이 작품은 RIKEN 뇌 과학 연구소 (BSI), 인간 프론티어 과학 프로그램 (HFSP) (TS에게 수여) 및 RIKEN 주니어 리서치 연결 프로그램 (JRA, MK에게 수여) RIKEN 뇌 과학 연구소 (BSI), 인간 프론티어 과학 프로그램에 의해 투자되었다 (HFSP) (TS에게 수여) 및 RIKEN 주니어 리서치 연결 프로그램 (JRA, MK에게 수여)이 작품을 후원.

1. 분사에 대한 모세관과 DNA 솔루션의 준비 <ol><li> 맥시 - 준비 또는 이와 동등한 방법으로 플라스미드 DNA를 정화, 후 2-3 μg / μl의 최종 농도합니다. 나누어지는 DNA의 100 μg가 빠르고, 녹색 염료 (1 % 주식) 및 TE의 10μl를 추가 (10mM 트리스베이스, 1mM EDTA (에틸렌 다이아 민 테트라 초산) 솔루션, 산도 8.0)와 1μg /에 분사 혼합의 최종 DNA 농도를 100 μl로 음량을 조절 μl. 플라스미드 DNA의 농도를 변경할 수있다는 개별적으로 테스트해야 자사의 transfection 효율 또한 플라스미드의 크기에 따라 달라집니다합니다. </li><li> 실온에서 14,000 rpm으로 5 분 DNA 솔루션을 스핀하고 잔류물을 제거 뜨는 수집합니다. </li><li> micropipette의 풀러를 사용하여 유리 모세관 tubea 유리 모세관 튜브를 당겨. 팁는 20-30 μm의 직경을 가지고 만들어 포셉와 팁을에서 핀치. 끝에서 800 μm의 - 1mm를 측정하고 외부 직경 미만 60 μm의 (그림 1A)입니다 확인하십시오. </li><li> micromanipulator에 모세를 연결하고, 미네랄 오일과 함께 입력합니다. DNA 솔루션 잠수함 솔루션에 팁으로 모세관을 입력하고 DNA 용액을 빨아합니다. </li></ol> 2. 백금 전극을 스틱 여기에는 피질 (그림 1 층)로 두뇌의 표면 지역에 electroporate에 충실 플래티넘 전극 (Nepe 유전자, CUY611P2 - 1)을 사용합니다. 3. 마이크로 전극의 준비 두뇌 (즉, 시상과 시상 하부) (그림 2B - E)의 깊은 부분으로 electroporated에 마이크로 전극을 사용하십시오. <ol><li> 하나 텅스텐 끝 (부정적인 전극) 및 백금 (긍정적인 전극)을 전선은 금도금 핀에 코일과 parafilm로 고정됩니다. 면봉의 줄기에 철사를 삽입하고 parafilm (그림의 1B)와 양쪽을 포장. </li><li> 그것이 800 20-30 μm의 외부 직경 60 μm의 μm의 - 1 끝의 mm (그림 1C 및 D)를 도달할 때까지 균일하게 사포와 와이어의 끝부분을 날카롭게. </li><li> 절연을위한 와이어 매니큐어가 손톱의 얇은 외투를 적용하십시오. 매니큐어가 건조되면, 팁 (끝부분에서 약 200 μm의)에서 제거하는 아세톤 묻은 면봉을 사용합니다. 중요 참고 : 마우스의 자궁에 손상을 방지하기 위해, 800 시간 부분 μm의 - 1 끝의 mm 직경에 더 이상 70 이상 μm의 (그림 1E)해야합니다. </li></ol> 4. 수술 준비 <ol><li> 임신 마우스 (E9.5 - E15.5)을 마취. 우리는 제약 학년 나트륨 pentobarbital로 anesthetization을 증명 (표 II, g 체중 당 50 μg은 intraperitoneally을 주입하고 5 분 기다). 마취로 pentobarbital 나트륨에 대한 대안은 케타민을 / Xylazine 주입 또는 가스 anesthetics을 (가스 짧은 기간 절차에 대한 것이 바람직합니다)가 있습니다. 주사 어떤 기관을 피하기 위해주의와 함께 이루어져야합니다, 그렇지 않으면 그것은 자궁가 손상되거나 동물을 죽이는 원인이됩니다. 성공적인 IP가 완료되면 생존율 이상 95%입니다. 또한 수술은 야외 환경에서 수행할 수 있습니다. 전에 마취를 확인하기 위해 발가락을 곤란하게하기 위해 동물의 응답의 부족에 대한 작업, 테스트를 시작했다. </li><li> 면도날과 50 % EtOH를 사용하여 복부에서 머리를 면도. betadine과 알코올의 스크럽 (70 %)을 교대로 피부를 준비합니다. </li><li> 고급 가위로 복부 정중선에서 절개를하고 37에 신중하게 모든 자궁 뿔을 꺼내 ° C 사전 예열 인산 버퍼 호수 (PBS) moistened 상처 주위에 배치됩니다 코튼 거즈를. PBS 당시의 모든 자궁 촉촉한 보관하십시오. </li><li> 색인과 중간 손가락 사이 유연한 광섬유 케이블을 잡고 자궁 경적 아래로 놓으십시오. 사용하기 전에 70 % EtOH로 청소 케이블. 아무도 현미경이나 돋보기가 시각화가 필요하지 않습니다. 위치 광섬유 조명 광 케이블과 엄지손가락 사이에 자궁과 가까운 자궁 벽에 배아를 밀어 부드럽게 쥐어 짜기. </li></ol> 5. DNA와 electroporation의 분사 <ol><li> 태아가 위치하면, 유리는 대상 꽂혔어 조심스럽게 모세관과 DNA 솔루션의 약 1 μl (그림 2A)를 주입 삽입합니다. </li><li> electoporation에 대한 스틱 백금 전극과 함께 사용하여 brainIf의 표면 부분 electroporation 들어, 자궁의 외부 전극을 배치하고 (표 I) 제조의 지시에 따라 제안 평방 파 전류 펄스를 적용합니다. 두뇌의 깊은 부분에 usingFor의 electroporation은 nmicro의 electroporation은 eedle 입력 전극을 사용하는 경우. Iinsert DNA에 잘 텅스텐 음극은 평방 파 전류 펄스 (표 I) (그림 2B)를 제안 적용 후, 두 전극 사이에 자궁과 장소 대상 지역으로 뇌실과 백금 전극을 주입. </li></ol> 6. 포스트 electroporation <ol><li> 는 원본보다 실적이있는 자궁 호른 다시 플레이스inal과 위치와 37 ° C 사전 예열 PBS 500 μL를 추가합니다. </li><li> 9mm autoclipauto 클립과 수술 봉합과 가까운 바깥쪽 레이어와 내부 레이어를 봉합해. </li><li> 2 시간 동안 가열 패드 플레이스 동물은 마취에서 회복을 허용합니다. 이러한 carprofen 또는 buprenorphine 같은 진통제는 통증에 대한 게시물 수술 시행하실 수 있습니다. </li></ol> 7. 대표 결과 : pCAG - EYFP의 대뇌 피질의 electroporation의 몇 가지 예입 다른 시간 지점에서 스틱 백금 전극을 사용하는 구성은 그림 그림 32에 표시됩니다. 두뇌는 E13.5, E14.5 E15.5과에서 electroporated하고, 출생 후의 일 (P) 6 수확하고 있습니다. RORc의 AThe ntibody의 얼룩은 레이어 4 EYFP transfected 세포의 위치의 위치가 GFP의 항체 염색법에 의해 밝혀 아르 공개, 두 이미지 (그림 3A와 B) 슈퍼 부과됩니다. 라벨 피질 세포는 electroporated 대뇌 피질 세포의 계층이 명확하게 시간 의존 electroporation은 대뇌 피질의 특정 계층 고프 / LOF 가능하게 제시 각 실험 (그림 32)에 차이가 있습니다. 세포 transfection의 배아 및 효율성의 생존 능력이 배아의 연령 electroporation의 위치에 따라 다릅니다. 샘플 표 1에 나열되어 있습니다, 그러나, 조건이 무대와 두뇌의 한 부분에 따라 각각의 electroporation을 위해 최적화해야합니다. 이러한 스틱 백금 전극을 이용하여 시상과 시상 하부로 깊은 조직으로 Electroporation은 종종 낮은 효율성을 (표 1) 제공합니다. 이 문제는 마이크로 전극에게 두뇌의 깊은 부분에 도달합니다 whichelectrodes를 사용하여 해결할 수 있습니다. 그림 3 개발 diencephalon에 pCAG - EYFP electroporation 4 보여주는 예제. 플라스미드 DNA 솔루션은 E11.5에서 제 3 뇌실로 주입 및 마이크로 electroporation (그림 3C4C)이옵니다. electroporated 영역은 시상 제한됩니다 어디 피질 (그림 3B4B)에 axons 프로젝트의 대부분. 피질의 레이어 4 액슨 투영은 전체 신경 세포를 fillesfills EYFP에 의해 또한 시각하실 수 있습니다. (그림 3A4A). <img src="/files/ftp_upload/3024/3024fig1.jpg" alt="그림 1"/> 그림 1. 모세관의 정확한 모양을 보여주는 만화의 약도. (A) 유리 모세관 특정 형태를 만들기 위해 micropipette의 풀러를 사용하여 뽑아 있습니다. 팁는 20-30 μm의를 만들기 위해 집게로 슬쩍 꺼져 있습니다. 팁 (레드 브래킷)에서 800 μm의 - 1mm를 측정하고 외부 직경 미만 60 μm의 (녹색 브래킷)되어 있는지 확인합니다. 텅스텐 또는 백금 와이어 (B) 한쪽은 금도금 핀에 코일과 parafilm로 고정됩니다. 그런 다음 끝의 1mm 20-30 μm의 외부 직경 60 μm의되기 위해 자신의 팁을를 만들기 위해 모래 종이를 사용합니다. 텅스텐 와이어 (C) 이미지 구체화하기 전에. 성형 후 텅스텐 와이어 (D) 이미지. 매니큐어의 얇은 외투를 적용 후 텅스텐 와이어 (E) 이미지. E 규모 막대 CE 10 μm의 (작은 규모)입니다. (F) 스틱 백금 전극 (NEPA 유전자. CUY611P2 - 1). F 규모 막대는 2mm입니다. <img src="/files/ftp_upload/3024/3024fig2.jpg" alt="그림 2"/> 수술의 그림 2. 만화 스키마. 측면 뇌실이나 대형 배아 (왼쪽)로, 3 차의 뇌실 또는 작은 태아 (오른쪽)로 분사의 (A) 다이어그램. 백금 스틱 전극 (왼쪽)와 바늘 용접봉 (오른쪽)으로 electrporation의 (B) 다이어그램. <img src="/files/ftp_upload/3024/3024fig3.jpg" alt="그림 3"/> 그림 3. 마우스 telencephalon 개발은 E13.5 (A), E14.5 (B)와 E15.5 (C)에서 pCAG - EYFP와 electroporated와 P6에서 수확했다. 뇌는 따로 코로나 sectioned 및 GFP 항체 또는 항체 RORc 물들일되었고 이미지가 병합됩니다. E13.5 transfect 많은 계층 4 뉴런에서 pCAG - EYFP 플라스미드의 (A) Electroporation. 어느 4 마커 RORc 레이어와 중복됩니다. 레이어 4 뉴런보다 표면 아르 E14.5 transfect 세포에서 pCAG - EYFP 플라스미드의 (B) Electroporation. 이러한 계층 3분의 2 등 (C) E15.5 electroporation 레이블 많은 천박한 뉴런. 스케일 바 200 μm의. <img src="/files/ftp_upload/3024/3024fig4.jpg" alt="그림 4"/> 그림 4. thalamo - 대뇌 피질의 axons (TCA)에 EYFP의 Transfection. (A) 대뇌 피질의 레이어 4 TCA 터미는 GFP의 항체 염색법에 의해 드러났습니다. RORc 항체의 얼룩은 레이어 4의 위치를​​ 공개하는 데 사용됩니다. 두 이미지는 동일한 섹션에서 촬영한과 병합됩니다. (B) 시상에 electroporation의 위치는 표현이 시상 11 제한되어 GFP의 항체 얼룩과 RORc 염색법에 의해 표시됩니다. (C) thalamic electroporation과 TCA 프로젝션을위한 만화의 약도가 표시됩니다. 스케일 바 200 μm의.

<ol>
	<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=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+Visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240, 237-246 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Kataoka, A., 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=Fgf8+controls+regional+identity+in+the+developing+thalamus.">Fgf8 controls regional identity in the developing thalamus.</a> Development. 135, 2873-2881 (2008).</li><li>Imayoshi, I., Shimogori, T., Ohtsuka, T., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hes+genes+and+neurogenin+regulate+non-neural+versus+neural+fate+specification+in+the+dorsal+telencephalic+midline.">Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline.</a> Development. 135, 2531-2541 (2008).</li><li>Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signaling+centers+expressing+BMP,+WNT+and+FGF+proteins+interact+to+pattern+the+cerebral+cortex.">Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex.</a> Development. 131, 5639-5647 (2004).</li><li>Shimogori, T., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+application+with+in+utero+electroporation+in+mouse+embryonic+brain.">Gene application with in utero electroporation in mouse embryonic brain.</a> Development Growth &#38; Differentiation. 50, 499-506 (2008).</li><li>Takahashi, Y., Watanabe, T., Nakagawa, S., Kawakami, K., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+Embryology.">Avian Embryology.</a> Methods in Cell Biology. Vol 87, 271-271 (2008).</li><li>Feil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-activated+site-specific+recombination+in+mice.">Ligand-activated site-specific recombination in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 10887-10890 (1996).</li><li>Indra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally-controlled+site-specific+mutagenesis+in+the+basal+layer+of+the+epidermis:+comparison+of+the+recombinase+activity+of+the+tamoxifen-inducible+Cre-ERT+and+Cre-ERT2+recombinases.">Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases.</a> Nucleic Acids Research. 27, 4324-4327 (1999).</li><li>Nakagawa, Y., O'Leary, D. D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+patterned+expression+of+orphan+nuclear+receptor+genes+ROR+alpha+and+ROR+beta+in+developing+mouse+forebrain.">Dynamic patterned expression of orphan nuclear receptor genes ROR alpha and ROR beta in developing mouse forebrain.</a> Developmental Neuroscience. 25, 234-244 (2003).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 프로토콜에서는, 우리는 서로 다른 모양의 전극을 사용하여 작은 배아 두뇌의 제한 구역에 pCAG - EYFP transfection의 기술의 장점을 설명했다. 다른 발기인에 의해 transfection 효율 비교는 이전에 설명한 경우, 이것은 세포 유형 특이성 1 보여줍니다. 그러나, transfection 등 기술에 대한 제한은 일시적이고 플라스미드에 따라 차이가있다, 그것은 모든 세포 특이성을하지 않으며 그것은 transfected 세포의 숫자를 제어하기 어렵다. ThereforeHowever은 다른 기법이 프로토콜을 결합하면 더욱 다양한 조작 방법에 대한 가능성을 제공합니다. 첫째, 플라스미드 DNA에 특정 셀 모터를 통합하면 이러한 뉴런, glial 세포 및 astrocytes 같은 셀 타입의 특정 transfection을하실 수 있습니다. 둘째, transfection이 과도이기 때문에, 그것은 나중에 유전자의 기능을 분석하려는 과학자에 적합하지 않습니다. 따라서 플라스미드 DNA가 게놈에 통합하기 위해 transposase와 조합은 안정적인 transfection 8로 변환됩니다. tet - 또는 tet - 오프 시스템의 다음, 채택은 가능한 8 신경 세포의 타이밍이나 세포 유형 특정 유전자 발현을 조작하는 것입니다. 또는, 하나는 tamoxifen - inducible Cre 호텔 - ER (T) recombinases 9 기자 마우스 10이 프로토콜을 조합하여 사용할 수 있습니다. 조직의 광범위한 접근은 크게 신경 과학에서 사용되는 실험 설계 변경됩니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 베어 텅스텐 와이어 직경 125 μm의 </td><td> AM 시스템 </td><td> 797600 </td></tr><tr><td> 베어 백금 와이어 직경 125 μm의 </td><td> AM 시스템 </td><td> 767000 </td></tr><tr><td> 스틱 전극 </td><td> NEPA 유전자 </td><td> CUY610P4 - 1 </td></tr><tr><td> 유리 모세관 </td><td> 스토엘팅 </td><td> 50,611 </td></tr><tr><td> micromanipulator </td><td> KD 과학 </td><td> KDS310 </td></tr><tr><td> 가위 </td><td> ROBOZ </td><td> RS - 5865 </td></tr><tr><td> 펄스 발생기 </td><td> AM 시스템 </td><td> 모델 2100 </td></tr><tr><td> 9mm autoclip </td><td> ROBOZ </td><td> RS - 9260 </td></tr><tr><td> 금도금 핀 </td><td> WPI </td><td> 5482 </td></tr><tr><td> RORc 항체 </td><td> 페르세우스 Proteomics </td><td> H3925 </td></tr></tbody></table>

mouse in utero electroporation: controlled spatiotemporal gene transfection

신호 분자와 축삭지도 분자, 지방 유전자 전송 또는 노크 아웃이 필요합니다을 포함하여 개발 두뇌의 특정 영역에서 표현 유전자의 기능을 이해하기 위해서. 지역 지역에 안으로 또는 노크 아웃 타겟 유전자는 특정 힘드는, 비용이 많이 드는 CRE 라인, 그리고 시간이 소요와 함께 함께 수행할 수 있습니다. 따라서, 간단한 transfection 방법, 짧은 시간에 수행할 수 있습니다에서 utero의 electroporation 기술은, 사전에 유전자 변형 동물의 1,2 세대의 후보 유전자의 가능한 함수를 테스트하는 것이 편리할 것입니다. 이 외에도, utero의 electroporation에 특정 CRE 라인은 존재하지 않는 두뇌의 영역을 대상으로하고, 배아 파괴 장면을 보여 주죠에게 3,4를 제한합니다. 여기, 우리는 개발 두뇌의 대상 영역으로 간단하고 편리한 유전자 전달을위한 전극의 두 가지 종류의 결합 utero의 electroporation에의 방법을 제시한다. 첫째, 광학 광섬유 조명 케이블을 사용하여 태아의 독특한 지주 방법은 심실 및 타겟 뇌 영역 5,6에 바늘 형태 전극 삽입에 타겟 DNA 용액 주입을위한 보이는 작은 배아 (E9.5에서) 할 것입니다. 같은 피질 영역으로 뇌의 patterning 따라서, E9.5에서 이러한 초기 electroporation은 전체 지역 patterning 이벤트를 이해하기 위해 큰 공헌을 초기 배아 단계에서 발생합니다. 둘째, 모세관의 정확한 모양은 모세 삽입하여 구멍을함으로써 자궁 손상을 방지합니다. 또한, 바늘 전극의 정확한 모양은 텅스텐과 백금 와이어로 만든 모래 종이를 사용하여 날카롭게하고 매니큐어 7이 프로토콜 큰 자세히 설명되어있는 방법으로 절연하고 있습니다. 이 독특한 기술은 두뇌의 제한된 영역에 플라스미드 DNA의 transfection을 허용 작은 배아가 electroporated 수 있도록합니다. 이것은 아주 초기 배아 단계에서 세포 분화, 세포 이주, 축삭지도에 최선을 다하고 있습니다 많은 과학자를위한 새로운 창을 열고하는 데 도움이 될 것입니다. 또한,이 기술은 과학자들이 이러한 많지 않아 특정 지역과 관련된 CRE 라인 함수의 이득 (고프) 또는 기능 (LOF) 분석의 손실에 대해 존재하는 시상과 시상 하부로 개발 두뇌의 깊은 부분으로 플라스미드 DNA를 transfect 수 있습니다.

Watch this Scientific Journal Video about Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection at JoVE.com

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

개발 뇌 마우스로 유전자 전송 방법은 고유한 수술 방법과 전극의 특별한 모양을 사용하여 설명합니다. 이 독특한 기술은 두뇌 개발을 공부에 많은 도움이됩니다 neuroscientists temporally과 공간 플라스미드 DNA의 transfection 수 있습니다.

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 ...

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Research

JoVE Journal

Neuroscience

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

42.7K 조회수.  RIKEN Brain Science Institute. 개발 뇌 마우스로 유전자 전송 방법은 고유한 수술 방법과 전극의 특별한 모양을 사용하여 설명합니다. 이 독특한 기술은 두뇌 개발을 공부에 많은 도움이됩니다 neuroscientists temporally과 공간 플라스미드 DNA의 transfection 수 있습니다.

비디오: Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

utero에서 Electroporation은 두피 뉴런의 하위 집합의 유전자 기능을 학습에 대한 기본의 연결을 문화 뒤에

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

연구

신경과학

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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 ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

에 의해 개발 마우스 내이 (内耳)에 진 전송<em&gt; VIVO에서</em&gt; Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

사용하는 마우스 대뇌 피질과 해마의 시각화 및 Dendrites와 쪽이의 유전자 조작<em&gt; utero에서</em&gt; Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

몸에 붙이기 형질 전환 쥐의 발생에 의해<em&gt; 자궁 내</em&gt; 일렉트로 및<em&gt; 생체 내</em&gt; 생물 발광 상영

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

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 ...

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 ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

배아 마우스 두뇌의 organotypic 슬라이스 문화에서 마이 그 레이션 뉴런의 저속 공 촛점 이미징<em&gt; Utero에서</em&gt; 일렉트로 포 레이션

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Cre lox 재결합 Electroporation Utero에 통해 마우스 대뇌 피 질에서 유도

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...