저자 Math1, Ngn1 위해 제인 존슨 감사 드리고, 그리고 Ptf1a의 항체와 pCAG위한 코니 Cepko :: GFP 플라스미드 것이다. 이 작품은 NIH R15 1R15HD059922-01에 의해 재정 지원되었다.

1. Electroporation 이전 준비 <ol><li> 맥시의 준비 (프라임 - 이건 또는 Qiagen)에 의해 electroporation을위한 DNA를 증폭. DNA의 농도는 효율적인 이해 1 밀리그램 / ML 최소이어야합니다. </li><li> microcentrifuge 튜브 1 X PBS (인산염 식염수 버퍼)의 0.01 % 빠른 그린의 5 μl로 DNA와 혼합 495 μL를 제거합니다. </li></ol> 2. 배아 하베스트 <ol><li> CD-1 생쥐 (할란)의 시간이 초과되었습니다 matings을 마련한다. 질 플러그의 존재를 확인하고 질 플러그가 배아 일 (E) 0.5으로 관찰되는 날짜를 생각한다. 태아도 11일 플러그 (E11.5)를 시각화 후에 수확한다. </li><li> 70 % 에탄올로 소독 도구를 놓습니다. 사용하기 전에 적어도 한 시간 동안 자외선과 판상 흐름 후드를 드셔보세요. 스프레이 후드, 해부 트레이, 그리고 70 % 에탄올로 범위를 해부. 37까지 사전 열 1 X PBS는 ° C. </li><li> E11.5에서 Institut의 승인 조건에 따라 여성을 안락사시켜야동물의 관리 및 사용 (ICCUA)에 대한 ional위원회. 층류 후드의 해부 트레이를 놓습니다. 70 % 에탄올과 여성의 복부를 스프레이. </li><li> 복막 캐비티를 열고 해부 트레이에 성벽을 달아주는. 그것이 복막 캐비티 내에 달려 있도록 자궁 뿔을 내밀어 봐요. </li><li> 조심스럽게 자궁 뿔의 벽을 열고 했네요. 부드럽게 멀리 태반에서 난황에서 배아를 제거하는 20 밀리미터 스푼 (파인 과학적 도구)를 사용합니다. 100 밀리미터 조직 배양 접시에 플레이스 배아 2.2에 preheated되었습니다 멸균 1 X PBS의 10 ML과 prefilled. </li><li> 현재 모든 배아에 대해 반복합니다. </li></ol> 3. E11.5의 태아의 Electroporation (그림 1) <ol><li> 20 밀리미터 스푼을 사용하여, 37에 preheated되었습니다 멸균 1XPBS의 10 ML로 prefilled 새로운 100mm 요리 ° C.에 최초의 배아를 전송 </li><li> 신중을 다시 0.05 X 0.02 밀리미터 팁 (파인 과학적 도구)로 11cm의 집게를 사용하여이동하고 난황을 버리고. </li><li> 위치 배아 그래서 그 지느러미 측면은 그림 1A에서 만화과 비슷한 정도로까지 직면하고 있다고. 부드럽게 배아를 개최하기 위해 7mm 전극 충격기 (하버드 장치)를 사용합니다. 전극은 hindbrain 넷째 뇌실의 수준에서 신경 튜브의 양쪽에 위치해야합니다. 뇌실은 맨눈으로 볼 수 있지만 해부 현미경을 사용하면 정확한 패들 배치를 용이하게하실 수 있습니다. 심폐 소의 위치는 electroporated DNA를받는 지역을 결정하기 위해 중요합니다. 지느러미 배아 hindbrain의 낮은 마름모꼴의 입술 (LRL)이 원하는 경우 심폐들이 직접 번째 뇌실 개구의 가장 넓은 부분을 측면되는 그러한 장소 여야합니다. </li><li> 0.01 % 빠른 그린과 혼합 플라스미드 DNA를 구축하도록 한 CC의 주사기를 사용하십시오. 혼합물 500 μL은 적어도 8-10 배아 (3.5 참조) electroporation 충분해야합니다. </li><li> 부드럽게 찔린 roofplate 오븐한 CC의 주사기에 부착된 25G 8분의 5 투베르쿨린 바늘로 네 번째 뇌실을 erlying 및 뇌실로 DNA를 염색 혼합물을 주입. 성공 주사는 전체 심실 시스템 (그림 1C)을 작성 DNA-염료 혼합물을 특징으로하고 있습니다. 일반적으로 주입 DNA-염료 혼합물의 양은 50 미만 μL입니다. 혼합물의 부분이 배아를 둘러싼 PBS로 뇌실 밖으로 새는 경향으로 정확한 금액은 배아 사이의 변수입니다. DNA-염료 혼합물을 전달하는 대체 수단 midbrain을 overlying 연구개를 펑쳐링하여 심실 시스템에 액세스를 통해 것입니다. 다시 말하지만, 성공적인 주사는 전체 심실 시스템을 작성 DNA-염료 혼합물을 특징으로하고 있습니다. </li><li> 전기 펄스 발생기 (BTX) 및 7 밀리미터 전극 충격기를 사용하여 다섯 사각형 펄스를 제공합니다. 각각의 펄스는 각 펄스 사이의 500 MS와 펄스 당 50 V, 지속적인 다섯 MS입니다. 긍정적으로 충전 전극에 가장 가까운 조직 얘기가 그러면됩니다플라스미드 백업 E. </li></ol> 4. 태아의 문화 <ol><li> 층류 후드에서는 10 % 태아 소 혈청 5 % 말 혈청, 1 %의 글루타민, 1 % 페니실린 / preheated되었습니다 스트렙토 마이신과 보완 DMEM/F12 미디어 2 ML과 함께 12도 문화 접시의 바깥쪽 우물을 채울 37에 ° C. 문화 조건 드 샌디에고 및 동료에서 적응되었다. 15 </li><li> 포셉과 midsection (심장 아래)에서 층류 후드의 핀치 배아와 태아의 후부 부분을 제거합니다. 12 잘 배양 접시 가득 우물 중 하나로 앞부분 일부를 놓습니다. </li><li> 모든 배아에 대해 반복합니다. 문화의 오염을 피하기 위해 12 잘 판만을 외부 우물을 채웁니다. </li><li> 5% CO 2와 37 ° C 배양기에서 문화 배아. electroporated 플라스미드의 표현 24 시간 이내에 관찰할 수 있어야합니다. </li><li> 이상 배양 시간이 원하는되어야, 2 ML과 함께 새로운 12 잘 접시의 밖으로 우물을 채울미디어 4.1에 사용됩니다. 멸균 스푼으로 새 접시에 잘으로 배아를 전송합니다. 37 ° C 배양기에 다시 넣습니다. 최대 48 시간까지를위한 문화가 가능합니다. </li><li> 원하는 문화 시간에 도달하면 (아래) 분석을 위해 배아를 채운다. </li></ol> 5. 분석을위한 태아의 작성 <ol><li> 다섯 분에 대해 4 ° C에서 1 X PBS에서 배아를 씻어. 반복합니다. </li><li> 4 ° C.에 2 시간 1XPBS의 2 % paraformaldehyde의 분석 (PFA)의 배아를 고정 </li><li> 다섯 분에 대해 4 ° C에서 1 X PBS에서 배아를 씻어. 반복합니다. </li><li> 4에서 하룻밤 1 X PBS의 30 % 자당에 배아를 평형 ° C. </li><li> 드라이 아이스 / 에탄올 욕조를 사용하여 최적의 절삭 온도 (-10 월) 화합물의 배아를 포함시킵니다. 태아도 -20 ° C.에 저장될 수 </li><li> 30 μm의 섹션으로 그라 이오 스탯 (Leica)과 (VWR, Superfrost 플러스) 슬라이드에 마운트에 관한 섹션 배아. -20 ° C.에 저장 </li></ol> 6. Immunohistochemistry 분석 <ol><li> 16에 설명된대로 Immunohistochemistry이 수행되었다. 마우스 α-Mash1 (BD Biosciences) 1:100; 토끼 α-Ngn1 (제인 존슨) 1:5000, 토끼 α-Ptf1a (제인 존슨은이 연구에 사용되는 일차 항체 dilutions는 토끼 α-GFP (Invitrogen) 1:2500 포함 ) 1:2500, 토끼 Math1 (제인 존슨) 1:100. 4 ° C에서 하룻밤에 염색법 트레이에 슬라이드 평면, 표본 측면을 품어. </li><li> 슬라이드는 복합 현미경 (올림푸스 BX51)을 분석했다. </li></ol> 7. 대표 결과 그림 1A의 개략도 electroporation 실험을 묘사. 그림 1B는 조작 이전 E11.5 배아의 화살보기를 표시합니다. 0.01 % 빠른 그린에서 플라스미드 포함 배속 :: GFP의 주입에 따라 동일한 배아는 그림 1C 및 일방적 GFP를 전시 대표 고정되지 않은 배아에 나와 있습니다지느러미 hindbrain 24 시간 다음과 문화의 표현은 그림 1D에 표시됩니다. 성공적으로 electroporated되는 LRL의 영역의 범위는 변수이며 전극의 위치에 크게 의존 것 같습니다. 우리의 연구에 그것은 electroporated 배아 65 (80 %)의 52 밖으로 성공적으로 GFP를 표명 사실을 발견했습니다. 조직은 그것이 고정 및 immunohistochemical 분석 (아래)에 따라 여러 부분 이상의 지역화된 지역에서 GFP 양성 반응이 있었다면 성공적으로 electroporated로 간주되었다. 이 기준을 충족하는 데 실패 태아도 electroporation의 실패로 득점했다. electroporation 효율의 자세한 평가는 넷째 뇌실의 수준에서 electroporated 배아의 가로 부분에 GFP에 대한 immunohistochemistry를 수행하여 ascertained 될 수 있습니다. 그림 2는 일방적를 표시 배아의 대표 시리얼 섹션을 보여줍니다GFP를 표현. 그림 2A는 배아의 왼쪽은 긍정적인 전극쪽으로이라고 설명하고 이상적인 설계도를 보여줍니다. 가로 구역 (그림 2A의 중간 만화로 대표되는 수준에서) hindbrain 조직 (그림 2C와 2D)의 왼쪽에 독점적으로 지역화된 GFP 발현을 알 수있다. 그림 2C에 나타난 그 ~ 300 μm의 주동이의 이상 부분의 심사는 명시적 GFP (그림 2B)을하지 않으므로 LRL의 electroporated 면적은 신경 튜브의 전체 앞쪽에-후부 축을 연장하지 않았다. 유전자 표현의 조작이 분석의 유틸리티는 내생 단백질에 대한 표현 도메인의 안정성에 의존적일 수 밖에 없습니다. LRL는 proneural 전사 요인 차등 표현 (1 검토)을 특징으로 독특한 전구 도메인을 소지으로 특징되었습니다.이러한 요소의 하위 집합 (Mash1, Math1, Ngn1 및 Ptf1a)는 LRL, 미래 연구 16-18의 과목에 precerebellar 신경 subtypes의 사양에 그들의 제안 및 / 또는 특징 역할로 인해 분석에 선정되었습니다. 이들 네 단백질은 E11.5 16-18에서 꼬리 hindbrain에서 매우 특징적인 표현 도메인이 있습니다. 우리는 문화를 배치했다 배아 크기가 증가하는 데 실패하며 맥락막 얼기의 상피와 E11.5 및 E12.5 5 사이에 발생 LRL 및 roofplate, 형태학의 이벤트 invagination 생산을 시작하는 데 실패했음을 관찰했다. 이러한 관찰을 바탕으로 그것이 태아의 정상적인 개발이 중지 또는 조잡한 지연과 교양 배아에 대한 비교 가능한 컨트롤 E11.5에 미개한 배아있을 것을 결정했다. 문화와 electroporation은 내생 단백질의 수준을 방해하지 않게하기 위해, 우리는 immunohistoch하여 네 가지 단백질을 분석electroporated 그리고 최소 24 시간을위한 다음 교양했다 34 다른 배아에 emistry (IHC). 표 전 보관 정상적인 단백질 수준. 그림 3은 대표 IHC 데이터를 보여주는 것으로 분석 각 마커와 배아의 비율에 대한 분석 배아의 수를 보여줍니다 단백질의 두 분석에서 Mash1 (그림 3A 및 3B)와 Math1 (그림 3C 및 3D). 우리는 E11.5 (그림 3A와 3C)에서 제어 배아에 비해 배아의 대부분은 표현에 따라 electroporation과 문화 (그림 3B 및 3D)의 정상 수준을 유지 것을 관찰했다. 중요한 것은, 이러한 단백질의 특성 표현 도메인은 perturbed되지 않았습니다. <img src="/files/ftp_upload/3983/3983fig1.jpg" alt="그림 1"/> 1 그림. 회의 E11.5시 태아의 Electroporation. () 도식전자 electroporation 실험. E11.5의 배아는 격리되어 0.01 % 빠른 그린에서 플라스미드 표현식 번째 뇌실로 주입된다. 배아 그러면 전극 충격기의 어귀 사전 문화에 주입 이전 E11.5 배아의 (B) 화살 볼 수있게 된에 50 V 펄스를 받게됩니다. (C) 같은 E11.5의 배아는 0.01 % 빠른 그린에서 플라스미드의 주입에 따라. (D) GFP의 일방적 hindbrain 표현 문화의 24 시간에 따라 E11.5의 배아에서 관찰. 메가바이트-midbrain, 4V - 넷째 뇌실. <img src="/files/ftp_upload/3983/3983fig2.jpg" alt="그림 2"/> 그림 2. Electroporated 조직에서 GFP의 표정은. () 왼쪽에있는 만화는 E11.5 배아 주위에 전극의 위치를 묘사. 중동 만화는 배아의 왼쪽에있는 플라스미드 인코딩 GFP의 이해와 표현을 묘사. 오른쪽에있는 만화는 내 덕분에 배아를 통해 촬영한 이상 형인 가로 섹션의 개략도이다evels는 중간 만화의 검정색 라인으로 표시. 문화의 24 시간 이후 electroporated E11.5의 배아를 통해 가로 섹션에 GFP를위한 (B-D) Immunohistochemistry. 화살표는 roofplate (RP) 어떤 트랩 이차 항체를 나타냅니다. 이미지 10X 배율로 촬영하고 있습니다. 표시된 섹션의 상대적인 수준은 ()의 중간 만화를 통한 가로선으로 표시됩니다. 메가바이트-midbrain, 리터 4V-넷째 뇌실, RP-roofplate. <img src="/files/ftp_upload/3983/3983fig3.jpg" alt="그림 3"/> 그림 3. 로어 마름모꼴의 입술에 내생 단백질의 표현 Mash1 (A와 B) 또는 편대의 편대 장으로 electroporated 있었다 배아으로 (A, C) 교양 아니었 E11.5의 배아의 가로 섹션을 비교 Math1 (C와 D)에 대한 Immunohistochemistry. : : GFP, 24 시간 (B와 D)에 대한 교양. 이미지 10X 배율에서 찍은. <table border="1"><tbody><tr><td> Proneural Transcripti팩터에서 분석 </td><td> 태아의 수 분석 </td><td> 일반 표현식 패턴을 유지 비율 </td></tr><tr><td> Math1 </td><td> 15 </td><td> 86.7 % </td></tr><tr><td> Mash1 </td><td> 12 </td><td> 83.3 % </td></tr><tr><td> Ngn1 </td><td> 7 </td><td> 71.4 % </td></tr><tr><td> Ptf1a </td><td> 6 </td><td> 백퍼센트 </td></tr></tbody></table> LRL에서 정상 Proneural 전사 인자 도메인을 유지 Electroporated과 교양 태아의 표 I. 비율.

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rhombic+lip+and+early+cerebellar+development.">The rhombic lip and early cerebellar development.</a> Curr. Opin. Neurobiol. 11, 82-88 (2001).</li><li>Wingate, R. J., Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+rhombic+lip+in+avian+cerebellum+development.">The role of the rhombic lip in avian cerebellum development.</a> Development (Cambridge, England). 126, 4395-4404 (1999).</li><li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development of Cerebellar System: In relation to its evolution, structure, and function. , (1997).</li><li>Farago, A. F., Awatramani, R. B., Dymecki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+the+brainstem+cochlear+nuclear+complex+is+revealed+by+intersectional+and+subtractive+genetic+fate+maps.">Assembly of the brainstem cochlear nuclear complex is revealed by intersectional and subtractive genetic fate maps.</a> Neuron. 50, 205-218 (2006).</li><li>Wang, V. Y., Rose, M. F., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+expression+redefines+the+rhombic+lip+derivatives+and+reveals+novel+lineages+within+the+brainstem+and+cerebellum.">Math1 expression redefines the rhombic lip derivatives and reveals novel lineages within the brainstem and cerebellum.</a> Neuron. 48, 31-43 (2005).</li><li>Rodriguez, C. I., Dymecki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+the+precerebellar+system.">Origin of the precerebellar system.</a> Neuron. 27, 475-486 (2000).</li><li>Taber-Pierce, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histogenesis+of+the+nuclei+griseum+ponitis,+corporis+pontobulbaris+and+reticularis+tegmenti+pontis+(bechterew)+in+mouse.">Histogenesis of the nuclei griseum ponitis, corporis pontobulbaris and reticularis tegmenti pontis (bechterew) in mouse.</a> J. Comp. Neurol. 126, 219-240 (1966).</li><li>Dipietrantonio, H. J., Dymecki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zic1+levels+regulate+mossy+fiber+neuron+position+and+axon+laterality+choice+in+the+ventral+brain+stem.">Zic1 levels regulate mossy fiber neuron position and axon laterality choice in the ventral brain stem.</a> Neuroscience. 162, 560-573 (2009).</li><li>Takahashi, M., Sato, K., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+gene+expressions+by+electroporation+in+the+developing+brain+of+mammalian+embryos.">Manipulating gene expressions by electroporation in the developing brain of mammalian embryos.</a> Differentiation. 70, 155-162 (2002).</li><li>Taniguchi, H., Kawauchi, D., Nishida, K., Murakami, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classic+cadherins+regulate+tangential+migration+of+precerebellar+neurons+in+the+caudal+hindbrain.">Classic cadherins regulate tangential migration of precerebellar neurons in the caudal hindbrain.</a> Development (Cambridge, England). 133, 1923-1931 (2006).</li><li>Kawauchi, D., Taniguchi, H., Watanabe, H., Saito, T., Murakami, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+nucleogenesis+by+precerebellar+neurons:+involvement+of+ventricle-directed,+radial+fibre-associated+migration.">Direct visualization of nucleogenesis by precerebellar neurons: involvement of ventricle-directed, radial fibre-associated migration.</a> Development (Cambridge, England). 133, 1113-1123 (2006).</li><li>Okada, T., Keino-Masu, K., Masu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+and+nucleogenesis+of+mouse+precerebellar+neurons+visualized+by+in+utero+electroporation+of+a+green+fluorescent+protein+gene.">Migration and nucleogenesis of mouse precerebellar neurons visualized by in utero electroporation of a green fluorescent protein gene.</a> Neuroscience research. 57, 40-49 (2007).</li><li>de Diego, I., Kyriakopoulou, K., Karagogeos, D., Wassef, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+influences+on+the+migration+of+precerebellar+neurons+in+the+caudal+medulla.">Multiple influences on the migration of precerebellar neurons in the caudal medulla.</a> Development (Cambridge, England). 129, 297-306 (2002).</li><li>Landsberg, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindbrain+rhombic+lip+is+comprised+of+discrete+progenitor+cell+populations+allocated+by+Pax6.">Hindbrain rhombic lip is comprised of discrete progenitor cell populations allocated by Pax6.</a> Neuron. 48, 933-947 (2005).</li><li>Hoshino, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ptf1a,+a+bHLH+transcriptional+gene,+defines+GABAergic+neuronal+fates+in+cerebellum.">Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum.</a> Neuron. 47, 201-213 (2005).</li><li>Yamada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+climbing+fiber+neurons+and+their+developmental+dependence+on.">Origin of climbing fiber neurons and their developmental dependence on.</a> Ptf1a. J. Neurosci. 27, 10924-10934 (2007).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

본 연구에서 제시된 시험 관내 electroporation 기술의 효율적 회임 12 일 이내에 젊은 배아에서 유전자 발현을 조작하는 데 활용할 수있는 새로운 방법입니다. 문화에 대한 태아의 위치는 도입 유전자의 표현을 허용하고 electroporated 배아는 생체내에 남아 허용하는 경우 주죠 지켜 circumvents. 이 기술은 electroporation 기반의 연구 이전에 액세스할 수있었습니다 배아 progenitors에서 유전?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> 그라 이오 스탯 </td><td> Leica </td><td> CM-1850 </td><td></td></tr><tr><td> Biologie 팁 Dumoxel는 뒤몽 포셉을 취급 </td><td> 좋은 과학 도구 </td><td> 11252-30 </td><td></td></tr><tr><td> 20mm 모리아 다공 스푼 </td><td> 좋은 과학 도구 </td><td> 10370-17 </td><td></td></tr><tr><td> ECM 830 스퀘어 웨이브 Electroporation 생성기 </td><td> BTX (VWR) </td><td> 47745-928 </td><td></td></tr><tr><td> 하버드 장치 7mm Tweezertrodes * 전극 </td><td> BTX (피셔) </td><td> BTX450165 </td><td></td></tr><tr><td> 피셔 Isotemp CO 2 인큐베이터 </td><td> 어부 </td><td> 1325525 </td><td></td></tr><tr><td> NAPCO CO 2 가스 조정기 </td><td> 어부 </td><td> 15497020 </td><td></td></tr><tr><td> 12 음 조직 배양 플레이트 </td><td> BD 팔콘 (피셔) </td><td> 877,229 </td><td></td></tr><tr><td> HyClone 리퀴드 미디어 DMEM/F-12 (1:1), L-글루타민과 HEPES로, 500mL </td><td> 써모 과학 (피셔) </td><td> SH3002301 </td><td></td></tr><tr><td> HyClone * 기증자 말 혈청 </td><td> 써모 과학 (피셔) </td><td> SH3007402 </td><td></td></tr><tr><td> Inactivated 태아 소 혈청, 공인, 히트 </td><td> Invitrogen </td><td> 16140-063 </td><td></td></tr><tr><td> cellgro * 10,000 IU 페니실린 10,000 μg / ML 스트렙토 마이신 </td><td> Mediatech (피셔) </td><td> MT-30-002-CI </td><td></td></tr><tr><td> HyClone * L-글루타민, L 글루타민; 200.85 % NaCl에서 0mM </td><td> 써모 과학 (피셔) </td><td> SH3003401 </td><td></td></tr><tr><td> 빠른 그린 </td><td> 어부 </td><td> AC41053-0250 </td><td> 0.01 % </td></tr></tbody></table>

in vitro electroporation of the lower rhombic lip of midgestation mouse embryos

마름모꼴의 입술은 신경 튜브와 넷째 뇌실 (1 검토)의 roofplate 사이의 교차점에서 hindbrain에있는 배아 neuroepithelium입니다. 마름모꼴의 입술은 rhombomere 1 (R1)을 포함하고 소뇌의 뉴런의 연결을 다양한 brainstem의 lineages 2-4로 상승을주는 낮은 마름모꼴의 입술 (LRL)를 생성하는 상위 마름모꼴의 입술 (URL)로 세분화 할 수 있습니다. LRL 파생 상품은 청각 와우각 핵의 뉴런과 균형과 모터 제어 5-8를 규제에 관련된 precerebellar 핵 분들을 포함합니다. LRL에서 Neurogenesis은 배아 일 (E) 9.5-16.5 5, 9를 포함 대용량 시간적 창을 통해 발생합니다. 다른 lineages의 연결이 neurogenic 창을 동안 뚜렷한 발달 일간 postmitotic 세포 (또는 태어난다)로 LRL에서 나온다. 유전자 발현 구조의 Electroporation을하는 데 사용할 수있는LRL progenitors의 유전자 발현을 조작하고 잠재적으로이 지역에서 생산된 10-12 뉴런의 운명을 바꿀 수 있습니다. utero의 electroporation의를 통해 마우스의 LRL progenitors의 변화 유전자 발현은 배아 일 E12.5 이상 10, 12-14 일에 태어 lineages 조작을위한 큰 성공을 거두었습니다. utero의 electroporations에는 사전에 E12.5이 주로 인해 성공하지 못했습니다 보여 주죠는 네 번째 뇌실의 roofplate, LRL로 electroporated되는 외인성 DNA를 제공하는 필요한 단계를 펑쳐링과 관련된. 그러나 많은 LRL 파생된 lineages 일찍 E12.5 9보다 LRL에서 발생합니다. 이러한 이전 출생 lineages는 측면 망상을 구성하는 뉴런, 외부 cuneate 및 소뇌 5 척수와 대뇌 피질로부터 입력을 연결하는 기능 precerebellar 시스템의 열등 olivary 핵을 포함합니다. LRL 표현을 조작하기 위해서는E12.5 미만 배아의, 우리는 배아가 electroporation에 따라 문화에 배치되는 체외 시스템에서 개발. 이 연구 E11.5에서 LRL progenitors의 유전자 발현을 조작을위한 효율적이고 효과적인 방법을 제공합니다. 크게 활성 배속 모터에서 구동 녹색 형광 단백질 (GFP)을 electroporated 태아는 reproducibly 문화 24 시간 후 GFP를 표명했다. 이 분석의 핵심 측면은 유전자 발현에만 때문에 외인성 유전자의 표현이 아니라 인해 이차 효과 변경되는 것입니다 electroporation과 culturing 기법에 따른니다. 그것은 내생 유전자 발현 패턴 electroporated과 교양 배아에 그대로 남아있는 것으로 확인되었다. 이 분석은 overexpression에 대한 plasmids의 도입을 통해 E12.5 미만 배아의 LRL에서 신흥 세포의 운명을 변경하거나 (RNAi를 통해) 아래로 노크를 활용할 수 다른 프로신경 전사 요인.

Watch this Scientific Journal Video about In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos at JoVE.com

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

이 연구의 발전을 설명<em&gt; 체외에서</emmidgestation의 배아의 하단 마름모꼴의 입술에서 유전자 발현의 조작을 허용&gt; electroporation 기술.

Midgestation 마우스 태아의 아래쪽 사방 정계 립의 시험 관내 Electroporation에

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

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JoVE Journal

Neuroscience

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

10.3K 조회수.  University of Illinois at Springfield. 이 연구의 발전을 설명 체외에서 electroporation 기술.

비디오: In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

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 ...

연구

신경과학

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

의 활동을 Quantifying CIS</emExplant Electroporation하여 마우스 망막에서> - 규제 요소

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

개발 마우스 망막의 생체내 Electroporation에

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

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 ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

병아리 배아에서 축삭 궤적과 시냅스 대상을 추적하는 후뇌의 일렉트로

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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 ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

<em&gt; 체외</em성인 마우스 Myenteric 신경총에서 분리 된 장내 뉴런과 교세포의&gt; 준비

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.