Name: inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation
Uploaded: 2017-11-17T15:00:00.000+00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation at JoVE.com

저자는 제임스 매디슨 대학 생물학 부와 제임스 매디슨 대학교 빛 현미경 이미징 시설의 관대 한 지원을 감사합니다. 닥터 마크 L. 가브리엘 젊은 출생 후 조직 준비, 에 관한 유용한 조언 및 박사 저스틴 W. 브라운 코리 L. Cleland 수술 재료와 공간의 관대 한 조정에 대 한. 이 연구 4-VA, 전진 하는 연방의 버지니아 (G.S.V.)에 대 한 공동 제휴 및에 의해 버지니아 아카데미의 과학 작은 프로젝트 연구 부여 (G.S.V.) 공동 연구 교부 금에 의해 부분적으로 투자 되었다. 지원이 아낌없이 제공 되었습니다 베티 조 사랑 버틀러 ' 58 기부금으로 학부 연구 장학금 (K.M.B.), 파렐 여름 연구 장학금 (K.M.B.)을, (에 K.M.B.), 제임스 매디슨 대학 두 번째 세기 장학금에 대 한 한 제임스 매디슨 대학 센테니얼 장학금 (C.J.H.), 제임스 매디슨 대학 루시 로빈슨 검색 ' 30 기념 장학금 (Z.L.H.)을 및 과학 및 수학 교수 지원 그랜트 (G.S.V.)에 제임스 매디슨 대학 대학.

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저자는 공개 관심의 없습니다 충돌 있다.

여기, electroporation utero에 모자이크 뇌 조직의 생성을 floxed 마우스에 Cre recombinase와의 조합을 소개 합니다. 이 방법의 장점은 새로운 마우스 선 다른 셀룰러 하위는 타겟이 될 때마다 생성 될 필요는 없습니다: electroporation utero에서 흥분 성의 뉴런, 신경 억제, 또는 시간에 따라 명과 대상으로 사용할 수 있습니다 그리고 electroporation15,,16</s...

유전 모자이크를 생성 하는 것은 관심사의 유전자의 기능을 이해 하기 위한 고전적인 실험 패러다임 이다. 프로그램 유전자 세포 표현 형에 필요한 지 확인 하려면 가장 간단한 방법은 유기 체 (예 마네)에 걸쳐 유전자의 기능의 손실을 일으키는 것입니다. 그러나, 확인 하려면 유전자 특정 셀 종류에 특히 필요한 경우, 유기 체 전체 유전자의 녹아웃이 아니다 유효한 접근. 대신, 메서드는 필요한 wildtype (즉 유전자 교란) 조직으로 둘러싸여 동안 주어진된 셀에 유전자의 기능의 손실을 발생 합니다는-즉, 모자이크 조직 만들기. 만약 돌연변이 세포 돌연변이 표현 형을 표시 하지만 주변 wildtype 세포 안, 세포 자치 방법으로 유전자 기능. 돌연변이 세포 wildtype 조직에 의해 포위 된다 모자이크 조직의 분석 유전자, 특히 신경 및 명과 조직의 광대 한 상호 네트워크를 형성 하는 뇌의 세포 자치 기능을 이해 하는 데 이상적입니다.
여러 형태의 모자이크 뇌 조직의 강력한 모델 조사 유전자의 세포 자치 기능을 제공 합니다. 연구 신경 이식1, 여성 X 연결 mosaicism2,3,4에 집중 하 고 내 생 신체적인 mosaicism5,6 그린 모자이크에 따라 그들의 결론 뇌 조직입니다. 조건부 삭제 Cre lox 재결합 시스템을 통해 유전자의 유전자 변형 마우스 라인의 좋은 가용성을 최대한 활용 하는 방법입니다. 이 방법에서는, 2 개의 loxP 사이트 loxP 사이트 같은 방향으로 ("floxed")에서 두 얼굴을 있는 그것을 떠나 (예:는 exon), 유전자의 필요한 시퀀스의 양쪽에 소개 된다. Cre recombinase loxP 사이트7사이 시퀀스 excises. Cre 중재 재결합 Cre recombinase 셀 ("Cre 기자 선")의 하위 집합에서 형광 표식 함께 표현 하는 또 다른 마우스 라인을 건너 floxed 마우스에 의해 얻을 수 있습니다. 이 다양 한 방법으로 흥분 성의 뉴런 등 이다8셀의 하위 집합에서 유전자의 기능을 밝히기 위해 입증 되었습니다. Cre 기자 라인 Cre 중재 재결합 마약을 유도할 수 있는 (단일 신경 라벨 유도할 수 있는 Cre 중재 녹아웃 또는 매끄러운) 될 수 있도록 있다T2 를 표현할 수 있습니다9. 다른 전략 이라는 이중 표식 (MADM)10,11모자이크 분석, Cre 중재 interchromosomal 재결합 homozygous 돌연변이 heterozygous 조직 함께 만들 수 있습니다. 이러한 방식에서 쥐의 새로운 라인 각 후보 유전자 또는 세포 하위 테스트에 대 한 각 시간 제작 될 필요가 있다. 또는, Cre recombinase 소개 될 수 있다 postnatally 또는 바이러스 성 벡터 (예를들면 adeno 관련 바이러스13 또는 lentiviruses14 셀룰러 하위 형식 관련 운반 iontophoresis12 통해 발기인)입니다. 이 전략 강력 하 고 산 후 라벨을 만듭니다. 대뇌 피 질 뉴런을 띄엄띄엄 prenatally 개발 대상으로 이상적인 전략의 형광 표시와 함께 Cre recombinase electroporation utero에 입니다.
Cre lox 재결합 electroporation utero에서 생성을 통해 결합 뿐만 아니라 vivo에서조직 모자이크, 다른 게시 프로토콜15,,16에서 프로시저에 여러 각 색 한 소개 17,,1819,,2021. 우리는 사육 타임-임신 여성에서 성공을 개선 하는 정보를 제공 합니다. 우리 또한 우리의 두 전략 스파스 하 고 밝은 대뇌 피 질의 조직에서 뉴런의 라벨을 소개 하는 개요: 하나의 전략 Cre recombinase 및 형광 마커22단일 구문을 코딩의 수준을 적정 하는 것입니다. 또 다른 전략 마음23,24에 이러한 매개 변수를 특별히 설계 된 "초신성" 시스템을 사용 하는 것입니다. 또한, 생산 하는 일관 된 microinjection 펫 및 단순화에 utero electroporation 수술에 개선을 제공 합니다. 마지막으로, 우리는 더 얼룩 또는 immunohistochemistry 없이 모 수석 등뼈와 수지상 및 axonal 아 버의 분석을 허용 하는 간단한 조직학 준비에서 중요 한 단계를 개요.

<table><tbody><tr><td>C57BL/6J mice</td><td>The Jackson Laboratory</td><td>#000664</td><td>See &quot;1. Mouse set-up&quot; (step 1.1, &quot;wildtype mice&quot;)</td></tr><tr><td>GFP.Cre empty vector</td><td>AddGene</td><td>#20781</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;single DNA construct that codes for Cre recombinase as well as a fluorescent marker&quot;). GFP.Cre empty vector was a gift from Tyler Jacks.</td></tr><tr><td>pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova)</td><td>AddGene</td><td>#69138</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>pK031.TRE-Cre (Supernova)</td><td>AddGene</td><td>#69136</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK031.TRE-Cre (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova)</td><td>AddGene</td><td>#85006</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>EndoFree Plasmid Maxi Kit (10)</td><td>Qiagen</td><td>#12362</td><td>See &quot;2. DNA set-up&quot; (step 2.3 &quot;endotoxin-free plasmid purification kit&quot;)</td></tr><tr><td>Trypan Blue powder, BioReagent grade</td><td>Sigma</td><td>T6146-5G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;trypan blue&quot;)</td></tr><tr><td>Sodium Chloride, ACS, 2.5 kg</td><td>VWR</td><td>BDH9286-2.5KG</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;NaCl&quot;)</td></tr><tr><td>Potassium Chloride, ACS, 500 g</td><td>VWR</td><td>#97061-566</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;KCl&quot;)</td></tr><tr><td>Sodium phosphate dibasic, ReagentPlus, 100 g</td><td>Sigma-Aldrich</td><td>S0876-100G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&quot;)</td></tr><tr><td>Potassium phosphate monobasic, ReagentPlus, 100 g</td><td>Sigma-Aldrich</td><td>P5379-100G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&quot;)</td></tr><tr><td>Hydrochloric acid, ACS reagent, 500 mL</td><td>Fisher Scientific</td><td>A144-500</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;HCl&quot;)</td></tr><tr><td>P-97 Micropipette Puller</td><td>Sutter Instrument</td><td>P-97</td><td>See &quot;3. Pipette set-up&quot; (step 3.1 &quot;glass capillary puller&quot;)</td></tr><tr><td>3.0 mm wide trough filament</td><td>Sutter Instrument</td><td>FT330B</td><td>See &quot;3. Pipette set-up&quot; (step 3.1 &quot;glass capillary puller&quot;)</td></tr><tr><td>Thin Wall Glass Capillaries, 4&quot;, 1 / 0.75 OD/ID</td><td>World Precision Instruments</td><td>TW100-4</td><td>See &quot;3. Pipette set-up&quot; (step 3.1.1 &quot;glass capillary&quot;)</td></tr><tr><td>Single Ply Soft-Tech Wipes, 4.5&quot;</td><td>Phenix</td><td>LW-8148</td><td>See &quot;3. Pipette set-up&quot; (step 3.2.1 &quot;single-ply task wipe&quot;); other single-ply wipes (e.g. Kimwipes) can be used.</td></tr><tr><td>Graefe Forceps, 7 cm, Straight, 0.7 mm 1x2 Teeth</td><td>World Precision Instruments</td><td>#14140</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Graefe forceps&quot;)</td></tr><tr><td>Iris Scissors, 11.5 cm, Straight, 12-pack</td><td>World Precision Instruments</td><td>#503708-12</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;iris scissors&quot;)</td></tr><tr><td>Hartman Mosquito Forceps, 9 cm, Straight, 12-pack</td><td>World Precision Instruments</td><td>#503728-12</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Hartman mosquito forceps&quot;)</td></tr><tr><td>General Purpose Non-Woven Sponges, 2&quot; x 2&quot;, 4-ply</td><td>Medrepexpress</td><td>#2204-c</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;non-woven gauze sponges&quot;)</td></tr><tr><td>Ring Tipped Forceps, 10 cm, Straight, 2.2mm ID</td><td>World Precision Instruments</td><td>#503203</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;ring-tipped forceps&quot;)</td></tr><tr><td>Pyrex petri dishes complete, O.D. &amp;times; H 100 mm &amp;times; 20 mm</td><td>Sigma-Aldrich</td><td>CLS3160102-12EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Petri dishes&quot;)</td></tr><tr><td>Flat Type Instrument Tray, Stainless Steel, 13-5/8&quot; x 9-3/4&quot; x 5/8&quot;</td><td>Amazon</td><td>B007SHGAHA</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;stainless steel tray&quot;)</td></tr><tr><td>Platinum Tweezertrode, 5 mm</td><td>BTX</td><td>#45-0489</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 and 4.16 &quot;tweezer-type electrodes&quot;)</td></tr><tr><td>ECM 830 Foot Pedal</td><td>BTX</td><td>#45-0211</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 and 4.17 &quot;foot pedal&quot;)</td></tr><tr><td>ECM 830 Generator</td><td>BTX</td><td>#45-0052</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 &quot;generator&quot;)</td></tr><tr><td>Single Animal Isoflurane Anesthesia System with Small Induction Box</td><td>Harvard Apparatus</td><td>#72-6468</td><td>See &quot;4. In utero electroporation&quot; (step 4.4 and 4.6 &quot;nose cone&quot;, step 4.4 &quot;induction chamber&quot;)</td></tr><tr><td>Ophthalmic ointment</td><td>Hanna Pharmaceutical Supply Co</td><td>#0536108691</td><td>See &quot;4. In utero electroporation&quot; (step 4.7 &quot;veterinary ophthalmic ointment&quot;)</td></tr><tr><td>Space Gel (AIMS)</td><td>VWR</td><td>#95059-640</td><td>See &quot;4. In utero electroporation&quot; (step 4.8 &quot;sealed pouch filled with supersaturated salt solution&quot;)</td></tr><tr><td>Hair Remover Gel Cream, Sensitive Formula</td><td>Veet</td><td>#062200809951</td><td>See &quot;4. In utero electroporation&quot; (step 4.9 &quot;depilatory cream&quot;)</td></tr><tr><td>10ul Low Retention Tip Starter (960 tips/pk)</td><td>Phenix Research Products</td><td>TSP-10LKIT</td><td>See &quot;4. In utero electroporation&quot; (step 4.12 &quot;sterile 10 &amp;micro;L micropipette tip&quot;)</td></tr><tr><td>Aspirator tube assemblies for calibrated microcapillary pipettes</td><td>Sigma-Aldrich</td><td>A5177</td><td>See &quot;4. In utero electroporation&quot; (step 4.15 &quot;aspirator tube assembly&quot;)</td></tr><tr><td>Braided Absorbable Suture, 4-0, Needle NFS-2(FS-2), 27&quot;</td><td>Medrepexpress</td><td>MV-J397</td><td>See &quot;4. In utero electroporation&quot; (step 4.19 &quot;absorbable sutures&quot;)</td></tr><tr><td>&amp;ldquo;LiquiVet Rapid&amp;rdquo; Tissue Adhesive</td><td>Medrepexpress</td><td>VG3</td><td>See &quot;4. In utero electroporation&quot; (step 4.20 &quot;tissue adhesive&quot;)</td></tr><tr><td>Hypodermic syringes, polypropylene, Luer lock tip, capacity 1.0 mL</td><td>Sigma-Aldrich</td><td>Z551546-100EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.21 &quot;1 mL syringe&quot;)</td></tr><tr><td>BD Precisionglide syringe needles gauge 26, L 1/2 in.</td><td>Sigma-Aldrich</td><td>Z192392-100EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.21 &quot;26G, &amp;frac12;&amp;rdquo; needle&quot;)</td></tr><tr><td>Nestlets Nesting Material</td><td>Ancare</td><td>NES3600</td><td>See &quot;4. In utero electroporation&quot; (step 4.24 &quot;nesting materials&quot;)</td></tr><tr><td>Sunflower Seeds, Black Oil, Sterile</td><td>Bio-Serv</td><td>S5137</td><td>See &quot;4. In utero electroporation&quot; (step 4.24 &quot;sunflower seeds&quot;)</td></tr><tr><td>Paraformaldehyde, 97%</td><td>Alfa Aesar</td><td>A11313</td><td>See &quot;5. Histology&quot; (step 5.1.1 &quot;PFA&quot;)</td></tr><tr><td>Economy Tweezers #3, 11 cm, 0.2 x 0.4 mm tips</td><td>World Precision Instruments</td><td>#501976</td><td>See &quot;5. Histology&quot; (step 5.5 &quot;tweezers&quot;)</td></tr><tr><td>Agar powder</td><td>Alfa Aesar</td><td>#10752</td><td>See &quot;5. Histology&quot; (step 5.8.1 &quot;agar&quot;)</td></tr><tr><td>Single-edge razor blades, #9 blade</td><td>Stanley Tools</td><td>#11-515</td><td>See &quot;5. Histology&quot; (step 5.9 &quot;single-edge razor blade&quot;)</td></tr><tr><td>Specimen disc S D 50 mm</td><td>Leica</td><td>#14046327404</td><td>See &quot;5. Histology&quot; (step 5.9 &quot;vibrating microtome specimen disc&quot;)</td></tr><tr><td>Buffer tray S assembly</td><td>Leica</td><td>#1404630132</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;buffer tray&quot;)</td></tr><tr><td>VT1000 S Vibratome</td><td>Leica</td><td>#14047235612</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;vibrating microtome&quot;)</td></tr><tr><td>Double Edge Razor Blades</td><td>Personna</td><td>BP9020</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;blade&quot;)</td></tr><tr><td>Knife Holder S</td><td>Leica</td><td>#14046230131</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;knife holder&quot;)</td></tr><tr><td>Studio Elements Golden Taklon Short Handle Round Brush Set</td><td>Amazon</td><td>B0089KU6XE</td><td>See &quot;5. Histology&quot; (step 5.12.1 &quot;fine tipped paintbrush&quot;)</td></tr><tr><td>Superfrost Plus Slides</td><td>Electron Microscopy Services</td><td>#71869-11</td><td>See &quot;5. Histology&quot; (step 5.12.1 &quot;microscope slide&quot;)</td></tr><tr><td>ProLong Diamond Antifade Mountant, 10 ml</td><td>Thermofisher</td><td>P36970</td><td>See &quot;5. Histology&quot; (step 5.12.3-5.12.4 &quot;mountant&quot;)</td></tr><tr><td>Cover Glass, 24 x 50 mm, No. 1</td><td>Phenix Research Products</td><td>MS1415-10</td><td>See &quot;5. Histology&quot; (step 5.12.4 &quot;coverslip&quot;)</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td>See &quot;5. Histology&quot; (step 5.13.1 &quot;DAPI&quot;)</td></tr><tr><td>Fixed Stage Upright Microscope</td><td>Olympus</td><td>BX51WI</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;light microscope&quot;)</td></tr><tr><td>Laser Scanning Confocal Microscope</td><td>Nikon</td><td>TE2000/C2si</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;confocal microscope&quot;)</td></tr><tr><td>4x objective, NA = 0.20</td><td>Nikon</td><td>CFI Plan Apo Lambda 4X</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;low-power objective&quot;)</td></tr><tr><td>20x objective, NA = 0.75</td><td>Nikon</td><td>CFI Plan Apo Lambda 20X</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;medium-power objective&quot;)</td></tr><tr><td>60x objective, NA = 1.40</td><td>Nikon</td><td>CFI Plan Apo VC 60X Oil</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;high-power objective&quot;)</td></tr></tbody></table>

inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation

유전자의 세포 자율 신경 기능 손실이 발생 하 여 계시 될 수 있다 또는 뉴런의 작고 부족 인구에 있는 유전자의 기능을 얻을. 이렇게 하려면 손실 또는 이득 유전자의 기능의 신경 유전자 교란된 조직에 의해 포위 된다 모자이크를 생성 해야 합니다. 여기, 우리가 결합 Cre lox 재결합 시스템 electroporation utero에 모자이크 뇌 조직 신경에 있는 유전자의 세포 자치 기능을 공부 하는 데 사용할 수 있습니다을 생성 하기 위하여. DNA 구조 (저장소를 통해 사용 가능), 형광 라벨 및 Cre recombinase 코딩 유전자에 utero 를 사용 하 여 마우스 태아의 두뇌에 loxP 사이트와 측면을 포함 하는 대뇌 피 질의 뉴런 개발에 도입 electroporation입니다. 또한, 우리는 utero에 electroporation 메서드에 생존 및 재현성을 증가 하는 다양 한 적응을 설명 합니다. 이 방법은 또한 titer Cre 중재 재결합 뉴런의 스파스 또는 조밀한 인구에 대 한 설정 포함. (필요 하지 않습니다 하지만)을 적용할 수 있습니다 레이블이 뇌 조직의 조직학 준비 immunohistochemistry. 사용 하는 구문을 붙일 뉴런 수행 Cre recombinase에 대 한 유전자 표시 보장 합니다. 조직학 준비 수지상 및 axonal 아 버 및 모 수석 등뼈의 confocal 영상 통해 뉴런의 형태소 분석 허용. 있으므로 손실 또는 함수의 이득 스파스 모자이크 조직에서, 세포 자치 필요성의 연구와 유전자 제품에서 vivo에서의 자족이이 방법을 허용 합니다.

단일 GFP를 구성 합니다. Cre는 E15.5에서 electroporated 이었고 P14에서 시각 (재료의 목록 참조). 구성의 농도 주입의 볼륨에 따라 스파스 또는 밀도 결과22,26얻어질 수 있다. 예를 들어, 2 mg/mL GFP의 1 µ L의 주입. 레이블이 지정 된 셀, 일부는 밝은 (그림 1A), 그리고에서 지역화 레이어 II/III (그림 1B</...

Watch this Scientific Journal Video about Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation at JoVE.com

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

두뇌에 있는 유전자의 세포 자치 기능 손실을 유도 하 여 공부 될 수 있다 또는 셀의 스파스 인구에 있는 기능을 얻을. 여기, Cre recombinase 함수 비보의 손실에 floxed 유전자와 대뇌 피 질의 뉴런 개발의 부족 인구에 제공 electroporation utero에서 설명 합니다.

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 ...

inducing-cre-lox-recombination-mouse-cerebral-cortex-through-utero

Universidad de Cartagena UDEC

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JoVE Journal

Neuroscience

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

11.6K 조회수.  James Madison University. 두뇌에 있는 유전자의 세포 자치 기능 손실을 유도 하 여 공부 될 수 있다 또는 셀의 스파스 인구에 있는 기능을 얻을. 여기, Cre recombinase 함수 비보의 손실에 floxed 유전자와 대뇌 피 질의 뉴런 개발의 부족 인구에 제공 electroporation utero에서 설명 합니다.

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

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

단백질 삭제의 유도를 통해<em&gt; 자궁 내</em&gt; 일렉트로는 형질 전환 모델에서 신경 세포의 마이그레이션에 적자를 정의하려면

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

연구

발생학

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

E15 마우스 태아의 Utero의 심실 주입 및 Electroporation에

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

생물학

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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

사용하여 여러 개의 CNS 준주로 효율적인 유전자 전달 Utero에서 Electroporation

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

신경과학

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

마우스 Utero에 Electroporation : 통제 Spatiotemporal 진 Transfection

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

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

의 연결을 Morphogenesis의 연구를위한 방법 :<em&gt; 예 생체내</em배아 Murine 대뇌 피질에서&gt; RNAi의 Electroporation

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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 ...

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 ...

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

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

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

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

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

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

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

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

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

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

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

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

피질 마우스 성상세포를 개별화하기 위해 다각형 게놈 통합 색상(MAGIC) 마커의 자궁 전기포게에서

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

Cre lox 재결합 Electroporation Utero에 통해 마우스 대뇌 피 질에서 유도

요약

더 많은 비디오 탐색

이 비디오의 챕터

단백질 삭제의 유도를 통해

E15 마우스 태아의 Utero의 심실 주입 및 Electroporation에

사용하여 여러 개의 CNS 준주로 효율적인 유전자 전달 Utero에서 Electroporation

마우스 Utero에 Electroporation : 통제 Spatiotemporal 진 Transfection

의 연결을 Morphogenesis의 연구를위한 방법 :

사용하는 마우스 대뇌 피질과 해마의 시각화 및 Dendrites와 쪽이의 유전자 조작

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

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

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

피질 마우스 성상세포를 개별화하기 위해 다각형 게놈 통합 색상(MAGIC) 마커의 자궁 전기포게에서