이 작품은 독일 연구 재단 (도이치 Forschungsgemeinschaft)에 의해 재정 지원되었다.

1. 주사 DNA 유리 마이크로 피펫의 준비 <ol><li> 좋은 품질의 유리 마이크로 피펫은 양수의 손실로 인해 초기 높은 낙태 비율을 줄이기 위해 필수적입니다. 유리 마이크로 피펫을 당겨하는 절차가 잘 13,18,25를 기록하고있다. 열 = 300; 1.2 mm 직경의 모세관을 사용하여 설정 P = 500와 기존의 셔터에 P-97 장치를 뽑아 = 40을 당겨 속도 = 50; 시간 = 50. 3mm &quot;구유&quot;필라멘트 (셔터 악기 FT330B)와 풀러 맞 춥니 다. 2mm 크기의 필라멘트는 우리 덜 만족스러운 결과를 굴복했다. 한편, 마이크로 피펫 팁의 경사지는 것은 우리에 대한 결과를 개선하지 않는 것. </li><li> 1 ~ 2 ㎍ / μL의 최종 농도에 녹색 빠른 0.1 %를 포함하는 PBS의 정화, 독소 무료 플라스미드 DNA를 (세포 배양 학년) 녹입니다. 빠른 그린은 배아 뇌의 뇌실에서 볼 수 주입 솔루션을 만들 것입니다. </li><li> 유리 micropipette를에 DNA 용액 10 μl를로드합니다. </lI&gt; <li> 분사 시스템 (피코 펌프)이나 입 피펫에 유리 micropipette를 연결합니다. </li></ol> 2. 마취 가열 패드와 수술기구와 수술 테이블을 준비합니다. 배아의 시각화를 촉진하는 차가운 광원을 켭니다. 예를 유리 구슬 살균기에 사용 수술 도구를 소독. 세 가지 마취 절차는 (토론 참조)이 프로토콜에 대한 가능합니다. 여기에서 우리는 우리가 (유량 0.5 L / 분) 마취 유도를 위해 isoflurane을 흡입을 사용하여 최선을 고려해야 하나뿐만 아니라 유지 보수 (유량 1 L / 분)를 설명합니다. <ol><li> 튜브의 짧은 길이로 Komesaroff 마크 5 마취 장치에 연결 (그래서 마우스가 계속 표시) 투명한 작은 용기에 임신 마우스를 소개합니다. </li><li> isoflurane을 가진 기화기를 기입 한 후 장치에 연결된 산소 병을 엽니 다. isofl 중 하나 또는 두 개의 펄스를 날려urane / 마취 안으로 설정하는을위한 마우스와 시계를 들고 용기에 산소 (0.5 L / 분) </li><li> 그들을 건조 방지하고 즉시 머리에 흐름 마취 마스크를 맞게 연고 눈을 커버, 마우스를 꺼내. </li></ol> 3. 개복술 <ol><li> 가열 패드 (그렇지 않으면 그것의 몸의 온도가 회복의 기회를 감소, 매우 빠르게 내려 갈 것이다)에 최대 배꼽에 마우스를 놓고 테이프로 옆으로는 네 다리를 고정하여 제자리에 그 몸을 고정합니다. 반사 자극 (회사의 압력 예를 들어, 발가락이나 꼬리 핀치)에 대한 응답의 손실을 확인하여 마취의 깊이를 평가합니다. </li><li> 복부 표면을 면도 iodophorpovidone 요오드 (Braunoderm)로 소독. </li><li> 복부 피부에 종 절개 (1-1.5 ㎝ 길이)를 확인합니다. 그런 복막를 잘라. 절개 주위에면 거즈를 놓습니다. 하나의 자궁을 표시하고 PBS-씻어서 gauz에 무딘 집게로 조심스럽게 잡아 당깁니다전자. 매우 자주 그것은 항상 (그림 2A와 2B) 촉촉한 유지하기 위해 PBS와 자궁을 씻어. 전체 절차를 수행하는 동안 자궁 내부의 높은 압력이 낙태의 결과로 주입시 유체의 손실의 가능성을 증가 배아를 전송합니다 때문에, 꽉 mesometrium 또는 자궁을 당겨 마십시오. </li></ol> 4. 뇌 심실로 DNA 주입 <ol><li> 뇌의 뇌실을 시각화 할 수있는 방식으로 자궁을 누릅니다. 광범위하게 불리한 위치에 놓여 태아의 위치를​​ 변경하지 마십시오 - 이것은 낙태의 가능성을 증가시킵니다. </li><li> 정상에서 배아의 머리를 보면, 왼쪽과 오른쪽 대뇌 반구 사이의 시각 차이 또는 균열을 집중. 반구 구별하기 쉽고 그 안에서 측면 심실은 (을 대상으로하지 않은) 보통 다소 어두운 모양으로 인식 할 수 있습니다. 배아 (넘겨 준다 DNA 주입을 위해 완벽 지향이 발견 된 경우에스 2C와 2D)는, 그것을 관통하여 자궁 벽에 가능하며 한 번에 세 번째 뇌실을 입력합니다. 약 1mm 대한 침투, 자궁 벽과 대뇌 피질 반구 사이의 간격의 주동이의 끝 부분에 구멍을 45 °로 유리 micropipette를 잡으십시오. 이러한 방법으로 유리 마이크로 피펫의 끝은 뇌 (안 뇌실) (그림 2C와 2D)의 세 번째 뇌실로 들어갑니다. 이 배아의 머리 근처에서 첫 번째 구멍을 뚫어 질 벽 (항상 45 °에서)에 유용 덜 호의적 중심의 배아에서 대뇌 피질 반구 사이의 올바른 위치에 micropipette의 팁을 배치하고 단지 그 후에 뇌를 관통. 세 번째 뇌실 (좋은 주사 녹색 액체가 뇌실을 채우고)에 DNA 용액 1 μL에 대해 주입. 하나의 자궁 뿔의 배아와 동일한 절차를 반복합니다. 이 DNA 솔루션에 대한 약간의 시간이 심실 유체로 균일하게 혼합 entir에 도달 할 수 있습니다전자 neuroepithelium. </li></ol> 5. Electroporation에 대한 시상을 대상으로 <ol><li> 에 electroporator를 전환하고 배아 나이 (E12.5 위해 우리는 5 구형파 펄스를 사용하여, 50 V, 50 밀리 ON/950 밀리 초 OFF)에 따라 설정을 조정합니다. 양극 스테인레스 스틸 바늘 전극 (CUY550-10)로하고 부정 극 원형 평면 전극 (CUY700P4L)로 사용합니다. (그것은 그렇지 않으면 현재 그냥하지만​​ 그것을 통해 배아 주위에 흐르고 있기 때문에 전극, 배아보다 작은 것이 중요합니다.) </li><li> electroporation에 대한 그의 지느러미 쪽 (즉, 실험을 향하고)까지이다 (입니다 대부분으로) 그 배아를 선택하고, 그 방향으로 유리한되지 않은 버립니다. 그것은 에탄올 소독 손가락이나 장갑으로 자궁을 만지지 지금은 안전합니다. 집게와 자궁을 잡고, 그러나, 일렉트로 동안 전류의 흐름을 방해한다. </li><li> 피어스 AMN 사이에 태아의 머리에 의해 자궁 벽바늘 전극의 끝으로 그것을 통해 아래로 밀어하여 iotic 주머​​니와 태반. 스러스트 블록으로 다른 손의 집게 손가락을 사용합니다. 전극의 끝 부분의 약 5 mm은 중뇌 (그림은 2E 및 2 층) 약 수준에서 양막 낭과 자궁 벽 사이에 지금 있어야합니다. 이 &quot;표적 전극&quot;, 그래서 그것의 위치는 시상 하부 neuroepithelium의 일부 형질을받을 가능성이 가장 높은 결정됩니다 것을 기억하십시오. 태아는 매우 부드럽게 전극 사이에 &quot;압박&quot;으로되어 있습니다. </li><li> 다른 한편으로, 위치에 태아의 머리 (그림의 2E와 2G)의 반대편에 자궁 벽의 외부 둥근 편평한 전극. </li><li> (950 밀리 초 OFF, 5 구형파 펄스 50 V, 50 밀리 초) 전압을 적용 페달 스위치를 사용합니다. </li><li> 다른 손의 검지 손가락으로 자궁을 다시 잡고 천천히 자궁의 바늘 전극을 당겨 - 만약 양수 검진TIC 액체가 구멍이 벽을 통해 손실, 일반적으로 배아 낙태를 받게 될 것이다. 모든 배아와 절차를 반복합니다. </li><li> 복부에 자궁 경적을 반환하고 남은 경적 주사와 일렉트로를 반복합니다. </li></ol> 6. 수술 마무리 <ol><li> 모든 배아의 주입과 electroporation을 한 후, 그들은 그것을 닫기 전에 식염수로 복강 전에 촉촉하고 있었다 정확하게 위치, 아주 조심스럽게 복부에 다시​​ 자궁을 배치합니다. </li><li> 외과 장선 (Vicryl Polyglactin 910, 5-0, ETHICON V4914)와 복막을 봉합. 피부 사용의 폐쇄 methodwith 대한 저항력이 봉합 (Supramid 나일론, 6-0, Serag Wiessner 07171L TO) &quot;스티치 중단&quot;. </li><li> 포비돈 요오드 (Braunoderm)와 복부 표면을 소독 비 스테로이드 피하 항염증제 (예 : 0.9 % 염화나트륨의 Rimadyl의 1:10 용액 100 μL) 또는, 더 나은, 부와 같은 아편 주사prenorphine (Temgesic, 0.9 % 염화나트륨의 0.05-0.1 ㎎ / ㎏ 체중)은 통증을 완화한다. </li><li> 마취 기계에서 어머니를 제거하고 가열 패드에 의해 가열 케이지에 넣습니다. 완전히 마취에서 회복 될 때까지 계속 마우스를 모니터링 할 수 있습니다. 나중에, 그들은 감염이나 고통의 흔적없이 프로 시저에서 복구하는 보장하기 위해 매일 동물을 확인합니다. </li></ol> 7. 결과 분석 <ol><li> 분석 원하는 날짜에 따라 배아 (또는 postnatals) 수확. 출생 마우스 (1 - 2 일 이전)는 잘린에 의해 살해된다. </li><li> 이전 electroporation의 주석에 따라 모든 배아를 분리합니다. 입체 현미경 하에서 뇌를 해부하고 해당 지역의 녹색 형광 신호 (GFP 기자에서)에 대한 현미경으로 확인합니다. </li><li> 선택한 두뇌는 &quot;신선한&quot;을 분석 할 수있다 : 4 % 파라 포름 알데히드에 짧은 시간 동안 조직을 수정하고 아가 4 % 또는 젤라틴에 포함lbumin 후 vibratome 형 장치 부분 (그림 3). 더 자세한 면역 분석 (그림 4), 10 설치 매체 (조직 테크)에 포함 30 % 자당 용액에 뇌를 극저온 보호는 그라 이오 스탯에 20 μm의 섹션을 잘라. </li></ol>

<ol>
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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

마취 소개 : 시상 하부에 자궁의 electroporation 기술적으로 험난 할과 긴 마취 시간을 필요로 할 수 있기 때문에, 우리는 산소와 isoflurane을의 혼합물의 관리를 통해 마취를 유도하고 유지하는 것을 선호합니다. 우리의 경험에서, 동물, 적어도 어머니의 회복이 매우 빠르고, 배아 생존율이 향상 1 시간까지의 기간 동안이 방법으로 적절한 마취 남아있을 수 있습니다. 마취 다른 방법도 사?...

배아 마우스 뇌의 유전자 조작은 개발 규제에 대해 배울 수있는 바람직한 방법입니다. 돌연변이 마우스 라인의 세대는 그러나 느리고 비싸다. 포유류의 뇌의 뉴런을 개발 특정 유전자 변화를 소개하는 하나의 강력한 방법은 자궁의 electroporation에 있습니다. 기본적으로, 기술은 그 배아는 일정 시간 동안 생존 뇌를 수집하고 가능한 한 소설, 정보 표현형을 위해 그 (것)들을 검사 할 수있는 전기 펄스에 의해 배아 뇌 neuroepithelium에 DNA를 형질로 구성되어 있습니다. 이러한 방법으로, 실험 마우스 돌연변이의 생산에 필요한 긴 대기 기간없이 즉시 가설을 테스트 할 수 있습니다. 개발 배아에 DNA의 형질 전환 병아리 배아 1 비켜 electroporation을에 시작했다. 마우스 증명 (proof-of-concept) 필수는 문화에 수행 된 <s2&gt;까지. 이것은 곧 자궁 3,4에서 마우스 기술의 첫 번째 설명으로 이어졌습니다. 주요 문제는 그 (것)들 또는 어머니를 죽이지 않고 자궁에서 개발 태아의 두뇌를 형질하는 것입니다. (개복술, 사출, 일렉트로) 필요한 수술을 배우는 것은 긴 훈련 기간이 필요합니다. 수술은 태아의 생존 비율이 허용되는 지점을 마스터하고 나면, 그 다음 중요한 질문은 뇌 구조에 액세스 할 수있는? 당연히 자궁의 electroporation에서 얻은 결과를 보여주는 첫 번째 발표 논문 대뇌 피질의 개발 5-9에 집중했다. 수술에 가장 접근 개발에 마우스 뇌의 영역은 피질 (그림 1)이기 때문에 이것은이 기술을 사용하여 출판물의 대부분을 여전히 사실이다. 피질에 자궁의 electroporation에 대한 절차는 설명했습니다인쇄 10 비디오 11~14인치 기술의 수정은 telencephalon의 복부 부분 기저 신경절 15를 대상으로 할 수 있습니다. telencephalon 이상, 간뇌는 (고전 시상과 시상 하부로 분할)에 도달하기 어려운 전뇌의 영역이다. 그 지느러미와 가장 접근 부분 시상 16-19의 대상 논문 보고서의 작은 숫자입니다. 시상 하부, 전뇌의 대부분의 복부 부분 때문에 하나의 지느러미 표면 (피질) (그림 1) 가장 깊이 지역화. 이 지역은 유전자 자궁에서 마우스 뇌를 조작하기 위해 최선을 다하고 연구자를위한 어려운 과제로 남아. 우리의 지식에, 극소수의 기사는 마우스의 시상 하부 20,21으로 자궁 형질에서의 결과를보고합니다. 그러나 시상 하부 canno의 기능적 중요성그것은, 짝짓기, 번식 및 양육 22 먹고 마시는 같은 행동을 조절 때문에 t은 아무리 강조해도 지나치지. 또한, 시상 하부 개발의 변경은 나중에 비만, 고혈압, 당뇨병 및 조숙 한 사춘기 23과 같은 생활 조건의 발생에 기여한다. 개발하는 동안 유전자 시상 하부를 변경할 수 있다는 것은 이해하기 위해 매우 강력한 도구를 제공 할 것입니다. 우리가 사용하는 임신 한 쥐의 개복 수술에 대한 기본적인 수술 프로토콜은 다른 프로토콜 11,13,14,24에서 사용하는 것과 비슷합니다. 우리는 완전성에 대해 간략히을 설명합니다. 우리의 절차의 핵심, 다른 한편으로는, 마취의 종류, 주입 위치, 전극의 종류와 삽입 및 태아의 머리에 대하여 긍정적 인 전극의 위치입니다. 전자는 대한 수 있기 때문에 우리는 간단한 복강 내 마취를 통해 가스 흡입 마취를 유도하고 유지하는 것을 선호마취의 다소 긴 기간은 어려운 수술이 필요합니다. 마취에서 빨리 회복 isoflurane을 흡입 결과, 일반적으로 어머니가 이미 수술 후 분 정상 동작을 보여줍니다 때문이다. 유리 micropipette를 가진 DNA 용액의 주입 쉬운 점은 그러나 시상 하부 electroporation을 위해 완전하게 적합하지 않은 측면 뇌실입니다. 직접 뇌실에 주입 참으로 깊은 diencephalic 구조를 대상으로 중요합니다. 이것은 표준, off-the-shelf 전극 E12.0 또는 E12.5에서 시상 하부 형질 할 수 있습니다. 우리는 특히이 목적에 적합 NEPA 유전자 (치바, 일본)에 의해 제조 된 전극의 일부를 발견했다. 우리의 절차를 우리는 전체 시상 하부 neuroepithelium 또는 전극 방향에 따라 부분, 지역 형질의 형질을 구하십시오. 여기에서 우리는 틀림없이, mammillary 몸 형질하여 기술을 보여깊은 모든 시상 하부 핵의 가장 오목한. 또한, 우리는 해상도 세포 수준에 형질 전환 세포의 자세한 조직 학적 분석을 보여줍니다. 자궁에서 마우스 두뇌 개발을 형질에 대한 다른 접근 방법 자궁의 electroporation에서의 비교 토론 섹션에서 찾을 수 있습니다.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Sanofi GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>HDG9623</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>Pharma GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Fluka</td>
			<td>44715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>&nbsp;</td>
			<td>non-steroidal anti-inflammatory</td>
		</tr>
		<tr>
			<td>Braunoderm</td>
			<td>Braun</td>
			<td>3887138</td>
			<td>povidone-iodine</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temgesic (buprenorphine)</td>
			<td>Essex Pharma</td>
			<td>&nbsp;</td>
			<td>opioid analgesic</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Pan-Ophtal</td>
			<td>7136926</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Anaesthetic Device Komesaroff Mark-5</td>
			<td>Medical Developments Australia</td>
			<td>ACN 004 903 682</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillary puller P-97</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compresstome</td>
			<td>Precisionary Instr.</td>
			<td>VF-300</td>
			<td>Vibratome-type device</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY21EDIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrode 1</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY550-10</td>
			<td>Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.</td>
		</tr>
		<tr>
			<td>Electrode 2</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY700P4L</td>
			<td>Cover Round Platinum Plate 4 mm diameter</td>
		</tr>
		<tr>
			<td>Fiberoptic cold light source</td>
			<td>Leica</td>
			<td>KL2500 LCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC120T-15</td>
			<td>1. 2 mm O.D. x 0. 94 mm I.D.</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>FST250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Harvard Apparatus</td>
			<td>py872-5272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Injection device</td>
			<td>World Precision Instruments</td>
			<td>Pneumatic Pico Pump PV820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Suture Thread Coated Vicryl</td>
			<td>Ethicon</td>
			<td>V4914</td>
			<td>Peritoneal Suture</td>
		</tr>
		<tr>
			<td>Suture Thr. Supramid</td>
			<td>Serag Wiessner</td>
			<td>TO07171L</td>
			<td>Skin Suture</td>
		</tr>
	</tbody>
</table>

genetic manipulation of the mouse developing hypothalamus through in utero electroporation

개발 포유류 뇌의 특정 영역의 유전자 변형은 매우 강력한 실험적인 방법입니다. 그러나 새로운 마우스 돌연변이를 생성하는 것은 종종 절망적 느립니다. 그것은 합리적인 포스트 치료실 생존 자궁에서 발전 마우스 뇌에 액세스 할 수 있습니다 것으로 나타났습니다. 하지만이 절차의 결과가 두뇌 개발의 가장 피상적 쉽게 접근 할 부분에 거의 독점적으로보고되었습니다 피질 즉. 시상, 좁은보다 중간 지역은, 대상에 대한 더 어려운 입증되었습니다. 특히 깊은 핵, 시상 하부의 사람들로 형질은 가장 도전하기 때문에 거의 결과가보고되었다 아마도. 여기에서 우리는 전체 시상 하부 neuroepithelium 또는 electroporation을 통해 형질 전환을위한 그것의 일부를 (시상 하부 지역) 대상으로하는 절차를 보여줍니다. 우리의 접근에 키는 t의 긴 마취 시간, 주입하다HIRD 뇌실, 적절한 종류와 전극의 위치. 또한, 우리는 대상과 가장 오목한 시상 하부 핵, mammillary 몸의 후속 조직 학적 분석 결과를 보여줍니다.

대부분의 시상 하부 뉴런은 다소 짧아 마우스 개발 27,28로 번역 쥐 26 출생 연대 분석에 따르면, E11.5 사이 E15.2에 태어난다. 시상 하부 신경의 피크 E12.5 29-31에 도달합니다. 따라서, 본 연구 (E12.5)에 대해 선택된 형질의 나이에, 시상 하부 뉴런의 큰 비율은 주어진 rostro - 꼬리 수준에서 표시 할 수 있습니다. 두꺼운 vibratome 유형 섹션에 E18.5에서 분석 <stro...

Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

시상 하부의 기능 및 의료 중요성에도 불구하고,<em&gt; 자궁 내</em개발의&gt; 유전자 조작은 거의 시도되지 않았습니다. 우리는 당신을위한 자세한 절차를 보여<em&gt; 자궁 내</em마우스 시상 하부 총과 부분 (지역) 시상 하부 형질의 쇼 대표적인 결과로&gt; 일렉트로.

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 ...

genetic-manipulation-mouse-developing-hypothalamus-through-utero

Research

JoVE Journal

Neuroscience

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

15.7K 조회수.  University of Heidelberg. 시상 하부의 기능 및 의료 중요성에도 불구하고, 자궁 내 유전자 조작은 거의 시도되지 않았습니다. 우리는 당신을위한 자세한 절차를 보여 자궁 내 일렉트로.

비디오: Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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 ...

연구

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 ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

소뇌 과립 신경 세포의 유전자 조작<em&gt; 체외</em&gt;와<em&gt; 생체</em&gt; 신경 세포의 형태와 마이그레이션을 연구하는

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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, ...

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

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

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

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

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

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

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

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

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

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

Cre lox 재결합 Electroporation Utero에 통해 마우스 대뇌 피 질에서 유도

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

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

신생아 마우스 두뇌의 Striatal 세포에서 유전자 조작에 대 한 stereotaxic 수술

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...