Name: stereotaxic surgery for genetic manipulation in striatal cells of neonatal mouse brains
Uploaded: 2018-07-10T12:30:00.000+00:00
Duration: 9 min 44 s
Description: Watch this Scientific Journal Video about Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains at JoVE.com

이 작품은 과학의 부에 의해 지원 되었다 및 기술 부여 MOST104-2311-B-010-010-MY3, MOST106-2321-B-010-012, 국립 건강 연구소 부여 NHRI-EX106-10429NI, 및 추천된 지역 연구 센터 프로그램 부여 뇌 연구 센터, 대만에서 국립 양 밍 대학 교육부의 박사 친목 부여 MOST106-2811-B-010-031 (S. Y.C.), MOST105-2811-B-010-036 및 MOST106-2811-B-010-030 (헤-영 규 과장).

여기에 설명 된 동물 프로토콜 동물 관리 및 사용 위원회의 국립 양 밍 대학에 의해 승인 되었습니다가지고.
1. 신생아 강아지 Stereotaxic 장치에 대 한 소유자의 준비
<ol><li>머리 트레이 만들기: 1.5 mL 원심 분리기 튜브 (길이 15 m m)의 하단 튜브의 벽의 1/5를 제거 하 여 신생아 강아지의 머리에 맞는 모양으로 잘라.</li>
	<li>피 펫 팁 상자 stereotaxic 기구의 받침대와 맞는 적당 한 크기와 고 상단 덮개를 제거. 머리 트레이 단계 1.1 및 뜨거운 용 해 접착제로 팁 쟁반의 기지에 조직 포함 카세트에 부착 조직 포함 카세트의 높이 7 m m, 머리 고정 위치에서 강아지의 목과 시체를 지 원하는 데 사용 되는입니다.</li>
	<li>표준 stereotaxic 장치에 전체 설치를 넣습니다.</li>
</ol>2. 30 G 주입 스테인리스 바늘의 준비
<ol><li>전 3 일 동안 클로 프롬에서 그들을 몸을 담글 하 여 30 G 주사기 바늘을 청소.</li>
	<li>집게를 사용 하 여 신중 하 고 천천히 당겨 바늘 화학 후드에서 폴 리 프로필 렌 그것의 허브에서.</li>
	<li>20 분 50 rpm에서 통에 70% 에탄올 린스 3 × 10 분 뒤 절대 에탄올과 바늘을 씻어. 사용까지 바늘 공기 건조, 그리고 청소 바늘 실 온 (RT)에서 깨끗 한 상자에서 저장을 하자.</li>
</ol>3. Microinjection 튜브 어댑터 준비
<ol><li>PE10 폴 리 에틸렌 튜브 (PE10 튜브) 30g 주사 바늘에 microliter 주사기를 연결 합니다. 브리징 microliter 주사기와 주사 바늘에 대 한 PE20 폴 리 에틸렌 튜브 (PE20 튜브) 어댑터를 준비 합니다. 어댑터의 사용 때문에 필요, PE10 튜브의 직경 26 G microliter 주사기의 바늘의 그것 보다 훨씬 작습니다.</li>
	<li>Microliter 주사기에 대 한 튜브를 준비: PE20 튜브 5 cm와 미리 청소 30g 주사 바늘을 연결 하 고 인스턴트 접착제로 접합 인감. 이 튜브 어댑터는 재사용 가능한. 참고: microinjection 주사기의 바늘 30 G 이면 준비 (3.1 단계) 및이 어댑터의 사용 (단계 3.3) 필요 하지 않습니다.</li>
	<li>Microliter 주사기 (10 µ L) (3.1 단계에서 준비) 튜브 어댑터를 연결 합니다.</li>
	<li>PE20 튜브 어댑터의 30 G 바늘에 (더 이상 60 cm) PE10 튜브의 한쪽 끝을 연결 합니다. PE10 튜브의 다른 쪽 끝에 새로운 30 G의 주사 바늘을 탑재 합니다.</li>
</ol>4. 부하 압력가 증류수, 염료와 바이러스 Microinjection 튜브
<ol><li>Microliter 주사기의 플런저를 제거 합니다. 25 G 주사기를 사용 하 여 로드 microliter 주사기 및 압력가 증류수는 배관에서 공기를 제거 하는 연결 된 PE 튜브.</li>
	<li>플런저 microliter 주사기를 다시 놓고 배럴에 증류수 남아 2 µ L까지 플런저를 밀어. 양의 증류수 2 µ L 보다 낮은 되 게 하지 마십시오.</li>
	<li>신중 하 게 마이크로 흐름 속도 주사기 펌프에 microliter 주사기를 탑재 합니다.</li>
	<li>적은 양의 0.1% 빠른 녹색 염료 (0.9% 식 염 수에 준비와 0.22 μ m 필터 필터링)와 parafilm 조각에 바이러스 액체 플라스틱</li>
	<li>Microinjection 튜브에 체액의 흐름을 테스트 하려면 30 G 주입 바늘과 튜브에 필터링 된 빠른 그린의 0.7 µ L을 로드 하 여 다음 PE10 튜브 사이 공기 방울이 볼 수 있도록 공기의 작은 금액을 철회 한다.</li>
	<li>또 다른 작은 양의 바이러스 액체 microinjection 튜브에 로드 하 여 다음 두 번째 공기 거품을 만들기 위해 공기를 철회 한다.</li>
	<li>연결 하 고 30 G microinjection 바늘 stereotaxic 기구의 팔을 확보.</li>
</ol>5입니다. 저체온증에 의해 신생아 쥐의 마 취
<ol><li>라텍스 장갑 소매에 강아지를 놓고 5 분 목까지 얼음에 몰두 합니다.</li>
	<li>되도록 그의 철수 응답 집게로 강아지의 발을 꼬집어.</li>
	<li>라텍스 장갑 소매와 강아지를 머리 트레이에 놓고 저 체온 마 취에 대 한 차가운 유지에 라텍스 슬리브 주위 일부 얼음을 넣어.</li>
	<li>가능 하면 마 취 동안 건조를 방지 하기 위해 강아지의 눈에 수 의사 연 고를 적용 합니다.</li>
</ol>6입니다. microinjection
<ol><li>70% 에탄올으로 깨끗이 stereotaxic 악기를 닦아 하 여 불 임 수술을 준비 합니다. 70% 에탄올에 그들을 immersing 하 여 외과 기구를 소독.</li>
	<li>70% 에탄올과 강아지의 머리를 제거 하십시오. 두개골에 랜드마크 람다를 찾아서 람다 마커 펜으로 표시 합니다. 람다에 바늘 끝을 조 준 하 고 0으로 이전 후부 (AP) 및 중간 옆 (ML) 좌표를 설정 합니다.</li>
	<li>대상 사이트의 대상 사이트의 X 및 Y 좌표에 따라 주입 팔을 이동 합니다. 출생 후 하루 (P) 2의가 대 한 강아지, 좌표는: AP, 람다; 앞쪽 +2.4 m m ML, ± 1.0 mm; 중간에서 측면 등-복 부 (DV), 두개골에서 −1.7 m m. 펜으로 PE10 관에서 빠른 녹색 염료의 위치를 표시 합니다.</li>
	<li>30g 주사 바늘 아래로 천천히 두개골, 피부를 통해 침투 하 가져오고 두개골의 표면에서 멈출 때까지 다음 바늘 팁을가지고. 0으로 DV 좌표를 설정 합니다.</li>
	<li>내릴 30 G 주입 바늘 천천히 대상 사이트의 DV 좌표에 도달할 때까지. 실질 정상적인 모양을 다시 시작 수 있도록 1 분 기다립니다. Microinjection 프로그램 (100 nL/min)를 실행 합니다.</li>
	<li>빠른 녹색 염료의 마크는 바이러스 액체 두뇌에 주입 되도록 PE 튜브에 움직이는 다는 것을 확인 하십시오.</li>
	<li>Microinjection, 종료 후 1 분 동안 대기 그리고 다음 천천히 그리고 점진적으로 30 DV 깊이의 1/2 높이에 바늘을가지고 s. 30 후 s, 천천히 강아지의 머리에서 바늘을 철회.</li>
	<li>모든 타겟된 사이트의 microinjections는 완료 될 때까지 단계 6.2 6.7를 반복 합니다.</li>
</ol>7. 강아지의 포스트 외과 복구
<ol><li>33 ° C 배양 기에 20 분 동안 강아지를 따뜻한. 강아지 sternal recumbency를 유지 하기 위해 충분 한 의식 회복 될 때까지 저 체온 마 취 매 5 분에서에서 강아지의 복구를 확인 하십시오.</li>
	<li>강아지는 완 치 후에을 다시 강아지를 반환 합니다.</li>
</ol>

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저자는 공개 없다.

현재 연구에서 신생아 마우스 두뇌가 AAV 바이러스 주입에 대 한 간단 하 고 안정적인 stereotaxic 방법을 보여 줍니다. 우리는 p 2에서 Ai14 기자 쥐가 AAV eGFP Cre 바이러스 microinjected 하 고 P14에서 리포터 유전자 발현 분석. 우리는 AAV 불리고 GFP-긍정적인 세포가 rostrocaudal 수준에 걸쳐 발견. 또한, 거의 모든 GFP-긍정적인 세포 공동 tdTomato 기자 유전자 striatal 셀에 제안 하는 성공적인 Cre loxP DNA 재조합 AAV 중...

현대 연구의 구조와 뇌의 기능 일반적으로 신경 세포에 특정 유전자의 유전자 조작이 필요합니다. 다른 유전자, 돌연변이 체 대립 유전자를 포함 하 여 운반 하는 유전자 변형 마우스의 기능을 녹아웃와 대립 노크에서 정기적으로 생성. Stereotaxic 뇌 수술 성인 설치류에 대 한 로컬 설치류 두뇌1,2의 특정 지역에 약물, 바이러스, 추적기 및 다른 시 약을 제공 하는 표준 방법입니다. 유전자 변형 마우스 stereotaxic 뇌 수술을 적용 한 유전자 조작 유전자 기능과 마우스 뇌의 특정 신경 인구에 신경 활동을 허용 합니다. 셀 형식 관련 조작 두뇌3,,45의 복잡 한 신경 회로에 신경 기능을 해독 하는 강력한 접근 방식을 제공 합니다.
신 경계의 신경 개발 초기 배아 단계에서 시작 하 고 개발 프로세스 청소년 기간까지 출산 후 계속. 신경 시스템의 출생 후 성숙 뇌6의 생리 및 인지 기능을 위한 필수적인 신경 회로의 정확한 시 냅 스 배선 포함 되어 있습니다. 따라서, 신생아 시간 윈도우에서 발생 하는 개발 이벤트를 공부 뿐만 아니라 정상적인 신경 발달을 이해 하는 데 중요 하지만 neurodevelopmental과7 정신병 무질서의 병 인에 대 한 통찰력을 제공할 수 있습니다. ,8. 성인 설치류 stereotaxic 뇌 수술의 방법으로는 쉽게 사용할 수 있는2,9, 비록 몇 가지 프로토콜 신생아 쥐10,11stereotaxic 뇌 수술에 대 한 인터넷에서 사용할 수 있습니다. 사실, 신생아 마우스 새끼의 두뇌에 약의 stereotaxic microinjections는 어렵다, 신생아 강아지의 머리는 너무 연약한 표준 stereotaxic 장치에 고정 하기 때문에. 그럼에도 불구 하 고, 유전자 변형 마우스 stereotaxic 뇌 수술의 응용 프로그램은 신생아 쥐12가능 합니다. 여기, 우리가 만든 설치 신생아 마우스 새끼 stereotaxic 뇌 수술을 수행 하는 간단한 방법 설명. 이 기술은 허용 하나 조건부로 AAV 표현 Cre DNA recombinase 기자 진 마우스와 조건에 따라 floxed 유전자 변형 쥐가 microinjecting floxed 유전자 삭제를 보여 줍니다. 이 기술은 또한 야생-타입 마우스의 신생아가에 시 약을 제공에 적용 됩니다.

<table><tbody><tr><td>30G PrecisionGlide Needle</td><td>Becton Dickinson</td><td>REF 305106</td><td></td></tr><tr><td>Chloroform</td><td>JT Baker</td><td>9180-03</td><td></td></tr><tr><td>Hamilton MICROLITER Syringe</td><td>Hamilton&amp;nbsp;</td><td>80300</td><td>30G needle fit for PE10 tube; 26G needle needs a PE20 adaptor</td></tr><tr><td>Polyethylene tubing PE20</td><td>Becton Dickinson</td><td>427406</td><td></td></tr><tr><td>Polyethylene tubing PE10</td><td>Becton Dickinson</td><td>427401</td><td></td></tr><tr><td>Micro Flow Rate Syringe Pump</td><td>Longer Precision Pump Co.</td><td>TJ-2A (Controller) and L0107-2A (Drive Unit)</td><td></td></tr><tr><td>25G syringe</td><td>Becton Dickinson</td><td>REF 302105</td><td></td></tr><tr><td>Fast green</td><td>Sigma-Aldrich</td><td>F-7252</td><td>0.1%</td></tr><tr><td>Standard Stereotaxic Instruments</td><td>RWD Life Science</td><td>68037</td><td>Without using 68030 Mouse/Neonatal Rat Adaptor</td></tr><tr><td>Anti-FOXP2 antibody</td><td>Abcam</td><td>ab16046</td><td>Rabbit polyclonal to FOXP2, 1:4,000</td></tr><tr><td>Anti-RFP antibody</td><td>Abcam</td><td>ab65856</td><td>Mouse monoclonal to RFP, 1:1,000</td></tr><tr><td>BX63 microscope</td><td>Olympus</td><td>BX63</td><td></td></tr><tr><td>LSM 880 confocal microscope</td><td>Zeiss</td><td>LSM 880</td><td></td></tr><tr><td>Goat anti-rabbit conjugated Alexa fluor594</td><td>Jackson lmmunoReserch Laboratories Inc.</td><td>111-585-003</td><td></td></tr><tr><td>AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40</td><td>Penn Vector Core</td><td>AV-9-PV1848</td><td>Lot # CS0987, 5.506x10&lt;sup&gt;13&lt;/sup&gt; (GC/mL)</td></tr><tr><td>AAV9.chicken actin-eGFP</td><td>AAV core, Institute of Biomedical Sciences, Academia Sinica, Taiwan</td><td>N/A</td><td>1x10&lt;sup&gt;14&lt;/sup&gt; (GC/ml)</td></tr><tr><td>B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td><td>The Jackson Labtorary&amp;nbsp;</td><td>007914</td><td>Ai14</td></tr><tr><td>B6(Cg)-Foxp2tm1.1Sfis/CfreJ</td><td>The Jackson Labtorary&amp;nbsp;</td><td>026259</td><td>Foxp2&lt;sup&gt;fl/fl&lt;/sup&gt;</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline</td><td>Corning&amp;nbsp;cellgro</td><td>21-030-CVR</td><td></td></tr></tbody></table>

stereotaxic surgery for genetic manipulation in striatal cells of neonatal mouse brains

많은 유전자는 미 발달 머리에 표현 하 고 그들 중 일부는 지속적으로 출생 후 뇌에 표현. 이러한 지속적으로 표현한 유전자에 대 한 그들은 발달 과정 및 신생아 두뇌의 생리 기능을 조절 하 작동 수 있습니다. 두뇌에 있는 특정 유전자의 neurobiological 기능 조사, 두뇌에서 유전자를 비활성화 필수적 이다. 여기, 우리 신생아 시간 창에서 유전자 변형 생쥐의 고가에 유전자 발현을 비활성화 하는 간단한 stereotaxic 방법을 설명 합니다. AAV eGFP Cre 바이러스는 출생 후 하루 (P) stereotaxic 뇌 수술에 의해 2에서 Ai14 기자 유전자 쥐가 microinjected 했다. TdTomato 기자 유전자 발현 P14가, 성공적인 Cre loxP 중재 DNA 재조합 AAV 불리고 striatal 셀에 제안에 발견 되었다. 우리는 더 AAV eGFP Cre 바이러스 P2Foxp2fl/플로리다 쥐로 microinjecting에 의해이 기술을 검증. GFP의 Foxp2 더블 라벨 GFP-긍정적인 세포 Foxp2 immunoreactivity P9가, AAV-eGFP-Cre 불리고 striatal 셀에서 Foxp2 단백질의 손실을 제안에 부족을 보여주었다. 함께 찍은, 이러한 결과 floxed 유전자 변형 생쥐의 신생아 두뇌에 있는 특정 신경 인구에 있는 stereotaxically microinjected AAV eGFP Cre 바이러스에 의해 효과적인 유전자 삭제를 보여 줍니다. 결론적으로, 우리의 stereotaxic 기술 신생아 마우스 두뇌에서 유전자 조작에 대 한는 쉽고 간단한 플랫폼을 제공합니다. 기술만 사용할 수 없는 신생아 두뇌의 특정 지구에 있는 유전자를 삭제 하 하지만 또한 사용할 수 있습니다 약물 약물, 신경 추적기, 유전자 변형된 optogenetics 및 chemogenetics 단백질, 신경 활동 지표를 삽입 하 고 신생아 마우스 두뇌의가에 다른 시 약.

실험의 첫 번째 집합에 대 한 우리 200 microinjected AAV9.hSynapsin.HI.eGFP Cre.WPRE.SV40 바이러스 (AAV-eGFP-Cre, Dulbecco의 버퍼링 인산 염 분에 1/10 희석) Cre DNA recombinase 표현의 nL Ai14 쥐의 P2가 GFP 융합. Ai14 마우스 loxP 형벌 (floxed) 정지 카세트 (그림 2F)의 Cre 중재 삭제 시 tdTomato 취재 원 유전자를 표현 한다. 두뇌는 GFP와 tdTomato의 immunostaining에 대 한 P14에서 수확 했다. ...

Watch this Scientific Journal Video about Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains at JoVE.com

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

신생아 마우스 두뇌의 Striatal 세포에서 유전자 조작에 대 한 stereotaxic 수술

우리는 신생아 마우스 두뇌의가에 microinjecting 시 약에 대 한 수 제 머리 고정 장치 stereotaxic 수술의 프로토콜을 설명합니다. 이 기술은 신생아 마우스 두뇌의 특정 영역의 신경 세포에서 유전자 조작 가능

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

stereotaxic-surgery-for-genetic-manipulation-striatal-cells-neonatal

Research

JoVE Journal

Neuroscience

14.8K 조회수.  National Yang-Ming University. 우리는 신생아 마우스 두뇌의가에 microinjecting 시 약에 대 한 수 제 머리 고정 장치 stereotaxic 수술의 프로토콜을 설명합니다. 이 기술은 신생아 마우스 두뇌의 특정 영역의 신경 세포에서 유전자 조작 가능

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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

신경의 분석을위한 성인 마우스 V-SVZ에서 세포의 안정적이고 효율적인 유전자 변형 줄기 세포 자율 및 비 자치 효과

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

연구

발생학

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

성인 쥐의 특정 두뇌 영역의 Excitotoxic 병변에 대한 Stereotaxic 외과

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

신경과학

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 ...

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

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

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

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 ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

마우스의 유전자 조작을 통해 시상 하부 개발<em&gt; 자궁 내</em&gt; 일렉트로

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

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 ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

코어 텍스, Hippocampus-, Thalamus-, Hypothalamus-, 횡 중격 Nucleus-과 줄무늬 별<em&gt; 자궁 내</em&gt; C57BL / 6 마​​우스에서 Electroporation에

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain regions still presents a challenge, particularly when targeting brain structures along the midline. These challenges include avoiding of the superior sagittal sinus and third ventricle and the ability to consistently target selective and discrete brain nuclei. In addition, more advanced neuroscience techniques (e.g., optogenetics, fiber photometry, and two-photon imaging) rely on targeted implantation of significant hardware to the brain, and spatial limitations are a common hindrance. Presented here is a modifiable protocol for stereotactic targeting of rodent brain structures using an angled coronal approach. It can be adapted to 1) mouse or rat models, 2) various neuroscience techniques, and 3) multiple brain regions. As a representative example, it includes the calculation of stereotactic coordinates for targeting of the mouse hypothalamic ventromedial nucleus (VMN) for an optogenetic inhibition experiment. This procedure begins with the bilateral microinjection of an adeno-associated virus (AAV) encoding a light-sensitive chloride channel (SwiChR++) to a Cre-dependent mouse model, followed by the angled bilateral implantation of fiberoptic cannulae. Using this approach, findings show that activation of a subset of VMN neurons is required for intact glucose counterregulatory responses to insulin-induced hypoglycemia.

Described here is a stereotactic procedure that can target challenging and difficult-to-reach brain regions (due to spatial limitations) using an angled coronal approach. This protocol is adaptable to both mouse and rat models and can be applied to diverse neuroscientific applications, including cannula implantation and microinjections of viral constructs.

다목적 신경 과학 기술을 위한 적응가능한 각진 입체 적 접근 법

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

Rodent Stereotaxic Surgery

Stereotaxic (or stereotactic) surgery is a method used to manipulate the brain of living animals. This technique allows researchers to accurately target deep structures within the brain through the use of a stereotaxic atlas, which provides the 3D coordinates of each area with respect to anatomical landmarks on the skull. After the skull is exposed, anesthetized animals are mounted on a specialized instrument known as a stereotaxic frame, which enables the precise placement of experimental tools at the defined coordinates. Stereotaxic surgery is a versatile approach that can be used to generate lesions, manipulate gene expression, or deliver experimental agents to the brain.This video-article provides a general overview of the principles behind stereotaxic surgery, including instructions for using a stereotaxic atlas and the stereotaxic frame, and an introduction to reading the Vernier scale for measurement of probe movements. The subsequent discussion outlines the steps required to perform the surgical procedure. Lastly, a broad range of technical applications are presented, such as the insertion of electrical probes to measure brain activity and genetic manipulation of brain tissue.

설치류 정위 수술

Stereotaxic (or stereotactic) surgery is a method used to manipulate the brain of living animals. This technique allows researchers to accurately target ...

신생아 마우스 두뇌의 Striatal 세포에서 유전자 조작에 대 한 stereotaxic 수술

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