma koordinatları (bregmadan 1,6) 1.6 mm ekleyin. Dorsal-ventral (DV) düzlem için, kaydedilen DV Bregma koordinatları (bregmadan -2.4) 2.4 mm çıkarın. </li><li> Son olarak, Medial Lateral (ML) düzlem için, kaydedilen ML Bregma koordinatları (bregmadan 2,6) 2.6 mm ekleyin. Bu ikili bir enjeksiyon olduğu gibi tüm koordinatlar için, hem sol ve sağ koordinatlarını kaydetmek için emin olun. </li></ol></li><li> İstenilen koordinatlarına iğne getir. iğneler sokulur ve bir matkap ile işaretlenmiş alanların matkap olacak nereye yerleri işaretleyin. </li><li> Bir pamuk uçlu aplikatör wi ileherhangi bir aşırı kan ya da kemik parçasını uzağa pe. </li><li> iğne uçları beynin yüzeyine dokunmadan kafatasına iğneler getirin. Sonra yavaş yavaş onları düşürücü istenilen koordinatlara iğneler indirin. </li><li> iğne istenen koordinatlara olduğunda, yavaş (toplam 0.5 ul) 5 dakika boyunca dakikada iğne 0.1 ul pistonu bükerek enjekte edilir. </li><li> Enjeksiyon yapıldıktan sonra başka bir 5 dakika kortekste şırınga bırakın. </li></ol></li><li> Up / kapama Bakımı <ol><li> sürekli ve yavaş yavaş fare korteks iğne çıkarın. Sonra stereotaksik çerçeve fare kaldırmak. </li><li> forseps cilt flep kapmak ve birlikte koyun ile daha sonra kesi deri taban kanatlarına siyanoakrilat (dermal yapıştırıcı) uygulayın ve. </li><li> siyanoakrilat kurumasını bekleyin. ısıtma yastığı Surg eğer isteğe bağlıdır (bir ısıtma yastığı üzerinde kafes içinde fare yerleştirinnik paketi 37 ° C oda sıcaklığında altında tutulur - Aksi gerekli değildir). yatak cerrahi bölgeye bağlı olmadığından emin olmak için bir kağıt havlu üzerine fare koyun emin olun. </li><li> İşlemden sonra 60 dakika - cerrahi işlemin süresine bağlı olarak, fareler 30 içinde anestezi dışında olduğundan emin olun. Ameliyatı takiben, hayvan kendi kafes yerleştirilir ve kurtarmak için kendi odasına dönmeden önce bilinçli hale gelene kadar izlenir. Onlar beyin biyokimyasal yollar bazı değiştirerek bizim deneylerin sonuçlarını değiştirebilir çünkü hiçbir analjezikler, postoperatif kullanılmaktadır. Ancak ameliyat günü anestezi kurtarmak kadar, hayvanlar izlenir, ve enfeksiyon ve ağrı / sıkıntı değerlendirilmesi belirtileri günlük operasyon sonrası. </li></ol></li></ol> 3. Kafa Seğirme Tepki Deneyi <ol><li> Kurmak <ol><li> 3 gün stereota sonra - 2 10:00 ve 02:00 PM arasındaki tüm davranış testleri yürütmekViral parçacıklar CTIC enjeksiyonu. </li><li> Çözündürün (±) -1- (2,5-dimetoksi-4-iyodofenil) 2.0 mg / kg bir% 0.9 salin çözeltisi -2-aminopropan hidroklorür (DOI). Ayrıca% 0.9 tuzlu su çözeltisi hazırlayın. </li><li> Herhangi bir yatak olmadan bir ev kafes (28 x 18 x 15 cm) hazırlayın ve video kamera görüntüle doğrudan ana kafes üstünde olacak şekilde bir tri-bölmesini kullanarak, bir kamera ayarlayın. </li><li> Deneyin başlamasından önce en azından 4 saat oda fareler alıştırarak. </li><li> Baş seğirme kaydetmek için kamera ayarlayın. </li></ol></li><li> deneme <ol><li> Bir evde kafes üzerinden doğrudan böylece kamerayı yerleştirin. Tüm ana kafes görüş alanında olacak şekilde kamera kalibre edin. </li><li> fare tartılır ve% 0.9 tuzlu su ya da DOI ya uygun dozu (0.01 ml / g) ile periton içine fare enjekte edilir. Not: Fare 25 gram ağırlığında durumunda, 0.25 ml lik bir toplam hacime dozunun uygulanması. </li><li> fo evlerine kafes geri her fare yerleştirinR 10 dk. Boş ev kafesin merkezine 10 dakika, yer fare sonra kameranın görüş alanında kör noktalar vardır olduğunu doğrulamak. Kamerada basın kaydı. odayı terk. NOT: Fare hareketleri ve orada çeşitli davranışsal tepkileri (... Kafa seğirme, kulak çizik vb DOI tarafından uyarılan davranışsal tepkilerin tam listesi için Gonzalez-Maeso ark 2007 ek veri tablosunun 1&#39;e bakınız) 3, kaydedilmiş olacak 30 dk. Nedenle, kaydedilen görüş alanında kör noktalar vardır önemlidir. </li><li> 30 dakika orijinal ev kafesine geri kamera ve yer fare kayıt durdurduktan sonra. Her fare için bu işlemi tekrarlayın. </li></ol></li><li> gözden geçirmek <ol><li> Bantlar fare deneysel koşullara kör her hakem inceleme var) (yani., Baş sırasında kullanılan ilaçlar deney veya intrakranial enjeksiyon sırasında kullanılan virüs seğirme). El ile her kafa Throug seğirme kayıthout videosu. NOT: Baş-seğirme bir fare (ek videosu) tarafından yapılan hızlı sarsıntı baş hareketi olarak tanımlanır. </li><li> Her fare için, kör hakemlerin üç toplamları nihai HTR yanıtını ortalama. Daha sonra grup, bu deneysel koşulu ile değerler ve istatistiksel analiz (yani., T-testi veya ANOVA) yürütmek. </li></ol></li></ol>

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The authors have nothing to disclose.

Birlikte mGlu2-KO farelerde 5 mi olacak biçimde kültürlenmiş hücrelerde 5-HT2A -mGlu2 reseptör kompleksini oluşturmayan mGlu2 ve mGlu2 / mGlu3 kimerik yapıları ile sonuçlar, 5-HT2A -mGlu2 in heteromerik reseptör kompleksi fare frontal korteks LSD benzeri halüsinojenik 5-HT2A reseptörü agonistleri tarafından baş seğirme davranışını uyarmak için gereklidir. Bu yöntemin bir sınırlaması yerli dokuda hücre içi düzeyde yakın moleküler yakınlığı öl...

LSD, psilosibin ve meskalin olarak halüsinojenler, insan bilinci, biliş ve duygu 7-9 önemli değişikliklere neden olmaktadır. Genetik ya da farmakolojik ya yaklaşımlarla serotonin 5-HT2A reseptörü sinyallemesinin inaktivasyonu belirgin iki kemirgen modellerinde 3,10 bölgesindeki halüsinojenler davranışsal tepkileri zayıflatılmış ve 11 insanlarda neden olur. Halüsinojenler, diğer reseptör alt-tipleri 8 bağlanan, ancak 5-HT2A reseptörü, bu kimyasal maddelerin benzersiz davranış aktivite için gerekli olarak kabul edilir. Grup II metabotropik glutamat reseptörleri (yani., MGlu2 ve mGlu3) halusinojenler moleküler mekanizması ve psikoz 12 altında yatan onların ayrılmaz rolüne ilişkin önemli ilgi hedef olmuştur. Daha önce, mGlu2 protein (mGlu2-KO fareleri) herhangi bir ifade ile farenin hal hücresel ve davranışsal etkilere karşı duyarsız olduğu gösterilmiştirlucinogens 5. Ayrıca, 5-HT2A ve mGlu2 reseptörleri serotonin ve glutamat ligandlan, canlı hücreler 1,2 G protein bağlama modelini modüle geçtiği belirli bir heteromerik kompleks oluşturduğunu ileri sürülmüştür. Yapısal olarak, zar-ötesi (TM) etki 4 ve mGlu2 5 5-HT2A reseptörü 5, heteromerik oluşumunda önemli bir rol oynamaktadır. Buna ek olarak, daha fazla araştırma mGlu2 bölgesinin TM4 hücre içi ucunda yer alan, üç kalıntıları hücreler 6 canlı 5-HT2A reseptörü -mGlu2 heterokompleks oluşturmak için gerekli olduğunu göstermiştir. Heterolog ifade sistemlerinde görülen Bu bulgulara dayanarak, burada test etmek için, vahşi tip mGlu2 ve mGlu2-KO farelerde frontal kortekste mGlu2 / mGlu3 Şimerik yapıların HSV-aracılı ifade kullanımını tarif 5-HT2A ile heteromerik oluşumu ve mGlu2 için gerekli olanhalüsinojenik 5-HT2A reseptörü agonistleri tarafından neden kafa seğirme davranışı.

<table><tbody><tr><td>mGlu2 bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>mGlu2&amp;Delta;TM4N bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>GFP bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Lloyd</td><td>List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Vedco</td><td>KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ophthalmic gel</td><td>Fisher Scientific</td><td>NC0550805</td><td></td></tr><tr><td>Burret clips</td><td>Fisher Scientific</td><td>NC9268369</td><td></td></tr><tr><td>Feather surgical blade</td><td>Fisher Scientific</td><td>NC9032736</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Fisher Scientific</td><td>19-898-919&amp;nbsp;</td><td></td></tr><tr><td>Hamilton syringe</td><td>Fisher Scientific</td><td>14815203</td><td></td></tr><tr><td>Hamilton&amp;trade; Small Hub Removable Needles (33 Ga)</td><td>Fisher Scientific</td><td>14816206</td><td></td></tr><tr><td>Cordless Micro Drill</td><td>Fisher Scientific</td><td>NC9089241</td><td></td></tr><tr><td>Dermabond Dermal Adhesive</td><td>Fisher Scientific</td><td>NC0690470</td><td></td></tr><tr><td>(&amp;plusmn;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td><td>Sigma-Aldrich</td><td>42203-78-1</td><td>Dissolved in 0.9% saline solution to the concentration of 2.0 mg/kg</td></tr></tbody></table>

hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

Önceki bulgular halusinojenler tarafından güvenilir ve sağlam ortaya bir fare davranış cevabı baş-seğirme olduğunu göstermektedir ve bu 5-HT 2A -KO fareler 3 yoktur. Ayrıca, DOI ve LSD halüsinojenik 5-HT2A agonistlerin kafa seğirme yanıtı anlamlı mGlu2-KO farelerde 5 azaldığını göstermiştir. Bununla birlikte, her ne kadar önceki bulgular ikna edici çözülemediğini canlı farelerde gibi bu yapısal düzenleme hareket i...

Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

Bu makalede, GPCR heteromerik oluşumunu gerektiren davranış testleri test etmek için fare frontal korteks içine viral vektörlerin enjekte açıklamaktadır.

Kimerik Yapılar HSV-aracılı Transgen İfade Farelerde GPCR Heteromers Davranışsal Fonksiyon Eğitim için

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

hsv-mediated-transgene-expression-chimeric-constructs-to-study

Research

JoVE Journal

Neuroscience

7.2K Görüntüleme.  Icahn School of Medicine at Mount Sinai.  Bu makalede, GPCR heteromerik oluşumunu gerektiren davranış testleri test etmek için fare frontal korteks içine viral vektörlerin enjekte açıklamaktadır. 

Video: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

Lentiviral aracılı transgenik fareler üretimi: doğrudan eğitim kurucuları basit ve son derece etkili bir yöntem

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Genetik

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Virüs tabanlı Cre Rekombinasyon kullanarak Postnatal Fare Beyin Gelişimi Gen Fonksiyonu Mosaic Analizi

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Nörobilim

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Koku Duyu Nöronlar lentivirüs aracılı Genetik Manipülasyon ve Görselleştirme In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Genetiği Tanımlı Nöral Devre arasında Tracing Viral

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Fare Omurilik Koşullu Gen Manipülasyon için bir Viral Vector Stereotaksik Enjeksiyon

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. However, the time and cost associated with colony creation and maintenance are high. Recent advances in CRISPR-mediated genome editing have allowed the generation of somatic GEMMs (S-GEMMs) by directly targeting the cell/tissue/organ of interest.
The oviduct, or fallopian tube in humans, is considered the tissue-of-origin of the most common ovarian cancer, high-grade serous ovarian carcinomas (HGSCs). HGSCs initiate in the region of the fallopian tube distal to the uterus, located adjacent to the ovary, but not the proximal fallopian tube. However, traditional mouse models of HGSC target the entire oviduct, and thus do not recapitulate the human condition. We present a method of DNA, RNA, or ribonucleoprotein (RNP) solution microinjection into the oviduct lumen and in vivo electroporation to target mucosal epithelial cells in restricted regions along the oviduct. There are several advantages of this method for cancer modeling, such as 1) high adaptability in targeting the area/tissue/organ and region of electroporation, 2) high flexibility in targeted cell types (cellular pliancy) when used in combination with specific promoters for Cas9 expression, 3) high flexibility in the number of electroporated cells (relatively low frequency), 4) no specific mouse line is required (immunocompetent disease modeling), 5) high flexibility in gene mutation combination, and 6) possibility of tracking electroporated cells when used in combination with a Cre reporter line. Thus, this cost-effective method recapitulates human cancer initiation.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

This protocol describes microinjection and in vivo electroporation for regionally restricted CRISPR-mediated genome editing in the mouse oviduct.

In Vivo Mikroenjeksiyon ve Elektroporasyon Kullanan Somatik Genom Mühendisliği Fare Modelleri

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. ...

Kanser Araştırmaları

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Embriyonik Fare beyninde İzleme Koşullu Genetik transsinaptik

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Fare Kornea ve Konjonktiva Canlı Görüntüleme için Özel Multifoton Mikroskop platformu

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

Biyomühendislik

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Omurilik genetik olarak tanımlanmış nöronlar Adeno ilişkili virüs-aracılı Transgene ifade

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

SerbestÇe Hareket Eden Hayvanlarda Nöral AktiviteYi Kaydetmek Için Çoklu Fiber Fotometri

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...