우리 감사 Henriette Bertemes 실험실 내 CGN 재배 프로토콜을 설정 하는 데 도움. 이 방법은 종이 DFG 투자 CRC992 의료 Epigenetics tv 자금으로 지원 했다. 저자 인정 프라이부르크 갤럭시 팀의 지원: Pavankumar Videm, 비 Grüning와 교수 Rolf Backofen, 생물 정보학, Freiburg의 대학, 독일 공동 연구 센터 992 의료 Epigenetics (DFG 부여 SFB 992/1 2012)에 의해 투자 독일 연방 교육부의 교육 및 연구 (BMBF 그랜트 031 A538A 로얄 (드. NBI))입니다.

<ol>
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저자 들은 아무 경쟁 금융 관심 선언

히스톤 수정, 녹음 방송 요인, 히스톤 코드 독자, 작가, 또는 지우개의 게놈 인 검출 하기 chromatin immunoprecipitation를 수행 하기 위해 두 가지 주요 방법이 있다. 하나는 nuclease 소화, immunoprecipitation에 대 한 네이티브 chromatin를 사용 하 여 네이티브 칩 방법 이며 다른 PFA 고정, 전단 chromatin, 있는 nucleosomes와 다른 DNA 연결 단백질 covalently에 바인딩된 DNA를 사용 하 여 제시 방법 39. 네이티?...

두뇌의 감각, 모터, 및 인식 함수는 매우 복잡 하 고 물리적, 환경 변화에 취약 합니다. 깊이 연결 되는 3 개의 일반적인 부품은 뒷 다리-, 중간-및 개, 두뇌에 의하여 이루어져 있다. 개, 내는 telencephalon 등 telencephalon (DT)와 복 부 telencephalon (VT)으로 나눌 수 있습니다. 6 대뇌 피 질의 레이어 E11.5와 E18.5 사이 "내부-아웃" 방식으로1에서 형성 되는 쥐의 DT에 의하여 이루어져 있다. 버몬트 나중 형성 기초 중추2,3개발에 절 정령을 포함 합니다. 여러 종류 나눌 수 있다 신경, 이다, 또는 oligodendrocytes4같은 포유류 중추에서5영역 공간으로에서 발전 하는. 첫째, 신경 조상 세포 (Npc)에 게 상승 다른 종류의 뉴런, 버몬트, 및 프로젝션 뉴런 수 DT에서 그리고 glial 세포 (예를 들면, 이다6) 나중에. 대뇌 피 질의 개발, 가장 표면 층 (전 층), 먼저 형성 된다 Cajal Retzius 셀 포함. 다음, E12.5 및 E14.5, 사이 NPCs 더 깊은 신경 층 (VI, V) 동안 14.5, 16.5, 창시자 줄 상승 상위 레이어 (4-II) 신경7,8사이 생성합니다. 신경 정체성 다른 morphogen 유발 영역 공간 transcriptional 프로그램 및 또한 epigenetic 프로그램2에 의해 지정 됩니다.
모터 조정에 연루는, 소 뇌는 hindbrain에 있으며, 쥐9E10 사이 대략 P20 개발. 그것은 소 뇌 피 질과 소 뇌 핵10포함 되어 있습니다. 3 레이어, 가장 뒤쪽 분자 층, Purkinje 세포 층 그리고 포함 하는 세부적인 신경10안쪽 세분화 된 계층 성인 소 뇌 피 질에 의하여 이루어져 있다. 소 뇌과 립 세포 작은 신경 이며11척 추가 있는 두뇌 모든 뉴런의 약 80%를 나타냅니다. 그들은 선구자 외부 새싹 영역에서 개발 하 고 그들의 대상12Purkinje 세포 층을 통해 이동. 처럼 telencephalon에서 소 뇌의 개발은 몇 가지 중요 한 morphogens에 의해 규제 됩니다, 어떤 특정 시간 및 공간 종속 기능을가지고 시작 정의 transcriptional 프로그램10.
대뇌와 소 뇌 레이어의 개발 특정 morphogens의 transcriptional 식으로 하 고, 따라서, DNA의 염색 질 상태에 의해 제어 됩니다. 단순화 된 보기에서 chromatin 상태 transcriptionally 활성화로 euchromatin와 섬으로 transcriptionally 자동 영역으로 나눌 수 있습니다. Chromatin의 기본 단위로 nucleosome의 복사본 두 개가 각 코어 히스톤 H2A, H2B, H3, H4, 둘러싸인 147 기본적인 쌍 DNA13의 포함 되어 있습니다. 히스톤 메 틸 화, acetylation, 인 산화, ubiquitination, sumoylation, ADP ribosylation, 탈, 그리고 프롤린 isomerization14,15post-translationally 높은 수정 됩니다. 히스톤 lysine 메 틸 화 녹음 방송, 복제, 재결합16, DNA 손상 응답17및 게놈 각 인18제어 하는 가장 안정적인 히스톤 수정 될 여겨진다. Lysines 모노, 디, 또는 트라이 갠19 하 고 액세스할 수 히스톤 꼬리에 뿐만 아니라 히스톤20의 구형 도메인 내의 표시 수 있습니다. H3K4 및 H3K36에 특정 methylations euchromatin와 주로 관련 된, 모든 잔류물은 히스톤 꼬리14, 내에 있는 특정 methylations H3K9, H3K27, 또는 H4K20에 주로 heterochromatic 지구에서 발견은 19,21. H3K79 메 틸 화는 히스톤 구형 도메인 내에 있는 이며 transcriptional 활동, 뿐만 아니라 transcriptionally 불활성 게놈 지역22와 연결 되었습니다. 수정이 효 모, 송아지 흉 선, 닭, 및 인간의23에서 관찰 되었습니다 때문에 진화론 보존 됩니다. H3K79 모노, 디, 및 trimethylation (H3K79me1, me2, me3) 히스톤 methyltransferases DOT1L,2425 와26핵 설정된 도메인에 포함 된 단백질 2 (Nsd2) 의해 촉매 된다. DOT1L 확산, DNA 수리, 및27reprograming 휴대에 연루 이다. 쥐에서 Dot1l 의 손실 발달 단계 E10.5,2829주위 태아 죽음에 이르게. 심장 개발 및 myocardiocyte 차별화, DOT1L 유전자 표현 규칙30에 대 한 필수적입니다. 중앙 신 경계에 DOT1L 기능 신경 튜브 개발31에 연루 수 있습니다, 그것은 Tbr1억제에 관여-개 개발32, 중 식 그리고 ER 스트레스의 규정에서 작동 수 있습니다 응답 유전자33. 부분적으로 이해 하는32만 맞는 활성화 또는 억압 작업과 H3K79me의 특히 중앙 신경 시스템의 개발 같은 경우 vivo에서 날짜입니다. H3K79 메 틸 화는 히스톤 3의 구형 도메인에 위치 하 고 있습니다, 이후 그것은 sterically 유연한 히스톤 꼬리23에 수정에 비해 액세스. H3K79 메 틸 화의 기능을 이해 하려면 그것의 위치 및 게놈 환경 결정을 신뢰성과 재현성 분석 방법 필요 합니다. 이 방법 종이에서는 다른 신경 창시자 (피 질에 대 한 Cpc) 및 소 뇌에 대 한 CGNPs, 효과적인 DOT1L 억제제 치료, 및 칩 메서드 분석 정량 또는 다른 시간에 시퀀싱을 통해 메 틸 H3K79를의 격리 방법 소개 대뇌와 소 뇌 개발 하는 동안 포인트입니다. 에 대 한 프로토콜 및 그것의 가능성의, 그림 1을 참조 하십시오.

<table><tbody><tr><td>Anti-GAPDH</td><td>Abcam</td><td>ab8245</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:5000</td></tr><tr><td>Anti-H3</td><td>Abcam</td><td>ab1791</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:3000</td></tr><tr><td>Anti-H3K79me2</td><td>Diagenode</td><td>pAb-051-050</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; ChIP antibody</td></tr><tr><td>Anti-H3K79me2</td><td>Abcam</td><td>ab-051-050</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:1000</td></tr><tr><td>Anti-rabbit-IgG</td><td>Diagenode</td><td>C15410206</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; ChIP Ctrl antibody</td></tr><tr><td>Anti-Tubulin alpha</td><td>Abcam</td><td>ab108629</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:3000</td></tr><tr><td>Apo-Transferrin (1 mg/ml)</td><td>Sigma-Aldrich</td><td>T1147</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>B27 Supplement (50x)</td><td>Life Technologies</td><td>17504044</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Bioanalyzer</td><td>Agilent technologies</td><td>G2940CA</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For analysis of sheared chromatin</td></tr><tr><td>Bioruptor NextGen</td><td>Diagenode</td><td>B01020001</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Ultrasonicator</td></tr><tr><td>Boric acid pH 8.4</td><td>Sigma Aldrich</td><td>B6768</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC culturing</td></tr><tr><td>CFX Connect RT PCR Detection System</td><td>Bio-Rad</td><td>1855201</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Detection system for qPCR</td></tr><tr><td>DMEM-F12</td><td>Life Technologies</td><td>11320-033</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGM</td></tr><tr><td>Dynabeads Protein A</td><td>Invitrogen</td><td>10001D</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Magnetic beads, for ChIP</td></tr><tr><td>EPZ-5676</td><td>Selleckchem</td><td>S7062</td><td>&lt;strong&gt;Category:&lt;/strong&gt; DOT1L inhibition&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For DOT1L inhibition in cell culture</td></tr><tr><td>Ethylenediamine tetraacetic acid</td><td>SERVA</td><td>39760.01</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; EDTA</td></tr><tr><td>Fetal Bovine Serum 10% (v/v)</td><td>Gibco</td><td>10082147</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC isolation and CGM</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGNP isolation</td></tr><tr><td>Glutathione (1.25 mg/ml)</td><td>Sigma-Aldrich</td><td>G4251</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Glycine</td><td>Carl Roth</td><td>3187</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For cell fixation</td></tr><tr><td>GoTaq mastermix</td><td>Promega</td><td>A6002</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; DNA polymerase master mix for qPCR</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Life Technologies</td><td>14025-100</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; HBSS</td></tr><tr><td>L-glutamine (200 mM)</td><td>Life Technologies</td><td>25030081</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC culturing</td></tr><tr><td>Lithium chloride</td><td>Sigma-Aldrich</td><td>L4408</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; LiCl</td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502048</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGM</td></tr><tr><td>NanoDrop 3300</td><td>Thermo Fisher</td><td>3300</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Fluorospectrometer for DNA quantification</td></tr><tr><td>NEB Next Ultra DNA Library Prep Kit for Illumina</td><td>NEB</td><td>E7645S</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Kit for Library preparation</td></tr><tr><td>NEBNext Multiplex Oligos for Illumina</td><td>NEB</td><td>E7335</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Oligos for Library preparation</td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>NP-40 Alternative</td><td>Calbiochem</td><td>492016</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP buffer</td></tr><tr><td>Paraformaldehyde</td><td>Carl Roth</td><td>335</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; PFA, for cell fixation</td></tr><tr><td>Penicillin-Streptomycin-Neomycin 1% (v/v)</td><td>Life Technologies</td><td>15640055</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; PSN, for CCM and CGM</td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>10010023</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; PBS, for CPC isolation</td></tr><tr><td>PicoGreen Kit</td><td>Thermo Fisher</td><td>P11496</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Visualizing dye for DNA quantification</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGNP isolation</td></tr><tr><td>Poly-L-ornithine hydrobromide</td><td>Sigma Aldrich</td><td>P3655</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC culturing</td></tr><tr><td>Potassium chloride</td><td>Thermo Fisher</td><td>AM9640G</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; KCl, for CGM</td></tr><tr><td>Protease inhibitor</td><td>Roche</td><td>4693159001</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>3115879001</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>Qiagen MinElute</td><td>Qiagen</td><td>28004</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Kit for DNA purification</td></tr><tr><td>RNAse</td><td>Sigma-Aldrich</td><td>R6513</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>SGC0946</td><td>Selleckchem</td><td>S7079</td><td>&lt;strong&gt;Category:&lt;/strong&gt; DOT1L inhibition&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For DOT1L inhibition in cell culture</td></tr><tr><td>Sodium bicarbonate</td><td>Carl Roth</td><td>8551.1</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; for Elution buffer</td></tr><tr><td>Sodium chloride</td><td>Carl Roth</td><td>9265</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; NaCl, for ChIP buffer</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>30970</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>Sodium dodecylsulfate</td><td>Carl Roth</td><td>183</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; SDS, for ChIP</td></tr><tr><td>Sonic hedgehock (SHH)</td><td>Sigma-Aldrich</td><td>SRP6004</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGNP isolation</td></tr><tr><td>Superoxide dismutase (1mg/ml)</td><td>Sigma-Aldrich</td><td>S7571</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Carl Roth</td><td>9090</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; TRIS, for ChIP buffer</td></tr><tr><td>Triton X-100</td><td>Carl Roth</td><td>X100</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP buffer</td></tr><tr><td>Trypsin-EDTA 0,05% (w/v)</td><td>Sigma Aldrich</td><td>59417C</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC isolation</td></tr><tr><td>Tween20</td><td>Carl Roth</td><td>28320</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For bead preparation</td></tr><tr><td>Other Lab devices: Neubauer counting chamber, Incubator, Rotator, Shaker, Disection set, Water bath</td><td></td><td></td><td></td></tr><tr><td>CCM: Cortical cell medium</td><td></td><td></td><td></td></tr><tr><td>CGM: CGNP cell culture medium</td><td></td><td></td><td></td></tr></tbody></table>

isolation and cultivation of neural progenitors followed by chromatin-immunoprecipitation of histone 3 lysine 79 dimethylation mark

두뇌 개발 transcriptional 프로그램과 다른 morphogens의 기울기로 영역 공간 방식으로 제어 되는 복잡 한 과정 이다. 또한, 히스톤 메 틸 화 처럼 epigenetic chromatin 수정 설치 하 고이 프로세스에서 특정 세포 운명을 유지에 대 한 중요 한 역할이 있다. 대부분 히스톤 메 틸 화는 히스톤 한정자, 지우개, 및 히스톤 리더 단백질 유연한 히스톤 꼬리에 발생 합니다. 대조적으로, H3K79 메 틸 화는 히스톤 3의 구형 도메인에 있으며 다른 발달 기능에 연루. H3K79 메 틸 화 봤을 보존 하 고 찾을 수 있습니다 다양 한 종에서에서 호모 사피엔스 에서 Saccharomyces cerevisiae. 수정 신경 창시자를 포함 한 유기 체 내의 다른 세포 인구에서 발생 합니다. 히스톤 3의 구형 도메인에서 H3K79 메 틸 화의 위치는 평가 하기 어려운 있습니다. 여기, 우리가 격리 하는 방법을 제시 하 고 문화 대뇌 피 질의 조상 세포 (Cpc) 배아 대뇌 피 질의 뇌 조직 (E11.5-E14.5) 또는 소 뇌과 립 상 신경 창시자 (CGNPs)에서 출생 후 조직 (P5-P7), 그리고 효율적으로 immunoprecipitate에서 정량 PCR (정량) 및 게놈 넓은 시퀀싱에 대 한 H3K79me2.

신경 조상 격리, 재배, H3K79me2 칩과 칩 분석 방법의 일반적인 계획: 그림 1 H3K79me2 칩 또는 소 뇌과 립 상 신경 창시자의 미 발달 두뇌 개발 하는 동안 다른 시간 지점에서 대뇌 피 질의 조상 세포의 출생 후 단계에서 수행 하는 순서도 보여 줍니다. 첫 번째 단계로 뇌 격리 하 고 telencephalon (E11.5 E14.5 사이) 또는 소 뇌 (P5-P7)을 검색할 수 있다. ...

Watch this Scientific Journal Video about Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark at JoVE.com

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

절연 및 신경 창시자 Chromatin-Immunoprecipitation 히스톤 3 리 79 Dimethylation 마크의 뒤의 재배

우리 신경 조상 세포의 히스톤 3 리 79 dimethylation (H3K79me2)-내에 있는 히스톤 마크 chromatin immunoprecipitation (칩)에 대 한 미 발달 및 출생 후 뇌 조직에서 문화를 격리 하는 효과적이 고 재현 가능한 방법을 제시는 히스톤 3의 구형 도메인입니다.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

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7.4K 조회수.  University of Freiburg. 우리 신경 조상 세포의 히스톤 3 리 79 dimethylation (H3K79me2)-내에 있는 히스톤 마크 chromatin immunoprecipitation (칩)에 대 한 미 발달 및 출생 후 뇌 조직에서 문화를 격리 하는 효과적이 고 재현 가능한 방법을 제시는 히스톤 3의 구형 도메인입니다.

비디오: Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

뇌실 주위 백질 성인 쥐의 지역 및 인간의 척수에서 신경 줄기 / 전구 세포의 분리

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

연구

발생학

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2D 및 3D 환경에서 신경 전구 세포의 전기장 제어 감독 마이 그 레이션

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

생체공학

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

네이티브 Chromatin Immunoprecipitation Murine 뇌 종양 Neurospheres를 사용 하 여

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

암 연구학

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

3D 신경 줄기 세포 문화 번역하는 단백질 : 하이브리드 시각화 방법론

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

신경과학

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

배아 Murine 신경 튜브에서 신경 크레스트 세포의 분리 및 문화

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

절연 및 마우스 배아 신경 줄기 세포의 문화

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Native Chromatin Immunoprecipitation of Neurosphere Cells

<a href='https://app.jove.com/v/57016/native-chromatin-immunoprecipitation-using-murine-brain-tumor'>Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres.&#160;J. Vis. Exp.&#160; (2018).</a>This video demonstrates the isolation of protein-DNA complexes from neurosphere cells derived from a mouse brain tumor using native chromatin immunoprecipitation. It details the steps for chromatin preparation, immunoprecipitation, washing, and protein digestion to release DNA for downstream analysis.

Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres.&#160;J. Vis. Exp.&#160; (2018).This video ...

JoVE Encyclopedia of Experiments

Neuropathology

Tumors

Isolating Induced Neural Progenitor Cell Colonies Derived From Adult Human Fibroblasts

Source: Meyer, S. et al., <a href="https://app.jove.com/v/52831">Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for isolating induced neural progenitor cells (iNPCs) derived from adult human fibroblasts. It outlines the steps involved in isolating the iNPC colonies and culturing them in laminin-coated multi-well plate wells in the presence of suitable culture media that facilitate the continued proliferation and expansion of the iNPC population.

성체 인간 섬유아세포에서 유래한 유도 신경전구세포 군집(Induced Neural Progenitor Cell Colonies)

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Putative Induced ...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

Source: Bovio, P., et al., <a href="https://app.jove.com/v/56631">Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark.</a> J. Vis. Exp. (2018)
This video demonstrates the isolation and cultivation of cerebellar neural progenitor cells from a mouse pup&#39;s cerebellum. Cerebellar granule neuron progenitors are isolated and purified through differential adhesion, effectively removing astrocytes. These cultured progenitors subsequently differentiate into cerebellar granule neurons.

Isolating and culturing murine Cerebellar Granular Neuron Progenitors(쥐 소뇌 과립 뉴런 전구 세포)

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1.&#160;Preparations

	Preparations ...

Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat

Source: Mothe, A., et al., <a href="https://app.jove.com/v/52732">Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord.</a> J. Vis. Exp. (2015).
This video presents a method for isolating neural stem/progenitor cells from the periventricular tissue of the spinal cord in adult rats. The technique includes incubating the tissue fragments in a solution containing proteolytic enzymes to disrupt the extracellular matrix and release the cells. Subsequently, the released cells are plated in a medium containing specific growth factors that enhance neural stem/progenitor proliferation and promote aggregation into three-dimensional neurospheres.

성체 쥐의 척수 뇌실 주위 영역에서 신경 줄기 전구 세포의 격리

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of Dissection ...