우리는 기술 지원을 Bakhos A. Tannous, 박사, rAAV-eGfp 벡터으로 우리를 제공 매사추세츠 종합 병원, 찰스 타운, 매사추세츠,의 신경 과학 센터에서 벡터 개발 및 생산 이사, 존 강타 감사드립니다. 이 작품은 신경 질환 및 뇌졸중의 국립 연구소에서 교부금으로 R01 NS050408을 (JS까지) 지원되었습니다.

설명 수술 절차는 컬럼비아 대학 (Columbia University)의 기관 동물 케어 및 사용위원회 (IACUC)에 의해 승인되었습니다. 1. 장비 및 바이러스 입자 현탁액의 준비 <ol><li> 장비를 청소하고 소독, 수술기구와 척추 뼈의 L1 문제를 해결하는 데 사용됩니다 V 노치 스파이크를 소독. </li><li> 당기 및 베벨 유리 피펫. 우리는 40 μm의 팁 직경이 20 °의 각도로 beveled 아르 피펫을 사용합니다. 유리 피펫 멸균을. </li><li> stereotaxic 프레임을 설정, 머니퓰레이터에 microsyringe 분사기를 장착하고 컨트롤러에 주입기를 연결합니다. </li><li> 압축 피팅 키트를 사용하여 microsyringe에 유리 피펫 중 하나를 첨부합니다. </li><li> microsyringe에서 플런저를 제거하고 미네랄 오일과 주사기를 입력합니다. 오일 레드 O (1 - (2,5 - 디메틸-4-(2,5-dimethylphenylazo) phenylazo) -2 - 나프톨은)는 v를 높이기 위해 미네랄 오일에 추가 할 수 있습니다isibility. 플런저를 다시 삽입하고 팁의 모든 방식을 밀어. 조심스럽게 기포를 생성하지 마십시오. </li><li> 바이오 안전성 캐비닛의 바이러스 입자 현탁액을 준비합니다. 얼음에 얼어 붙은 바이러스를 해동하고, 단지 사용하기 전에, 원하는 입자 농도에 생리 멸균 인산 버퍼로 희석. </li><li> 주입기의 홀더에 microsyringe를 삽입합니다. </li><li> 작은 플라스틱 시트로 바이러스 정지 피펫 5 μl, 예를 들어 Parafilm. 드롭에 유리 피펫 팁을 절감하고 microsyringe을 채우기 위해 플런저를 당긴다. 막힘 않도록하기 위해 피펫 팁의 작은 기포를 만듭니다. </li></ol> 2. Laminectomy <ol><li> 벤치와 살균제로 소독 가열 패드를 닦고하여 수술 영역을 준비합니다. </li><li> 마우스를 마취. 우리는 isoflurane과 흡입 마취를 사용하여 (3 %를 유도, 유지 보수 기간 동안 2% -3 % 중). </li><li> 동안 건조에서 눈을 보호하기 위해 각 눈에 윤활제를 배치작동. </li><li> 허리에서 마우스의 목에 털을 면도하고 chlorhexidine 또는 povidone-요오드와 70 %의 에탄올과 같은 국소 살균의 잎사귀 교대로 피부를 소독. 수술 드레이프와 무균 준비된 사이트를 격리하고 bupivacaine (0.25 % 생리 식염수에 희석 1시 10분)로 절개 사이트를 침투. </li><li> 중간 선 (2-3 ㎝)을 따라 흉곽의 꼬리 끝에있는 피부를 절개하고 척추를 덮고있는 근막을 분리합니다. </li><li> 척추는 척추 열보다 출생 후의 개발 기간 동안 이전의 성장을 중지하기 때문에, 척추 세그먼트 L4는 첫 번째 요추 척추 (L1) 아래에 자리 잡고 있습니다. 척추 L1은 갈비뼈의 마지막 쌍을 보유하고있는 척추에 꼬리에 자리 잡고 있습니다. 확인하고 작은 척추 근육과 등쪽 표면에 부착 인대를 제거하여 척추의 L1을 쉽게받을 수 있습니다. </li><li> 약간 Adson의 포셉과 함께 척추 뼈의 L1를 들고십시오. 등쪽의 portio를 제거하는 전용 laminectomy의 집게를 사용하여N 척추 (척추와 얇은 판)와 척수를 노출의. 척수를 손상하지 마십시오. </li><li> stereotaxic 프레임에 가열 플레이트에 마우스를 전송합니다. 후속 작업 중에 마우스의 온도를 모니터링 할 수 있습니다. </li></ol> 3. 주입 <ol><li> V 노치 스파이크와 척추 L1 문제를 해결했습니다. 척추 뼈가 호흡하는 동안 움직이지 않도록 스파이크는 척추를 안정화해야합니다. </li><li> 가까이 있으므로 피펫 팁은 laminectomy 사이트 위에있는 microsyringe 가져와. 당신은 피펫을 종료 바이러스 정지가 나타날 때까지 플런저를 낮 춥니 다. 멸균면 팁으로 작은 물방울을 제거합니다. </li><li> 노출 된 척수의 뱃 부리 장식이있는 - 가장 부분에 피펫 팁을 배치합니다. 센터 뒤쪽 중간 고랑 위의 피펫 다음, 500 μm 측면 팁을 이동합니다. 코드와 펑크 경질 한 학교의 표면에 팁을 낮추거나, 당신이 unbeveled 유리 피펫 작업을하는 경우, D에게 주사에 beveled 철강 캐뉼라를 사용하여우라. 척수에 유리 피펫 300 μm의 팁을 낮 춥니 다. </li><li> 200 NL / 분의 속도로 바이러스 정지 1 μl를 삽입. </li><li> 분사의 끝에서 천천히 retracting 피펫을하기 전에 최소한 2 분 정도 기다리십시오. </li><li> 척추 세그먼트 L4에서 바이러스 벡터의 전체 분포를 달성하기 위해 노출 된 척수의 꼬리 - 대부분의 부분에 3.5 단계 3.3를 반복합니다. 두 사출 사이트는 대상 지역의 조직 손상을 방지 할 수 뱃 부리 장식이있는 및 L4 세그먼트의 꼬리에 위치해 있습니다. </li></ol> 4. 상처 폐쇄 <ol><li> 릴리스 척추 V 노치 클램프의 L1하고 stereotaxic 프레임에서 마우스를 제거합니다. </li><li> 5.0 vicryl과 근막을 봉합 해. 폐쇄 근막은 laminectomy 사이트에 대한 커버를 제공합니다. </li><li> 나일론 봉합이나 수술 스테이플로 피부를 닫습니다. </li></ol> 5. 수술 후 관리 <ol><li> 부드러운 nonparticular 침구 복구 케이지에 마우스를 전송합니다. t에 배치그는 편안한 호흡을 위해면. 이 걸을 수있는, 완벽하게 경고하고 술을 시작할 때까지 동물을 모니터링 할 수 있습니다. </li><li> 우리는 carprofen 매일 피하 주사 (5.0 밀리그램 / kg)와 72 시간에 postsurgical 진통제를 제공합니다. </li><li> 처음 3 일간 매일 검사하여 postsurgical 복구를 모니터링 한 다음 실험까지 매일 3 일에 한 주이 완료됩니다. </li><li> 상처 치유가 완료되면, 7-10일 수술 후 피부 봉합 또는 스테이플을 제거합니다. </li><li> 실험 완료 후 동물을 안락사시켜야. </li></ol>

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제작자들은 더 경쟁 금융 이익이 없다는 점 선언합니다.

Stereotaxic 벡터 주입은 neurodegeneration 11 예방 또는 치료에 대한 상해 9,10, 또는 유전자 치료에서 재생하는 동안 6,7 또는 optogenetic의 해부 8, 축삭 안내를 확산 transsynaptic 바이러스에 따라 이러한 neuronal 네트워크 매핑과 같은 응용 프로그램에 대한 척추의 뉴런을 대상으로 할 수 있습니다 12. 바이러스 벡터는 somatosensory, 모터 및 자율 경로주세요 9,10...

마우스 조건부 유전자 조작의 고급 기술은 중추 신경계의 신경 경로와 기능 연결의 탐구에 다각적 인 접근을 가능하게합니다. Transgenes은 에스트로겐 수용체 (1)의 변이 리간드 결합 도메인을 인식 억제 또는 유전자 전사의 활성화, 또는 tamoxifen으로 작동하도록 설계 할 수있는 테트라 사이클린 제어 transactivator에 행동 등 doxycycline와 같은 작은 분자 effectors에 의해 규제 될 수 있습니다 . 돌이킬 수없는 transgene 수정은 일반적으로 데 옥시 리보 핵산 (DNA) recombinases에 의해 달성된다. Cre (원인 재조합) 및 Flp (flippase 재조합 효소)는 절단, 반전 또는 각각 1 loxP (교차로 X의 궤적 이상, P1) 또는 Frt (flippase 인식 대상) 사이트로 둘러싸인 아르 DNA 조각의 translocation을 catalyze. 응용 프로그램은 유전자 활성화 또는 입을과 inducible ribonucleic 산성 (RNA) 간섭을 포함 <sup&gt; 2. 이러한 β-갈 락토 또는 알칼리성 인산 가수 분해 효소와 같은 형광 또는 효소 기자 조건부 표현식은 뉴런에 라벨 및 국소 조직과 연결 심사 3하는 데 사용할 수 있습니다. 북미 (의 대규모 mutagenesis 프로젝트 <a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a> )와 유럽 ( <a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm가</a> )와 마우스 배아 줄기 세포 클론의 라이브러리를 생산하고 있습니다 결국 전체 마우스 게놈을 충당 할 조건부 유전자 목표 및 트랩. 이 클론에서 생성 된 쥐들이 선택적 유전자 조작에 대한 뉴런의 특정 인구 (에 특정 발기인 또는 loci에서 DNA의 recombinases을 표현하는 마우스 라인의 확장 번호로 교차 될 수 있습니다 <a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index . PHP</a> ). <p class= &quot;jove_content&quot;&gt; 그러나, 뉴런 또는 관심 분야의 특정 지역의 고유 한 사람들에게 유전자 조작을 제한하는 것은 유전자 관심의 신경 세포 인구에 특정 발기인이 알려져 있지 않은 경우 혼자 타겟팅 또는 지역의 모든 뉴런으로 표현하지함으로써 달성 할 수 없습니다 관심. 척수에서 실험 디자인은 하나 또는 두 개의 craniocaudal 세그먼트에 유전자 조작의 공간적 제한을 요구할 수 있습니다. Cre 또는 Flp을 표현 바이러스 벡터의 Stereotaxic 주입 floxed 또는 flrted 대립 유전자 소위, DNA 조각이 loxP 또는 Frt 사이트으로 둘러싸인되어있는 마우스의 척수에 지역에 유전자 재조합을 제한 할 수 있습니다. 제정 DNA를 다시 정리하기는 달리, 이는 recombinase 표현 생쥐와 이종 교배하는 동물에서 나타날 전략이 또한 유전자 활성화 또는 입을 통해 일시적으로 제어 할 수 있습니다. 인코딩 바이러스 성 벡터는 floxed 또는 transgenes이 correspo을 표현하는 쥐 유전자 조작의 역 옵션을 제공 경솔한 짓을뉴런 특정 발기인의 recombinase의 하류를 nding. 뉴런에 대한 친화력과 여러 재조합 벡터는 4 이용하실 수 있습니다. 높은 용량 (베짱) 아데노 바이러스, adeno - 관련 바이러스, 헤르페스는 바이러스를 단순하고 lentivirus는 일반적으로 neurotropic 벡터를 사용합니다. 연구 질문에 해당하는 바이러스를 선택하면 실험 설계의 중요한 부분입니다. transgene의 크기, 배달 경로, 염증과 독성 부작용 4 간주해야 glial 세포, 감염 효능, 반대로 뉴런에 감염의 특이성. 여기 우리는 척추의 등 뿔, 우리는 고통의 신경 생물학에 대한 우리의 연구에서 조건부 유전자 조절을위한 고용 한 기술로 바이러스 벡터의 stereotaxic 주사를 설명합니다. 등쪽 뿔은 nociceptive 뉴런을 포함한 기본 somatosensory 뉴런에서 수입 성의를 입력받습니다. 프로젝션 뉴런이에서 전달하기 전에 지역 interneurons는 정보를 처리뇌 5 등쪽 뿔. 우리는 constitutively 활성화 사이토 메갈로 바이러스 프로모터에서 더욱 녹색 형광 단백질 (eGfp)를 표현하는 neurotropic 재조합 adeno - 관련 바이러스 (rAAV)와 척추 segmental 수준 L4의 등쪽 뿔 뉴런의 감염을 보여줍니다.

<table border="1"><tbody><tr><td> 재료 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td></td></tr><tr><td> 척추베이스 플레이트 </td><td> 데이비드 Kopf 악기 </td><td> 912 </td><td></td></tr><tr><td> 작은 동물 stereotaxic 악기 </td><td> 데이비드 Kopf 악기 </td><td> 900 </td><td></td></tr><tr><td> 마우스 가스 마취 헤드 홀더 </td><td> 데이비드 Kopf 악기 </td><td> 923-B </td><td></td></tr><tr><td> 조정 기본 장착 </td><td> 데이비드 Kopf 악기 </td><td> 982 </td><td></td></tr><tr><td> V 노치 스파이크 </td><td> 데이비드 Kopf 악기 </td><td> 987 </td><td></td></tr><tr><td> 작은 동물 온도 제어 시스템 </td><td> 데이비드 Kopf 악기 </td><td> TCAT - 2LV </td><td></td></tr><tr><td> Adson 포셉 </td><td> 정밀 과학 도구 </td><td> 11006-12 </td><td></td></tr><tr><td> Laminectomy의 포셉 </td><td> 정밀 과학 도구 </td><td> 11223-20 </td><td></td></tr><tr><td> SYS-Micro4 컨트롤러와 UltraMicroPump (한) </td><td> 세계 정밀 계측기 </td><td> UMP3-1 </td><td></td></tr><tr><td> Microsyringe, 65RN </td><td> 해밀턴 </td><td> 7633-01 </td><td></td></tr><tr><td> RN 압축 피팅, 1mm </td><td> 해밀턴 </td><td> 55750-01 </td><td></td></tr><tr><td> 붕규산 유리 모세 혈관 </td><td> 세계 정밀 계측기 </td><td> 1B100F-4 </td><td></td></tr><tr><td> Microgrinder </td><td> Narishige </td><td> EG-44 </td><td></td></tr></tbody></table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

바이러스 벡터의 Intraparenchymal 주사가 뉴런 또는 중추 신경계의 특정 지역의 독특한 인구에 조건부 유전자 조작을 할 수 있습니다. 우리는 마우스 척추의 등 뿔의 대상으로 유전자 발현이나 입을 수 stereotaxic 분사 기술을 보여줍니다. 수술 절차는 간단합니다. 그것은 동물과 척추 손상되지 않은 운동성의 빠른 복구를 위해 제공하는 하나의 척추 뼈의 laminectomy이 필요합니다. beveled 유리 캐뉼라와 microsyringe의 낮은 속도와 사용의 작은 벡터 정지 볼륨의 조절 주입은 조직 병변을 최소화합니다. 벡터에 대한 현지 면역 반응은 고용 바이러스의 고유 특성에 따라, 우리의 경험으로는, 재조합 adeno - 관련 바이러스가 사용될 때 미성년자 수명이 짧은 것입니다. 이러한 강화 된 녹색 형광 단백질 등의 리포터 유전자는 벡터의 모니터링 공간 배포를 용이하게하고, 효능 및 세포 specitransfection의 ficity.

성공적으로 transfection 수율 contralateral 측면, 배면의 뿔과 등쪽 루트 신경절의 등 뿔을 구해 주신 주입 등쪽 뿔 (그림 1)의 뉴런에 강력한 유전자 표현. <img src="/files/ftp_upload/50313/50313fig1.jpg" alt="그림 1" /> 1 그림. 등쪽 뿔 뉴런의 Transfection. (A) L4 척추, rAAV-eGfp의 stereotaxic 주입 후 2 주 (혈청 형 AAV2 / 8 / 10 9 게놈 사본의 왼쪽 등쪽 뿔에있는 형광 ?...

Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

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

바이러스 성 벡터는 타겟 유전자 조작을 위해 수 있습니다. 우리는 등쪽 뿔에 바이러스 벡터의 stereotaxic 주입, 기본 somatosensory afferents 및 중추 신경계의 뉴런 사이의 시냅스 접촉의 눈에 잘 띄는 사이트를 사용하여 조건부 유전자 발현 또는 마우스 척추 절제하는 방법을 보여줍니다.

마우스 척추 조건부 유전자 조작을위한 바이러스 성 벡터의 Stereotaxic 주입

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

stereotaxic-injection-viral-vector-for-conditional-gene-manipulation

Research

JoVE Journal

Neuroscience

30.1K 조회수.  Centre National de la Recherche Scientifique (CNRS). 바이러스 성 벡터는 타겟 유전자 조작을 위해 수 있습니다. 우리는 등쪽 뿔에 바이러스 벡터의 stereotaxic 주입, 기본 somatosensory afferents 및 중추 신경계의 뉴런 사이의 시냅스 접촉의 눈에 잘 띄는 사이트를 사용하여 조건부 유전자 발현 또는 마우스 척추 절제하는 방법을 보여줍니다.

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

척수 전기 생리학 II : 세포 흡입 전극 제작

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

연구

신경과학

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

두 광자 현미경을 사용하여 마우스 척수의 생체내 이미징에

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

의 배아와 성인 신경 줄기 세포의 확장<em&gt; 자궁에</em&gt; Electroporation 또는 바이러스 성 Stereotaxic 주입

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

영구와 광범위한 신경 세포의 형질 도입에 대한 신생아 마우스 뇌의 뇌실 바이러스 감염

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

종의 척추 상공 회의소를 주입하는 절차<em&gt; 생체</em&gt; 마우스 척수의 영상

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

마우스 척수의 세포 역학의 두 광자 이미징

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

신생아 마우스 두뇌의 Striatal 세포에서 유전자 조작에 대 한 stereotaxic 수술

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

마우스에 있는 Laminectomy 및 척수 창 이식

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

쥐와 마우스 두뇌에 신경 줄기 세포의 동맥 내 납품: 대뇌 허혈에 응용 프로그램

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...