Bu çalışma, Burroughs Wellcome Fonu, Whitehall Vakfı ve Sloan Vakfı (AKM) Biyomedikal Bilimler Kariyer Ödülü NIH (EY012977) bağışları ile mümkün olmuştur.

1. Virüs İşleme ve Depolama <ol><li> Uygun koruma ve kullanım teknikleri kullanılacak virüs biyogüvenlik düzeyi dayalı seçilmelidir. Bu uygulamalar CDC web sitesinde mevcut Mikrobiyolojik ve Biyomedikal Laboratuvarlar 5 th edition, Biyogüvenlik bulunabilir ( <a href="http://www.cdc.gov/od/OHS/biosfty/bmbl5/bmbl5toc.htm">http://www.cdc.gov/od/OHS/biosfty/bmbl5/bmbl5toc.htm</a> ). AAV vektörleri Biyogüvenlik Seviye 1 (BSL-1) kullanım için onaylanmıştır. Virüs işlerken deney burada gösterildiği için, bir laboratuvar önlüğü ve eldiven BSL-1 agentları için prosedürler uyarınca, aşınmış olacak. </li><li> Virüs aktivitesini korumak için tekrarlanan dondurma ve çözme önlemek için küçük alikotları içine bölmek en iyisidir. </li><li> Biyogüvenlik Kabini (BSC) Sınıf II, herhangi bir gereksiz nesneler BSC takas ve% 70 EtOH yüzey sterilizasyon virüs aliquoting için hazırlayın. AAV-kontamine atık toplama kaputu% 10 çamaşır suyu çözeltisi içeren bir behere koyun. Ayrıca, steril 0.5mL tüpler ve kuru buz bir kap BSC yerleştirilir. </li><li> BSC dışında buz üzerinde viral stok çözülme. </li><li> Vorteks virüs ve BSC içinde tüp açık. </li><li> Flaş dondurucu virüs 0.5mL bir tüp içine istenen kısım hacim (örneğin 5 mcL) Pipet, tüpün kapağı kapatılır ve kuru buz koyun. </li><li> Tüm virüs aliquoted edildiğinde, virüs tüp ve pipet uçları,% 10 çamaşır suyu içeren atık kabını atmayın. </li><li> Kaputun atık kabını çıkarın ve% 10 oranında ek çamaşır suyu ekleyin, daha sonra lavaboya çamaşır suyu dökün. Enstitünün biyogüvenlik memuru tarafından talimat olarak biohazard bir kap içinde plastik atık bertaraf edin. </li><li> % 10 çamaşır suyu ile virüs ile temas geldi herhangi bir ekipman veya yüzeyleri temizleyin. Eldiven atın. </li><li> -80 ° C derin dondurucuda saklayın alikotları. </li></ol> 2. Cerrahi <ol><li> Emici laboratuarlarında kağıt cerrahi alanı kapsayacak. Cerrahi aletler kurumun biyogüvenlik ve hayvan kullanım yönergelerine uygun aseptik koşullarda steril ve cerrahi yapılmalıdır. </li><li> Mikropipetler virüs yükleme adanmış olacaktır cerrahi alanına bitişik bir alanda seçin ve emici laboratuvar tezgah kağıt ile örtün. Virüs, bu alanda buz bir kap içinde bir kısım yerleştirin ve izin ameliyat yapılmaktadır virüs buz üzerinde çözünme. </li><li> Pipet uçları, vb. Atılmasının virüsü ile temas halinde geldiği için adanmış bir virüs taşıma alanında% 10 çamaşır suyu atık konteyneri Kur. </li><li> Bir bardak ucu çapı yaklaşık 20 mikron Wiretrol mikropipet çekin. Mikropipet künt ucunda küçük bir damla mineral yağ koyun ve mikropipetler tel piston takın. </li><li> Micropump kol kelepçesi mikropipet sabitleyin. </li><li> Buprenorfin ile 0.5 mg / kg dozunda subkutan bir fare enjekte edilir. Intraperitoneal enjeksiyon yoluyla 200 mg / kg dozu ile Avertin fare anestezi ve cerrahi makas ile başın üst saç kaldırmak, saç kesi girmesini engellemek için yeterince büyük marjları bırakmak emin olun. </li><li> Vücut ısısı 37 ° C ameliyat boyunca korumak ve stereotax fare güvence altına almak için, bir stereotax taban ısıtma battaniye takın. </li><li> Etanol ve betadin alan sterilize etmek için üç alternatif scrubs ile baş batırın. </li><li> Ameliyat sırasında gözleri nemli tutmak için her gözün Tobradex göz merhemi bir damla yerleştirin. </li><li> Baş orta hatta aşağı bir kesi yapın ve cilt kafatası açığa çıkarmak için geri çekin. </li><li> Ince uçlu forseps kullanılarak kafatası fasya dikkatlice çıkarın. </li><li> Stereotaksik koordinatları kullanılarak enjekte edilir alanını bulun ve cerrahi bir kalem ile kafatası işareti. </li><li> Birkaç parça halinde inceltilerek alan bölme, yaklaşık 2mm kafatası kafatası çatlak kadar çapı 1.4mm bir çapak, ince bir alana sahip bir diş matkap kullanma. </li><li> Bu işlem boyunca, steril tuzlu su uygulamaları ile kafatası nemli tutmak. </li><li> Ekstra ince uçlu forseps kullanarak inceltilmiş kafatası parçaları hafifçe kaldırarak kraniotomi gerçekleştirin. </li></ol> 3. Enjeksiyon Hazırlık <ol><li> KimWipe kapağı üzerine yerleştirilen bir virüs virüs ve pipet 1.5uL (1uL bir enjeksiyon için), parafilm küçük bir parça üzerine tüp açın. </li><li> Mikropipet ucu viral stok içine yerleştirin ve piston elle çekilmeye. Mikropipet viral stok çizim güçlük yaşıyorsanız, ucu biraz daha küçük bir bölümünü cam kırmak için mikropipet KimWipe piercing büyütülmüş olabilir. </li><li> Mikropipet ucu kadar piston üzerine küçük bir miktar virüs Alt micropump kol effect. Idin bir pamuk uçlu aplikatör ve iptal aplikatör ile bu küçük bir damla çıkarıne kap. </li><li> Mineral yağ mikropipet mikropipet beyin içine düşürdü olarak tıkanmasını önlemek için ucuna bir damla uygulayın. </li></ol> 4. Virüs Enjeksiyon <ol><li> X ve Y stereotaksik koordinatları kullanarak mikropipet, enjekte edilecek alanın üzerine yerleştirin. Bu deneyde, stereotaksik orta hatta lateral 2.7mm bregma posterior ve 2.5mm birincil görsel korteks bulmak için kullanılır koordinatları. Çok yavaş doğru Z konumuna mikropipet 1mm/1minute yaklaşık bir oranda daha düşük. </li><li> SYS Micro4 micropump denetleyici kutuya istenilen enjeksiyon parametreleri girin ve enjeksiyon başlatabilir. Bu deney için, 10 dakika içinde 1 mikrolitrelik enjeksiyon oran olarak kullanılacaktır. </li><li> Enjeksiyon bittiğinde, çıkarılması sırasında virüs efflux önlemek için bir-iki dakika dinlenmek için pipet bırakın. Bu dönemden sonra, çok yavaş beyin (yukarıdaki gibi aynı oranda) mikropipet çıkarın. </li><li> Kafa derisi sütür ve doku yapıştırıcısı ile yapıştırın. Sonraki 72 saat içinde 0.1 mg / kg her 8-12 saatte bir doz hayvan buprenorfin ile subkutan enjekte edilir, veya hayvan olarak sürece ağrı belirtilerini sergiliyor. Yürüyebilen ve kendi kafesine iade edilmesi için hazır olana kadar bir ısı lambası altında hayvan kurtarmak için izin verin. Görüntüleme deneyler istenen inkübasyon süresi (viral enjeksiyondan sonra hafta gün) sonra başlanmalıdır. </li></ol> 5. Temizleme <ol><li> Mikropipet% 10 çamaşır suyu ile durulayın ve bir dikiş iğnesi kap içinde atın. </li><li> Bölüm 1 aynı şekilde atık konteyneri atın. </li><li> Laboratuarlarında kağıt biohazard bin içine atın ve tüm yüzeylerin ve aletlerin% 10 çamaşır suyu ile virüs ile temas gelmiş aşağı silin. </li><li> Kullanılmayan virüs tekrarlanan donma / çözülme döngüsü virüs düşmesine neden olduğunu göz önünde tutarak, tekrar donmuş olabilir. </li></ol> 6. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2140/2140fig1.jpg" alt="Şekil 1" /> Şekil 1. CMV Promotor kontrolü altında yeşil floresan proteininin (GFP) taşıyan çift iplikli adeno-ilişkili virüs serotip 1 (dsAAV S1) enjeksiyonundan sonra nöron Transduced. Epifluorescent bir mikroskop kullanılarak görüntülü yanı sıra hücre gövdesi proksimal ve distal dendritler sabit bölümlerde açıkça görülebilir. Ölçeği bar = 100 mikron. <img src="/files/ftp_upload/2140/2140fig2.jpg" alt="Şekil 2" /> Şekil 2. intrakranial bir virüs gibi viral yayılma derecesi etiketli nöronlar, glia ve süreçleri gösteren dsAAV S1 kullanarak birincil görsel korteks enjeksiyonu tipik Etiketleme. Ölçeği bar = 250 mm. <img src="/files/ftp_upload/2140/2140fig3.jpg" alt="Şekil 3" /> Şekil 3 dsAAV S1 kullanarak hipokampus hücrelerinin Etiketleme. Ölçeği bar = 250 mm. Bu rakamlar Lowery ve ark uyarlanmıştır. 2009 1

<ol>
	<li>Kaplitt, M. G., Leone, P., Samulski, R. J., Xiao, X., Pfaff, D. W., O'Malley, K. L., During, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nat Genet. 8, 148-154 (1994).</li><li>Harding, T. C., Dickinson, P. J., Roberts, B. N., Yendluri, S., Gonzalez-Edick, M., Lecouteur, R. A., Jooss, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+gene+transfer+efficiency+in+the+murine+striatum+and+an+orthotopic+glioblastoma+tumor+model,+using+AAV-7-+and+AAV-8-pseudotyped+vectors.">Enhanced gene transfer efficiency in the murine striatum and an orthotopic glioblastoma tumor model, using AAV-7- and AAV-8-pseudotyped vectors.</a> Hum Gene Ther. 17, 807-820 (2006).</li><li>Lo, W. D., Qu, G., Sferra, T. J., Clark, R., Chen, R., Johnson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+transfer+to+the+brain:+duration+and+modulation+of+expression.">Adeno-associated virus-mediated gene transfer to the brain: duration and modulation of expression.</a> Hum Gene Ther. 10, 201-213 (1999).</li><li>Lowery, R. L., Zhang, Y., Kelly, E. A., Lamantia, C. E., Harvey, B. K., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+long-term+labeling+of+cells+in+the+developing+and+adult+rodent+visual+cortex+using+double-stranded+adeno-associated+viral+vectors.">Rapid long-term labeling of cells in the developing and adult rodent visual cortex using double-stranded adeno-associated viral vectors.</a> Dev Neurobiol. 69, 674-688 (2009).</li></ol>

Hayvan ve deneysel protokolleri PHS, Laboratuvar Hayvanları İnsani Bakım ve Kullanım Politikası uyarınca Hayvan Kaynakları (UÇAR), University of Rochester Üniversitesi Komitesi tarafından kabul edildi.

Viral-aracılı gen teslim nörolojik süreçleri ve beyin bozuklukları 1,2,3 tedavi çalışmaları için büyük bir potansiyele sahiptir . Bu tekniğin çok yönlülüğü de hücreleri in vitro ve in vivo 4 hem de görüntüleme için floresan etiket yararlanılabilir. Burada nöron ve gelişmiş yeşil flüoresan protein ifade eden bir çift iplikli adeno-dernek virüs kullanarak fare görsel korteks glia transdüksiyon için ayrıntılı bir prosedür ortaya koymak...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Stoelting Mouse and Neonatal Rat Adaptor</td>
<td>&nbsp;</td>
<td>Stoelting</td>
<td>51625</td>
<td>Regular stereotax for securing animals for surgery may be substituted</td>
</tr>
<tr>
<td>Extra Fine Bonn Scissors, 8.5cm, straight tip, cutting edge 13mm</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14084-08</td>
<td>&nbsp;</td>
<tr>
<td>Eye Dressing Forceps, 10cm, tip width 0.5mm, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11152-10</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5/45 Forceps- Dumoxel Standard Tip, 11cm, angled</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11251-35</td>
<td>Extra-fine tipped forceps for performing craniotomy</td>
</tr>
<tr>
<td>Standard Pattern Forceps, straight, 2.5mmx1.35mmtip, 12cm</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11000-12</td>
<td>&nbsp;</td>
<tr>
<td>Microtorque Control Box and Tech2000 handpiece</td>
<td>&nbsp;</td>
<td>Ram Products, Inc.</td>
<td>TECH2000ON/OFF</td>
<td>Dental drill</td>
</tr>
<tr>
<td>Micro Drill Stainless Steel Burrs 1.4mm tip diameter</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>19008-14</td>
<td>&nbsp;</td>
<tr>
<td>Wiretrol micropipettes, to deliver 1-5 Ul</td>
<td>&nbsp;</td>
<td>VWR International/ Drummond</td>
<td>5-000-1001 or 53480-287</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>29447-338</td>
<td>&nbsp;</td>
<tr>
<td>Manual Micromanipulator and Tilting Base (right-handed)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>M3301-M3-R</td>
<td>Used for determining stereotaxic co-ordinates</td>
</tr>
<tr>
<td>UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>UMP3-1</td>
<td>&nbsp;</td>
<tr>
<td>Sutures</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>95056-952</td>
<td>&nbsp;</td>
<tr>
<td>P-97 Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Tobradex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Available from your institution&#x2019;s veterinary services</td>
</tr>		</tbody>
		</table>
		</div>

intracranial injection of adeno-associated viral vectors

İntrakranial floresan proteini ifade etmek için tasarlanmış viral vektörlerin enjeksiyonu in vivo ve beyin bölümlerde iki farklı beyin bölgelerindeki hücrelerin belirli altkümelerini görünüm için çok yönlü bir etiketleme tekniği. Enjeksiyon floresan boyaların aksine, viral etiketleme bireysel hücre tipleri hedef sunar ve transgenik fare hatları kurulması daha tüketen daha az pahalı ve zaman. Bu teknikte, adeno-ilişkili virüs (AAV) vektörü, çevredeki dokuya minimum zarar ile intrakranyal istenen alana stereotaksik koordinatları, bir mikropipet ve AAV hassas teslimat için otomatik bir pompa kullanarak enjekte edilir. Enjeksiyon parametreleri, enjeksiyon enjeksiyon, enjeksiyon yeri, enjeksiyon hacmi, hızı, AAV serotip ve gen ekspresyonu sürüş organizatörü hayvan yaş ayarlayarak bireysel deneyler için uygun olabilir. Seçilen koşullara bağlı olarak, viral indüklenen transgen ifadesi, dendritik dikenleri düzeyini aşağı hücreleri, tek tek hücreler veya ince hücresel süreçlerin grupları görselleştirme, izin verebilirsiniz. Burada gösterilen deneyi fare birincil görsel korteks nöronlar ve glia etiketleme için yeşil flüoresan protein ifade çift iplikli AAV bir enjeksiyon gösteriyor.

Watch this Scientific Journal Video about Intracranial Injection of Adeno-associated Viral Vectors at JoVE.com

Intracranial Injection of Adeno-associated Viral Vectors

Burada görsel korteks nöronlar ve glia floresan etiketlenmesi için AAV vektörleri intrakranial enjeksiyon sunuyoruz.

Adeno-ilişkili Viral Vektörler İntrakranial Enjeksiyon

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

intracranial-injection-of-adeno-associated-viral-vectors

Research

JoVE Journal

Neuroscience

47.1K Görüntüleme.  University of Rochester. Burada görsel korteks nöronlar ve glia floresan etiketlenmesi için AAV vektörleri intrakranial enjeksiyon sunuyoruz.

Video: Intracranial Injection of Adeno-associated Viral Vectors

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

İlaçlar, Viral Vektörler, veya Hücre Transplantasyon teslimi için Microinjections Derin Beyin Yapıları Hedefleme

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

Nörobilim

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

Fareler Retina Pigment Epitel Gen Tedavisi için Viral Vektörler Limbal Yaklaşım-Subretinal Enjeksiyon

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 &#181;L; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

Yetişkin Farelerde Subpial Adeno-ilişkili Virüs 9 (AAV9) Vektör Teslimatı

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

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 ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

Rekombinant Adeno ilişkili virüs yetişkin farelerde doğrudan intratekal enjeksiyon

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Üretim, arıtma ve kalite kontrol için virüs tabanlı vektörel çizimler Adeno ilişkili

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

Online MRG görüntüleri rehberlik altında Rhesus Macaque korteks için optogenetik Adeno-ilişkili Viral vektör konveksiyon geliştirilmiş teslim

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

In vivo doğrudan reprogramming Resident glial hücreler Interneurons viral vektörler ıntracerebral enjeksiyon ile

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

Farelerde Cofilin Hedef Alan Adeno İlişkili Bir Virüs Kullanılarak Manipüle Edilen Serebral Korteks Alanlarının Elektrokortikografik Kaydı

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.