Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

윤리 선언문 : 마우스 및 실험 절차의 처리는 동물 보호에 대한 유럽 국가 및 기관의 지침에 따라 실시 하였다. 1.에서 자궁 Electroporation에 참고 : 이전에 (12, 14)을 발표 한 자궁 전기에서 수행되었다. 따라서, 상기 방법은 이하의 (도 1)에 간단히 설명 만한다. <ol><li> 준비 <ol><li> 이전 12 설명 된대로 (1,5는 pCAGGS를 포함) 빠른 그린 컬러 독소없는 고급 형질 급 플라스미드 솔루션을 준비합니다. </li><li> 주사를위한 (35 ° 각도) 붕규산 유리 모세관 (0.8-0.9 직경)를 당겨 갈기. </li><li> 악기를 소독. </li></ol></li><li> 외과 <ol><li> 수술을 시작하기 전에 진통제 (카프로 펜, 4 ㎎ / ㎏ 체중, SC, 24 시간 저장소) 적어도 30 분을 관리합니다. </li><li> 의 길이를 유지(- 수술 마취의 단계 Guedel 15에 따라 - 단계 III가 최대 25 ~ 30 분) 35 ~ 45 분의 최대 마취. </li><li> (: 마스크를 통해 2.8 %, 수술 : 2.5 % 유도 챔버) 이소 플루 란과 시간이 초과 임신 쥐를 마취. 발가락 핀치에 응답하지 않는 문제로 마취를 확인합니다. </li><li> 마취 동안 눈 건조를 방지하기 위해 눈 연고를 적용합니다. </li><li> 7.5 % 프로 비돈 - 요오드 용액으로 수술 영역을 소독 노출 만 수술 영역 (0.9 % 벤질 알코올 식염수와 습기) 멸균 거즈와 마우스를 커버한다. </li><li> (: 1.5 cm, 근육 절개 : 1.5 cm의 피부 절개) 메스로 복강을 열고 잘라. </li><li> 수의학 직원 또는위원회의 지시에 따라 예열 0.9 % 벤질 알코올 식염수 또는 다른 멸균 등​​장 성 용액과 가습에 의해 건조에서 열린 복강을 보존합니다. </li><li> 조심스럽게 손가락으로 자궁을 만지지 마십시오, 링 집게 사용하여 자궁 뿔 (추출에스). </li></ol></li><li> DNA와 전기 사출. 참고 : 특정 지역을 대상으로하는 정확한 세부 사항은 섹션 2에 표시됩니다. <ol><li> 준비된 붕규산 모세 혈관을 통해 그림 1과 2 절에 표시된대로 천천히 뇌실에 색깔의 DNA 용액을 주입. </li><li> 해당 배아 단계에 해당하는 전압을 적용 (예. E14를위한, 36 ~ 38 V) 전문 집게 형 백금 전극 (간격주기 길이 50 밀리 초, 간격 일시 정지 950 밀리 초)를 통해. 질 벽의 손상을 최소화하기 위해 전압의인가시 전기 충격기 펄스. </li></ol></li><li> 포스트 전기 / 수술 후 관리 <ol><li> 조심스럽게 복강에서 자궁 뿔을 교체합니다. </li><li> 근육과 별도로 피부 절개 봉합. </li><li> 복구를위한 열 지원 장치에 마우스 나머지를 보자. </li><li> 복구 중에 마우스 (행동, 공급을 감독하고물 소비), 4 시간, 24 시간 및 수술 후 48 시간. 필요한 경우, 추가로 2 일간 추가 진통제 (카프로 펜, 4 ㎎ / ㎏ 체중, 24 시간 저장소)에 적용됩니다. </li></ol></li></ol> 특정 뇌 영역 2. 형질 주 : 양극의 위치는 DNA 용액 (오른쪽 또는 제 3 뇌실)와 양극의 위치를​​ 가득 뇌실 통해 수직 중심선 사이의 각도로 표시된다. 좌심실을 채울 때 위치는 거울 반전. 도 4에 이어 원시의 위치는 중요한 기준점을 제공하는 나타낸다. <ol><li> 피질 영역의 형질 감염 (도 2A, 표 1). <ol><li> 1.2 절에 설명 된대로 수술을 수행합니다. 대뇌 피질의 영역에서 자궁 전기의 경우 E14 배아를 사용합니다. </li><li> 우측 뇌실 4 μg의 DNA를 함유하는 DNA-용액 1.5 μL를 주입준비된 붕규산 모세 혈관을 통해. <ol><li> 신중 천자 부위 위에 보이는 혈관이 없도록 링 집게를 이용하여 태아의 위치. </li><li> 천천히 lambdoidal 봉합에서 약 0.75 mm의 관상면과 시상 봉합 (그림 3)에서 0.5 mm 측면의 DNA 용액을 주입. 삽입 깊이는 1.2 mm (자궁 벽에서 측정 E14가) 있는지 확인하십시오. 날카로운 경계와 파란색 - 녹색 색소를 확인합니다. </li></ol></li><li> 전문 집게 형 백금 전극을 통해 전압 (간격주기 길이 50​​ 밀리 초, 간격 일시 정지 950 밀리 초)를 적용합니다. <ol><li> 3 5mm 전극 패들을 사용합니다. </li><li> 36 ~ 38 V.에 이르기까지 전압을 사용하여 </li><li> 전극 귀 primordia의 바로 앞쪽의 중심을 놓습니다. </li><li> , 운동 피질 0-40 °를 형질 형질, 체성 감각 피질 40-95 °를 형질, 330-0 °에서 retrosplenial 피질의 사용 각도를 형질배 모양의 피질 95-110 ° (그림 2A). </li></ol></li><li> 1.4 절에 설명 된대로 포스트 전기 / 수술 후 관리를 수행합니다. 쥐를 희생과 3 장에 기술 된 바와 같이 전기 (E18) 후 배아 4 일 분석 할 수 있습니다. </li></ol></li><li> 해마 (도 2B, 표 1)의 형질 전환. <ol><li> 1.2 절에 설명 된대로 수술을 수행합니다. 해마 형성의 자궁에 전기를 들어 E15 배아를 사용합니다. </li><li> 준비된 붕규산 모세 혈관을 통해 오른쪽 측면 뇌실에서 4 μg의 DNA를 포함하는 DNA-솔루션 μL 2를 주입. </li><li> 전문 집게 형 백금 전극을 통해 전압 (간격주기 길이 50​​ 밀리 초, 간격 일시 정지 950 밀리 초)를 적용합니다. <ol><li> 3 5mm 전극 패들을 사용합니다. </li><li> 38-40 범위의 전압을 사용합니다. </li><li> 음극 귀 primordium의 바로 앞쪽의 중심과 중심 (O)를 배치했다귀 primordium의 중간에서 양극 F. </li><li> 치아 이랑은 CA1 250-275 ° (그림 2B)를 형질, CA2 230-250 °를 형질, cornu의 ammonis (CA) 3 210-230 °를 형질, 190-210 °에서 각도를 사용하여 형질합니다. </li></ol></li><li> 1.4 절에 설명 된대로 포스트 전기 / 수술 후 관리를 수행합니다. 3 장에 설명 된대로 (출생, P21 후) 완전한 해마 형성 한 후 분석을 수행합니다. </li></ol></li><li> 횡 중격 핵 선조체의 형질 감염 및 (도 2C, 표 1) 참고 : 각도는 해마를 형질에 대한 그와 중첩된다. 효과적으로 측 중격 핵 원치 않는 해마 형질 전환 이전 배아 단계 (E12)없이 선조체를 형질하는 데 사용되어야한다. <ol><li> 1.2 절에 설명 된대로 수술을 수행합니다. 횡 중격 핵과 striat의에서 자궁 전기 용음 E12 배아를 사용합니다. </li><li> 준비된 붕규산 모세 혈관을 통해 오른쪽 측면 뇌실에서 4 μg의 DNA를 포함하는 DNA-솔루션 μL (1)을 주입. </li><li> 전문 집게 형 백금 전극을 통해 전압 (간격주기 길이 50​​ 밀리 초, 간격 일시 정지 950 밀리 초)를 적용합니다. <ol><li> 0.5 mm의 전극을 사용합니다. </li><li> 33 (V)를 사용하여 </li><li> 이어 primordia의 수준에서 전극의 중심을 배치. </li><li> 선조체 170-240 ° (그림 2C)를 형질, 100-170 °에서 횡 중격 핵의 사용 각도를 형질합니다. </li></ol></li><li> 1.4 절에 설명 된대로 포스트 전기 / 수술 후 관리를 수행합니다. 쥐를 희생과 3 장에 기술 된 바와 같이 전기 (E18) 후 배아 6 일 분석 할 수 있습니다. </li></ol></li><li> 시상과 시상 하부의 형질 (그림 2D, 표 1) <ol><li> 1.2 절에 설명 된대로 수술을 수행합니다. U에 들어시상과 시상 하부 사용 E12-E13 배아의 테로의 전기. </li><li> 준비된 붕규산 모세 혈관을 통해 뇌실 (왼쪽 또는 오른쪽) 4 μg의 DNA를 포함하는 1.5 μL의 DNA 용액을 주입한다. 이 솔루션은 제 3 뇌실에 확산 될 때까지 2 ~ 3 분 동안 기다립니다. 다른 방법으로 직접 제 3 뇌실에 주입. 참고 :이 특이성을 강화하지만 감소 생존율 (손상의 위험이 높은)가 발생할 수 있습니다. </li><li> 전문 집게 형 백금 전극을 통해 전압 (간격주기 길이 50​​ 밀리 초, 간격 일시 정지 950 밀리 초)를 적용합니다. <ol><li> 0.5 3mm 전극을 사용합니다. </li><li> 33-35 V.에 이르기까지 전압을 사용하여 </li><li> 바로 귀 primordia 후부 전극의 중심을 놓습니다. </li><li> 시상 하부 90-180 ° (그림 2D)를 형질, 25 ~ 40 °에서 시상 사용 각도를 형질합니다. </li></ol></li><li> DES로 포스트 전기 / 수술 후 관리를 수행1.4 절에 cribed. 쥐를 희생과 3 장에 기술 된 바와 같이 전기 (E18) 후 배아 6 일 분석 할 수 있습니다. </li></ol></li></ol> 3. 관류 고정 <ol><li> 준비 <ol><li> 따뜻한 4 % 파라 포름 알데히드 (PFA)로 37 ° C. </li><li> PBS로 50 ML의 주사기 (4 ° C) 및 예열 PFA와 두 번째 주사기를 입력합니다. 거품을 피하십시오. </li><li> 세 방향 꼭지에 주사기를 연결합니다. 날개 달린 주입 세트 (나비)에 세 번째 끝을 연결합니다. </li><li> PBS로 날개 달린 주입 세트를 플래시합니다. </li></ol></li><li> 관류 <ol><li> 깊이 피하 케타민 염산염의 응용 프로그램 (60 ㎎ / ㎏)과 자일 라진 (7.5 ㎎ / ㎏)와 (임신 또는 출생 후의 형질) 마우스를 마취. </li><li> 발가락 핀치에 응답하지 않는 문제로 수술 허용 단계를 확인합니다. </li><li> 바로 흉곽 아래 복강을 엽니 다. </li><li> 조심스럽게 다이어프램을 잘라. </li><li> 조심스럽게 전자에 컷을 통해 흉곽을 열고쇄골까지 ACH 사이트. </li><li> (심장의 정점에서 시작) 왼쪽 심장 챔버로 나비의 끝을 삽입합니다. </li><li> 좌심방을 잘라. </li><li> PBS 천천히 (약 10ml)로 마우스를 관류. </li><li> 삼방 코크 스위치. </li><li> 5 ~ 10 ml의 4 % PFA와 마우스를 관류. </li><li> 가위를 사용하여 마우스를 목을 벨과 난원 매그넘에서 시작 뇌를 해부하다. 이전에 (16)이 발표 한 절개를 수행합니다. </li><li> 4 % PFA 2 일 동안 뇌를 Postfixate. 어둠 속에서 4 ° C에서 머리를 유지합니다. </li></ol></li></ol> 4. 면역 조직 화학 <ol><li> (vibratome와 예) 70 μm의 섹션을 생성합니다. </li><li> 옵션 : 항체 염색 형광을 향상시킬 수 있습니다. <ol><li> RT에서 1 시간 동안 차단 부 (0.1-0.2 % 트리톤 X-100, 5 % 정상 염소 혈청) </li><li> 적절한 항체 (1,000 방지 GFP, 1)로 O / N을 품어 </li><li> 워시 30.1 M 인산 완충와 -5 번. </li><li> 적절한 형광 - 복합 이차 항체 (1,000 알렉사 플 루어 488 복합 항 - 토끼 IgG를, 1)과 3-5 시간을 품어. </li><li> 선택적 : 1 분간 (0.1 M 인산 완충액 DAPI, 0.2 g / ㎖) 4-6-diaminodino -2- 페닐 인돌로 Counterstain과. </li><li> 적절한 형광 현미경으로 형광을 분석 할 수 있습니다. </li></ol></li></ol>

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D., Ikenaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+that+ventral+forebrain+cells+migrate+to+the+cortex+and+contribute+to+the+generation+of+cortical+myelinating+oligodendrocytes.">Direct evidence that ventral forebrain cells migrate to the cortex and contribute to the generation of cortical myelinating oligodendrocytes.</a> Dev Biol. 291 (1), 123-131 (2006).</li><li>Baumgart, J., Grebe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C57BL/6-specific+conditions+for+efficient+in+utero+electroporation+of+the+central+nervous+system.">C57BL/6-specific conditions for efficient in utero electroporation of the central nervous system.</a> J Neurosci Methods. 240, 116-124 (2015).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat Protc. 1 (3), 1552-1558 (2006).</li><li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+utero+electroporation:+controlled+spatiotemporal+gene+transfection.">Mouse in utero electroporation: controlled spatiotemporal gene transfection.</a> JoVE. (54), (2011).</li><li>Guedel, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inhalation anesthesia: a fundamental guide. , (1937).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> JoVE. (65), e3564 (2012).</li><li>Mizutani, K., Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitors+resume+generating+neurons+after+temporary+inhibition+of+neurogenesis+by+Notch+activation+in+the+mammalian+cerebral+cortex.">Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex.</a> Development. 132 (6), 1295-1304 (2005).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+embryonic+mouse+central+nervous+system+cells+by+in+utero+electroporation.">Labeling embryonic mouse central nervous system cells by in utero electroporation.</a> Dev, Growth &amp; Differ. 50 (6), 507-511 (2008).</li></ol>

The authors have nothing to disclose.

This protocol describes in detail how to transfect 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 of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific r...

3 개의 독립적 인 그룹 1-3에 의해 2001 년 처음 소개 이후 N 자궁 일렉트로는 설치류 중추 신경계에서의 유전자 발현을 분석하기 위해 널리 사용되는 표준 도구가되었다. 지속적으로 개선 기술, 아직 시간과 돈 단순하기 때문에, 자궁 내 전기 호소 소비에도 불구하고,이다 녹아웃 마우스의 세대에 비해. 따라서, 자궁 내 전기는 신속하고 효율적인 이득이 손실-의 기능 연구 (4)를 할 수 있습니다. 뇌 영역 트랜, 음으로 하전 된 플라스미드를 함유하는 용액이 뇌실 내로 주입된다. 전기 펄스 동안, 음으로 하전 된 DNA는 양극으로 이동한다 따라서 형질 영역은 양극의 위치를​​ 변경하여 간단하게 선택할 수있다. 그것은 자주 중추 신경계의 다양한 영역 (TA)이 될 수 있음을 보여왔다3,5-8를 rgeted. 예를 들어, 최근의 연구는 해마, 배 모양의 피질이나 줄무늬 9-11의 특정 형질을 보여줍니다. 그러나, 해당 위치에 대한 정보는 거의 종종 표준화되지 않고, 항상 상이한 마우스 균주로 전송할 쉽지 않다. 특정 배아 단계의 형질 사소한는 거리가 멀다. 자궁의 전기의 특정을위한 셋업을 선택할 때 많은 영향을 미치는 요인을 고려해야합니다. 먼저, 각각의 최적 배아 단계 형질, 적절한 전압에 대한 지식이 필요하다. 낮은 전압이 형질 전환 효율을 감소 2,3,12 반면 고전압은 생존율을 감소시킨다. 또한 전극 패의 크기가 중요한 역할을 감소 특이성에 너무 큰 결과 또는 인해 심장 리듬 4,12,13의 애정에 사망에이를 수 있습니다 전극 충격기를 사용하기 때문이다. 적용전압의 크기와 전극 패들의 위치가 고려하는 가장 중요한 특징이지만, DNA-용액의 도포량 같이 전기의 결과에 영향을 미치는 또 다른 요인들도있다. 우리는 C57BL / 6 마우스 12 다양한 뇌 영역의 빠르고 효율적인 형질 있도록 상세한 프로토콜을 개발했다. 이 프로토콜에서 전압에 대한 자세한 정보는 사용되는 향상된 특이성 전극 패들의 크기가 제공된다. 또한, 심실 대한 정보 플라스미드 용액의 양 전극의 위치에 대한 권장 사항이 공급과 함께 충전된다. 지도와 이러한 위치의 더 시각화의 자세한 위치 정보의 표시는 retrosplenial 피질, 운동 피질, 체성 감각 피질, 배 모양의 피질, T의 자궁의 전기에서 간단 구체적이고 효율적으로 할 수 있습니다그는 ammonis 1-3, 치아 이랑 (dentate gyrus), 선조체, 측 중격 핵, 시상과 시상 하부 cornu.

<table><tbody><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Fast Green</td><td>Roth</td><td>0301.1</td><td></td></tr><tr><td>pCAGGS</td><td>Addgene</td><td></td><td></td></tr><tr><td>borosilicate glass capillaries (0.8-0.9 mm diameter)</td><td>Wold Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott</td><td>PZN 4831850</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>approval number: 400684.00.00</td><td></td></tr><tr><td>eye ointment (Bepanthen Augen und Nasensalbe)</td><td>Bayer&amp;nbsp;</td><td>PZN 01578681</td><td></td></tr><tr><td>0.9% benzyl alcohol 0.9% saline solution</td><td>Pharmacy&lt;br/&gt; of the University Medical Center Mainz</td><td></td><td></td></tr><tr><td>gauze (ES-Kompressen)</td><td>Hartmann</td><td>407835</td><td></td></tr><tr><td>sterile 5-0 Perma-Hand Silk Suture</td><td>Ethicon Johnson &amp;amp; Johnson</td><td>K890H</td><td></td></tr><tr><td>ring forceps 1/ 1.5 mm</td><td>Fine Science Tools</td><td>11101-09</td><td></td></tr><tr><td>ring forceps 4.8/ 6 mm</td><td>Fine Science Tools</td><td>11106-09</td><td></td></tr><tr><td>ring forceps 2.2/ 3 mm</td><td>Fine Science Tools</td><td>11103-09</td><td></td></tr><tr><td>Adson Forceps-Serrated Straight 12 cm</td><td>Fine Science Tools</td><td>1106-12</td><td></td></tr><tr><td>IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Mayo-Stille Scissors-Straight 15 cm</td><td>Fine Science Tools</td><td>14012-15</td><td></td></tr><tr><td>Dumont #5 Forceps-Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Elektroporator CUY21 SC&amp;nbsp;</td><td>Nepa Gene Co.</td><td></td><td></td></tr><tr><td>FST 250 Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Microgrinder EG-44</td><td>Narishige</td><td></td><td></td></tr><tr><td>P-97 Micropette Puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Platinum electrodes 650P 0.5 mm</td><td>Nepagene</td><td>CUY650P0.5</td><td></td></tr><tr><td>Platinum electrodes 650P 3 mm</td><td>Nepagene</td><td>CUY650P3</td><td></td></tr><tr><td>Platinum electrodes 650P 5 mm</td><td>Nepagene</td><td>CUY650P5</td><td></td></tr><tr><td>Platinum electrodes 650P 10 mm</td><td>Nepagene</td><td>CUY650P10</td><td></td></tr><tr><td>Anesthesia system</td><td>Rothacher-Medical GmbH</td><td>CV-30511-3 Vapor 19.3</td><td></td></tr><tr><td>Heating plate</td><td>Rothacher-Medical GmbH</td><td>HP-1M</td><td></td></tr><tr><td>Temperature Controller 220V AC</td><td>Rothacher-Medical GmbH</td><td>TCAT-2LV</td><td></td></tr></tbody></table>

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.

그림 2, retrosplenial 피질로 개발 지역의 자궁 전기의 특정에 대한 예를 보여줍니다, 운동 피질, 체성 감각 피질, 배 모양의 피질, cornu가 1-3 ammonis, 치아 이랑 (dentate gyrus), 선조체, 횡 중격 핵 , 시상과 시상 하부. 형질 감염의 결과는 권장 각도 (도 2) 다음에 나타낸다. 생체 내에서 각의 더 나은 시각화를위한 전극 (0.5 mm)의 위치도 (양막 낭 그림 5, ...

Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

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 ...

cortex-hippocampus-thalamus-hypothalamus-lateral-septal-nucleus

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

28.0K 조회수.  University Medical Center, JGU Mainz. 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.

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

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15 마우스 태아의 Utero의 심실 주입 및 Electroporation에

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

연구

생물학

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

사용하여 여러 개의 CNS 준주로 효율적인 유전자 전달 Utero에서 Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

신경과학

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

마우스 Utero에 Electroporation : 통제 Spatiotemporal 진 Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

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 ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

마우스 망막 신경절 세포의 유전자 발현에 대한 utero 및 전직 생체내의 Electroporation에

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

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 ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

두피의 interneurons의 하위 유형 선택적 일렉트로

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

자궁 내 일렉트로는 신경 세포의 부분 집단의 흥분 및 단일 세포의 연결을 연구에 접근

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

자궁에서 두 배의 자궁 전기화는 시간적으로 그리고 공간적으로 분리된 세포 인구를 표적으로 합니다

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

쥐 In Utero 전기 천공

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...

코어 텍스, Hippocampus-, Thalamus-, Hypothalamus-, 횡 중격 Nucleus-과 줄무늬 별

요약

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