이 작품은 NIH (EY012977), 버로우즈 웰컴 기금, 화이트홀 재단과 슬로언 재단 (AKM)에서 의생명 과학의 경력 수상의 부여에 의해 가능하게되었다.

1. 바이러스 처리 및 저장 <ol><li> 적절한 보호와 처리 기술을 사용하는 바이러스의 biosafety 레벨에 따라 선택하여야한다. 이러한 관행은 CDC 웹사이트에서 사용 가능한 미생물 및 바이오 메디컬 연구소 5 일 버전에 Biosafety에서 찾을 수 있습니다 ( <a href="http://www.cdc.gov/od/OHS/biosfty/bmbl5/bmbl5toc.htm">http://www.cdc.gov/od/OHS/biosfty/bmbl5/bmbl5toc.htm</a> ). AAV 벡터의 사용은 Biosafety 레벨 1 (BSL - 1)에 대한 승인. 바이러스를 다룰 때 실험을 여기에 표시된 들어, 실험 복과 장갑은 BSL - 1 에이전트를 처리하기위한 절차에 따라 착용한다. </li><li> 바이러스의 활동을 유지하기 위해서는 반복 동결 융해를 피하기 위해 작은 aliquots로 나누어하는 것이 최선입니다. </li><li> 불필요한 개체의 BSC를 지우고 70 % EtOH로 표면을 살균하여 바이러스를 aliquoting위한 Biosafety 내각 (BSC) 클래스 II를 준비합니다. AAV - 오염된 폐기물을 수집하기 위해 후드의 10 % 표백제 솔루션을 포함하는 비커를 놓습니다. 또한 무균 0.5mL 튜브와 드라이 아이스의 컨테이너가있는 BSC에 배치. </li><li> BSC 외부 얼음에 바이러스 재고를 녹여. </li><li> 소용돌이 바이러스와 BSC 내부 튜브를 엽니다. </li><li> 0.5mL 튜브에 원하는 나누어지는 볼륨 (예 : 5 μL)를 피펫, 튜브를 닫고하는 드라이 아이스에 배치 바이러스를 플래시 동결. </li><li> 모든 바이러스가 aliquoted되면, 바이러스 튜브와 10 %의 표백제가 들​​어있는 폐기물 용기에 피펫 팁 처분. </li><li> 후드에서 폐기물 컨테이너를 제거하고 추가로 10 % 표백제를 추가한 다음 싱크대 아래 표백제를 부어. 귀하의 연구소의 biosafety 임원의 지시에 따라 생물 학적 용기에 플라스틱 폐기물의 배출. </li><li> 10 % 표백제로 바이러스와 접촉에 와서 어떤 장비 또는 표면을 청소하십시오. 장갑을 폐기하십시오. </li><li> -80 ° C의 냉동고에 저장 aliquots. </li></ol> 2. 외과 <ol><li> 흡수 실험실 벤치 종이로 수술 영역을 커버. 수술 도구는 귀하의 기관의 biosafety과 동물 사용 지침에 따라 무균 조건 하에서 살균 및 수술이 완료되어야합니다. </li><li> 바이러스와 micropipettes 로딩에 전념하게 될 외과 지역에 인접한 지역을 선택하고, 흡수 실험실 벤치 종이로 커버. 이 분야에 얼음 용기에 바이러스 나누어지는를 삽입하고, 수술이 수행되고 있기 때문입니다이 바이러스가 얼음위에 해동 수 있습니다. </li><li> 바이러스에 접촉되는 피펫 팁 등의 처분을위한 전용 바이러스 처리 영역에서 10 % 표백제의 폐기물 컨테이너를 설치합니다. </li><li> 약 20 미크론의 팁 직경에 유리 Wiretrol의 micropipette을 당기세요. micropipette의 뭉툭한 끝에 광유의 작은 방울을 놓고 micropipettes와 함께 제공된 와이어 플런저를 삽입합니다. </li><li> micropump 팔 클램프의 micropipette를 확보. </li><li> 0.5 MG / kg의 투여에 buprenorphine와 subcutaneously 마우스를 주사. 200 MG / kg의 투여와 함께 intraperitoneal 주사를 통해 Avertin와 마우스를 마취하고 절개를 입력부터 머리카락을 방지하기 위해 충분히 큰 여백을 떠나 확인하고, 수술 가위로 머리의 상단에서 머리를 제거합니다. </li><li> 37 수술에 걸쳐 ° C의 체온을 유지하고 stereotax에 마우스를 확보 stereotax의 기본에 난방 담요를 연결합니다. </li><li> 에탄올과 지역을 소독하기 위해 betadine 세 교대 스크럽으로 머리를 목욕. </li><li> 수술 중에 눈을 촉촉한 유지하는 눈은 Tobradex 아이 연고의 드롭 놓습니다. </li><li> 머리의 정중선 아래 절개를하고 두개골을 노출로 피부를 당기십시오. </li><li> 조심스럽게 세부적으로 밀고 집게를 사용하여 두개골에서 근막을 제거하십시오. </li><li> stereotaxic 좌표를 사용하여 주입하는 지역을 찾아 수술 볼펜으로 두개골을 표시합니다. </li><li> 여러 세그먼트로 thinned 영역을 나누어, 두개골의 균열까지 직경의 두개골 약 2mm의 1.4mm 버, 얇은 지역과 치과 드릴을 사용합니다. </li><li> 이 절차를 통해, 멸균 생리의 응용 프로그램과 두개골이 촉촉한 유지. </li><li> 부드럽게 추가 - 좋아 밀고 집게를 사용하여 thinned 두개골 세그먼트를 제거하여 craniotomy을 수행합니다. </li></ol> 3. 사출 준비 <ol><li> 뚜껑 위에 위치 KimWipe으로 parafilm의 작은 조각에 바이러스의 바이러스 및 피펫 1.5uL (1uL 주사)의 튜브를 엽니다. </li><li> 바이러스 주식에 micropipette의 팁을 놓고 수동으로 플런저를 철회. 어려움이 micropipette에 바이러스 주식을 그리는 경험하는 경우, 끝 부분이 약간 유리의 일부를 깰 수있는 micropipette과 KimWipe을 피어싱 수 확대하실 수 있습니다. </li><li> 바이러스의 소량 때까지 플런저로 로우어 micropump 암은 micropipette의 일각에서 무너 뜨 리신 것입니다. 와스트에서 면화 팁 작은 주걱 및 폐기 작은 주걱이 작은 방울을 제거E 컨테이너. </li><li> micropipette이 두뇌에 넣어 져서으로 막히는 것을 방지하기 위해 micropipette의 끝부분에 미네랄 오일 한 방울을 적용합니다. </li></ol> 4. 바이러스 사출 <ol><li> X와 Y 좌표 stereotaxic을 사용하여 주입하는 영역 micropipette 위치를. 본 실험에서는 stereotaxic은 정중선을 가로 2.7mm bregma에 후부와 2.5mm 아르 기본 시각 피질의 위치를​​ 사용하는 좌표. 아주 천천히 적절한 Z의 위치로 micropipette을 (1mm/1minute의 대략적인 비율) 낮아집니다. </li><li> SYS Micro4 micropump 컨트롤러 상자에 원하는 사출 매개 변수를 입력하고 주사를 시작합니다. 본 실험에서는 10 분 이상 1 microliter는 주입의 속도로 사용됩니다. </li><li> 주입이 완료되면 제거하는 동안 바이러스의 유출을 방지에 한 2 분 동안 휴식을 피펫을 두십시오. 이 기간 후에, 아주 천천히 두뇌 (위의 동일 요금)에서 micropipette를 제거합니다. </li><li> 두피를 봉합하고 조직 접착제로 밀봉. 72 시간 동안 0.1 MG / kg 모든 8~12시간의 복용에 buprenorphine와 subcutaneously 동물을 주사하거나, 한 동물로 고통의 흔적을 전시하고있다. 그것이 ambulating 및 케이지로 돌아 준비가 될 때까지 동물이 가열 램프에 따라 복구할 수 있습니다. 이미징 실험은 원하는 배양 시간 (바이러스 주입 후 주 일) 이후에 시작하실 수 있습니다. </li></ol> 5. 대청소 <ol><li> 10 % 표백제로 micropipette를 씻어 샵스 컨테이너에서 삭제. </li><li> 제 1과 같은 방식으로 폐기물 컨테이너 처리. </li><li> 생물 학적 bin에 실험실 벤치 종이를 버리고 10 % 표백제와 바이러스에 접촉 할 수도 있습니다 모든 표면과 악기를 닦으십시오. </li><li> 사용하지 않는 바이러스는 반복 동결 / 해동 사이클이 바이러스 저하의 원인이 점을 유념해 다시 냉동 수 있습니다. </li></ol> 6. 대표 결과 <img src="/files/ftp_upload/2140/2140fig1.jpg" alt="그림 1" /> 그림 1. CMV promotor의 통제하에 녹색 형광 단백질 (GFP)을 들고 두 번 좌초 adeno - 관련 바이러스 혈청형 1 (dsAAV S1)의 주사 후 신경 세포를 Transduced. 전지 본체뿐만 아니라 근위 및 말초 dendrites 고정 섹션에서 분명히 볼 수있는가 epifluorescent 현미경을 사용하여 몇 군데. 스케일 바 = 100 μm의. <img src="/files/ftp_upload/2140/2140fig2.jpg" alt="그림 2" /> 그림 2. 바이러스 확산의 범위뿐만 아니라 표시 뉴런, glia 및 프로세스를 보여주는 dsAAV의 S1을 사용하여 기본 시각 피질에 intracranial 바이러스 주입의 전형적인 라벨링. 스케일 바 = 250 μm의. <img src="/files/ftp_upload/2140/2140fig3.jpg" alt="그림 3" /> 그림 3. dsAAV의 S1을 사용하는 해마의 세포의 라벨링. 스케일 바 = 250 μm의. 이러한 수치는 어리 외에서 조정됩니다. 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>

동물 실험 프로토콜은 실험실 동물의 동물 애호 관리 및 사용에 PHS 정책에 따라 동물 자원 (UCAR)에서 로체스터 대학위원회의 대학에 의해 승인되었습니다.

Virally - 중재 유전자 전달은 신경 프로세스와 뇌 장애 1,2,3 치료 연구를위한 잠재력을 보유하고 있습니다. 이 기법의 가장 큰 융통성도 찬란 체외 및 생체내 4 두 이미징을위한 세포를 라벨에 악용될 수 있습니다. 여기 우리는 뉴런과 향상된 녹색 형광 단백질을 표현하는 이중 좌초 adeno - 협회 바이러스를 사용하여 마우스를 시각 피질에 glia의 도입에 대한 자세한 절차를 설?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
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intracranial injection of adeno-associated viral vectors

형광 단백질을 표현할 수 있도록 설계 바이러스 벡터의 Intracranial 주사는 생체내 및 뇌 부분에서 모두 서로 다른 뇌 영역의 세포의 특정 하위 집합의 시각화를위한 다양한 분류 방법입니다. 형광 염료의 주입과는 달리, 바이러스 라벨 개별 세포 유형의 대상으로 제공하며, 유전자 변형 마우스 라인을 설정하는 것보다 소비 저렴하고 시간입니다. 이 기법에서는, adeno - 관련 바이러스 (AAV) 벡터는 intracranially 주변 조직에 피해를 최소화하여 원하는 지역 stereotaxic 좌표, micropipette와 AAV의 정확한 배달을 위해 자동 펌프를 사용하여 주입합니다. 사출 매개 변수가 주입의 주입, 주입 위치, 분사의 볼륨, 속도, AAV 혈청형 및 유전자 발현을 구동 모터에있는 동물 연령을 조정하여 각각의 실험에 맞출 수 있습니다. 선택한 조건에 따라 virally 유발 transgene 표현은 아래로 돌기 쪽이 수준으로 세포, 개별 셀 또는 미세 세포 프로세스 그룹의 시각화를 허용할 수 있습니다. 여기에 표시된 실험은 마우스 기본 시각 피질의 뉴런과 glia의 라벨에 대한 녹색 형광 단백질을 표현하는 이중 좌초 AAV의 주사를 묘사.

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

Intracranial Injection of Adeno-associated Viral Vectors

여기서 우리는 시각 피질의 뉴런과 glia의 형광 라벨에 대한 AAV 벡터의 intracranial 주사를 제시한다.

Adeno - 관련 바이러스성 벡터의 Intracranial 분사

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 조회수.  University of Rochester. 여기서 우리는 시각 피질의 뉴런과 glia의 형광 라벨에 대한 AAV 벡터의 intracranial 주사를 제시한다.

비디오: 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.

마약, 바이러스성 벡터, 또는 세포 이식 전달 Microinjections 깊은 뇌 구조의 타겟팅

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

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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.

마우스 망막 색소 상피의 유전자 치료에 대한 바이러스 벡터의 윤부 접근 - 망막 주입

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.

성인 쥐의 Subpial Adeno-associated Virus 9 (AAV9) 벡터 전달

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.

척수의 유전으로 정의 된 신경에 있는 Adeno 관련 바이러스 중재 Transgene 식

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.

성인 쥐에 있는 재조합 형 Adeno 관련 바이러스의 직접 Intrathecal 주입

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.

생산, 정화, 및 Adeno 관련 바이러스 기반 벡터에 대 한 품질 관리

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.

대류 온라인 MRI 이미지의 지도하에 Rhesus Macaque의 피질에 광유전학 아데노 관련 바이러스 벡터의 향상된 납품

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.

바이러스 벡터의 intracerebral 주입에 의해 인터뉴런으로 상주 신경교 세포의 생체 내 직접 재프로그래밍

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.

쥐에서 코필린을 표적으로 하는 아데노 관련 바이러스를 사용하여 조작된 대뇌 피질 지역의 전기 코르티코그래피 기록

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.