We would like to acknowledge David O'Brien for his support with data analysis, and all the members of the Canto-Soler lab for their critical discussions. This work was supported by NIH grants EY004859 and EY022631 (MVCS), Core Grant EY1765, and an unrestricted departmental grant from Research to Prevent Blindness, Inc.

이 작품에 설명 된 모든 절차는 존스 홉킨스 대학에서 동물 관리 및 사용위원회에서 권장하는 지침에 따라 수행 하였다. 미리 1 :기구, 시약 및 요리의 준비 <ol><li> 계란의 준비 : 계란 배양기에서 5 일간 37.5 ℃, 상대 습도 60 %에서 수정 된 백색 레그혼 닭 알을 품어. 참고 : 표준화 및 배아 발달을 동기화 도움이 될 수있는 능력을 흔들하지만, 락과 보육이 프로토콜의 결과에 대해 엄격하게 할 필요가 없습니다. </li><li> 플라스미드 준비 : <ol><li> 필요에 따라 설계 플라스미드 (1,34의 RNAi 기술을 사용하여 예를 들어) 유전자 과발현 또는 하향 조절을 위해 구축. 가능한 한 구조에서 리포터 유전자 (예 : EGFP, RFP 또는 LacZ를 등)를 포함한다. 참고 : 같은 거대 세포 바이러스와 같은 일부 일반적으로 사용되는 프로모터는, 시간이 지나면 침묵이 될 수 있습니다. CAG의 PROMOT어 (닭 베타 - 액틴 CMV 인핸서와 프로모터) 관심 35-37의 유전자를 효율적으로 장기 식을 제공하는 아주 좋은 대안이다. </li><li> 멸균 PBS 1.5 μg의 / μL의 농도에서 플라스미드 솔루션을 준비합니다. 옵션 : 시간 ​​앞서 플라스미드 솔루션을 준비하고 냉동 사용할 준비가 될 때까지 저장할 수 있습니다. 주 : 높은 플라스미드 농도를 사용하는 반면 낮은 농도는 플라스미드 트랜 스펙 션 효율을 낮추는 1, 효율의 현저한 증가를 초래하지 않는다. </li></ol></li><li> 전극 : <ol><li> 양극 사용을 위해 두꺼운 금 (특정 재료 / 장비의 표 참조) 전극을 냈습니다. 70 % 에탄올로 닦아. 주 : 금 절연 노출 약 0.5 mm를두고 전극 스쳐, 전류의 누출을 최소화하는 것이 바람직하다. 절연 재료에 대한 정보는 특정 재료의 표에서 찾아 볼 수있다. </li><li> 2.5 mm의 정사각형 B에서 음극을 확인(일반적으로 마이크로 피펫 끌어 당기는에서 사용) 4.5 mm 폭 황소 필라멘트. 집게의 도움으로, 사각형 상자를 취소하고 긴 스트립에 필라멘트를 펴. 그 후, 해부 현미경 및 가이드로자를 사용 하에서, 기지 3mm 긴 사각형 &#39;U&#39;자형으로 필라멘트를 구부리고 반대편 필라멘트의 나머지 길이 (도 1, 일측 2mm 높은 , C - D). 집게를 사용하여 96 %의 에탄올과 불꽃의 전극 소독 찍어. 그런 다음 전극의 긴 팔에 작은 악어 클립으로 전기 리드를 연결합니다. </li></ol></li><li> 전기 상공 회의소 설정 : <ol><li> 100mm 페트리 접시 사용하여 테이프 (그림 1E)에, 아래로, 평평한면을 멸균 1.5 ML의 microcentrifuge 관의 뚜껑을 절단 부착하여 전기 챔버를 준비합니다. 참고 : 일하는 것이 비슷한 크기의 잘의 형태로 다른 컨테이너; 그러나 microcentrifuge 관 덮개는 쉽게 접근하고 멸균한다. 잘플라스미드 솔루션의 낭비가 발생하는 등의 병아리의 눈과 음극 전극을 수용 할 정도로 큰,하지만 너무 큰되지해야합니다. </li></ol></li></ol> 2. 예 비켜 Electroporation에 절차 <ol><li> 악기의 제조 : <ol><li> 마이크로 해부 도구를 소독을 찍기로 (달걀 껍질을 열고 배아, 망막 색소 상피를 제거 뒤몽 핀셋을 꽂아 미세 팁 2 쌍을 떠서위한 큰 곡선 몰 로니 집게 2 쌍을 본 한 쌍은 제거 렌즈와 유리체 위해 집게를 톱니) 96 % 에탄올과 불타는에 팁. 70 % 에탄올에 담근 잎사귀를 사용하여 청소 해부 범위. 37 ° C의 물을 욕조에 멸균 칼슘 마그네슘 무료 행크의 균형 소금 솔루션 (CMF-HBSS)의 병을 따뜻하게. 따뜻한 CMF-HBSS 전체 3/4에 100mm 멸균 배양 접시를 채우십시오. </li><li> 다른 깨끗한 해부 현미경으로 전기 챔버를 놓고 플라스미드 솔루션의 120 μL로 입력합니다. , 전기 장치에 전극을 연결하고반드시 맞춤형 전극을 음극에 연결하고 금은 양극에 전극을 밀고있다. </li></ol></li><li> 배아 컬렉션 : <ol><li> 70 % 에탄올에 젖은 잎사귀와 껍질을 닦아 계란을 청소합니다. 문화에 오염을 운반 방지하기 위해 프로토콜의 나머지 부분에 걸쳐 멸균 절차를 유지한다. 큰 곡선 몰 로니의 집게를 사용하여 조심스럽게 부드럽게 (공기 실 이상) 달걀의 둥근 끝을 두드리고 다음 그립과 쉘 부분을 잡아 당겨 달걀 껍질에 구멍을 엽니 다. </li><li> 집게의 또 다른 한 쌍은 세포막을 제거하고 달걀에서 그것을 떠서하여 배아를 수집로 (조심 배아를 손상하지 말 것). CMF-HBSS와 함께 접시에 넣습니다. 주 : 2.4.4에 규정 된 전기에 대한 망막 컵의 수를 얻기 위해 필요한만큼 모아서 배아 </li><li> 중요한 단계 : 햄버거와 해밀턴 (38)에 따라 배아를 스테이지. 참고 : 최적의 효율 배아를 얻으려면단계 27 (그림 2A)에 있어야합니다. </li><li> 몰 로니의 집게를 사용 잘린 배아를 안락사. </li></ol></li><li> 망막 컵을 가져 오기 : <ol><li> 미세 뒤몽 핀셋을 사용하여 조심스럽게 눈을 명백히하다 새로운 CMF-HBSS 접시에 전송할 수 있습니다. </li><li> 시신경 유두에 가까운 각각의 눈의 후방 부분에서 시작, 2 개의 미세 뒤몽 핀셋을 사용하여, 신경에 손상을주지에 RPE는 신중 해부 신경 망막과 망막 색소 상피 조심스럽게 사이에 핀셋의 각 쌍 중 하나 팁을 소개합니다 망막 (그림 2B). 참고 : RPE 도움이 될 것 용액에서 칼슘과 마그네슘 2+의 부족은 더 쉽게 신경 망막에서 분리합니다. </li></ol></li><li> 전기 : <ol><li> 15 볼트, 50 밀리 초 지속 시간 950 밀리 초 간격의 5 제곱 펄스를 제공하기 위해 electroporator의 매개 변수를 설정합니다. 장소 &#39;U&#39;는 전기 챔버 (마이크로 원심 뚜껑 채우기 내부에 음극 모양플라스미드 함유 용액 120 μL)와 ED. </li><li> 사용 만곡 듀 핀셋 (아닌 떠서 누름) 일렉트로 포 레이션 챔버 한 망막 컵을 전송하고, 상기 전극의 아래쪽 및 위쪽으로 향하게 렌즈쪽으로 망막의 후방 부와, &#39;U&#39;형상의 전극 내부에서 배치 ( 그림 2D). </li><li> 그 다음 렌즈로, 눈의 앞쪽 부분을 터치하고 electroporator의 발 페달이 전기 펄스를 제공하는 사용하도록 양극을 놓습니다. </li><li> CMF-HBSS로 새로운 배양 접시에 전기 천공 망막 컵을 전송하고 나머지 망막 컵의 각각 전기의 절차를 반복합니다. 주 : 6 망막 컵까지는 형질 전환 효율이 크게 감소하지 않고 동일한 플라스미드 용액으로 전기 천공 할 수있다. </li><li> 본의 톱니 포셉으로 그들을 그립 부드럽게 T를 통해 당겨 각 일렉트로 망막 컵의 렌즈와 유리체를 제거곡선 뒤몽 핀셋의 다시 제자리에 눈을 유지하면서 그는 개방 학생. </li><li> 표준 절차에 따라 일렉트릭 망막 세포의 분리 및 배양을 진행합니다. 참고 : 병아리 망막 세포의 분리 및 문화에 대한 자세한 프로토콜의 벌 가라과 칸토 - 솔러, 2012 년 추가 파일 4 (21)을 참조하십시오이 프로토콜에서 세포가 폴리 오르니 틴 코팅 6 웰 플레이트 또는 35 낮은 밀도에서 씨앗을 품고있다. -mm 요리 (6 × 10 5 세포 / 35mm 직경의 우물 또는 접시). 사용 된 배지는 페니실린 / 글루타민, 10 % 소 태아 혈청 (FCS), 100 μg의 / ㎖에서 리놀산-BSA (199)와 매체이다. 문화는 5 % CO 2와 가습 37 ° C 배양기에서 유지된다. 이 과정을 사용하여 - 5 6 × 106 세포는 일반적으로 각 단계마다 27 일렉트로 눈을 얻을 수있다. 참고 : 이상적으로, 더 이상 3 시간이 배아 수집 및 세포 도금 사이를 통과한다, 좋은 세포 생존을 보장하기 위해스트레스를 최소화 할 수 있습니다. 관찰과 추가 분석을 위해 일반적인 타이밍이 시점에서 세포가 좋은 형태 학적 및 분자 차별화를 달성 생존이 여전히 높기 때문에, 문화 4 일 후에입니다. </li></ol></li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+primary+central+and+peripheral+nervous+system+neurons+by+electroporation.">Transfection of primary central and peripheral nervous system neurons by electroporation.</a> Methods Cell Biol. 71, 339-351 (2003).</li><li>Boatright, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+5'+flanking+regions+of+IRBP+and+arrestin+have+promoter+activity+in+primary+embryonic+chicken+retina+cell+cultures.">The 5' flanking regions of IRBP and arrestin have promoter activity in primary embryonic chicken retina cell cultures.</a> Exp Eye Res. 64 (2), 269-277 (1997).</li><li>Dudek, H., Ghosh, A., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+phosphate+transfection+of+DNA+into+neurons+in+primary+culture.">Calcium phosphate transfection of DNA into neurons in primary culture.</a> Curr Protoc Neurosci. Chapter 3, Unit 3.11 (2001).</li><li>Kumar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bZIP+transcription+factor+Nrl+stimulates+rhodopsin+promoter+activity+in+primary+retinal+cell+cultures.">The bZIP transcription factor Nrl stimulates rhodopsin promoter activity in primary retinal cell cultures.</a> J Biol Chem. 271 (47), 29612-29618 (1996).</li><li>Toy, J., Bradford, R. L., Adler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-mediated+gene+transfection+into+chick+embryo+retinal+cells+in+ovo+and+in+vitro.">Lipid-mediated gene transfection into chick embryo retinal cells in ovo and in vitro.</a> J Neurosci Methods. 104 (1), 1-8 (2000).</li><li>Werner, M., Madreperla, S., Lieberman, P., Adler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+transfected+genes+by+differentiated,+postmitotic+neurons+and+photoreceptors+in+primary+cell+cultures.">Expression of transfected genes by differentiated, postmitotic neurons and photoreceptors in primary cell cultures.</a> J Neurosci Res. 25 (1), 50-57 (1990).</li><li>Yuan, L., Kawada, M., Havlioglu, N., Tang, H., Wu, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+PRPF31+inhibit+pre-mRNA+splicing+of+rhodopsin+gene+and+cause+apoptosis+of+retinal+cells.">Mutations in PRPF31 inhibit pre-mRNA splicing of rhodopsin gene and cause apoptosis of retinal cells.</a> J Neurosci. 25 (3), 748-757 (2005).</li><li>Hughes, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RCAS+vector+system.">The RCAS vector system.</a> Folia Biol (Praha). 50 (3-4), 107-119 (2004).</li><li>Das, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+system+for+RNA+interference+in+the+chicken+using+a+modified+microRNA+operon.">A robust system for RNA interference in the chicken using a modified microRNA operon.</a> Dev Biol. 294 (2), 554-563 (2006).</li><li>Nguyen, A. T., Dow, A. C., Kupiec-Weglinski, J., Busuttil, R. W., Lipshutz, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+gene+promoters+for+liver+expression+by+hydrodynamic+gene+transfer.">Evaluation of gene promoters for liver expression by hydrodynamic gene transfer.</a> J Surg Res. 148 (1), 60-66 (2008).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice.">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li><li>You, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+promoter-containing+vector+for+direct+cloning+and+enhanced+expression+of+PCR-amplified+ORFs+in+mammalian+cells.">A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells.</a> Mol Biol Rep. 37 (6), 2757-2765 (2009).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Dev Dyn. 195 (4), 231-272 (1992).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

이 프로토콜의 성공을위한 가장 중요한 단계는 배아의 적절한 단계를 선택하는 것입니다. 이전의 출판물에서, 배아 단계의 범위는 일반적으로 배양 또는 배아 일의 일 (ED)에 의해 정의 된, 이러한 문화에 대한 주어진다; 따라서 보통 ED 6 배아 ED 5를 사용하여 동등한 결과를 얻을 수 있다고 가정한다. 그러나 우리는 전술 한 바와 같이 단계 (27) (ED 5)에 형질 전환 효율, 전체 세포 인구의 약 22 %가 될 ?...

배아 병아리 망막에서 해리 된 세포 배양 널리 생존 2-9, 차별화 10-12, 신경 돌기의 파생물 (13) 등을 포함한 광 수용체 세포 생물학의 다양한 측면을 연구하는 데 사용되었다. 루벤 애들러와 공동 작업자에 의해 1980 년대에 개발하고 자신과 다른 그룹 (14-20)에 의해 완성이 시스템의 장점은, 동물 모델 (21)와 여자의 고유 특성에 있습니다. 심지어 배아 단계에서 병아리 눈의 큰 크기는, 문화에 대한 자료의 많은 양을 제공한다. 배아는 배양 일 (ED) 5 이용하여 수행 될 때 더욱이, - 6 망막, 55 -이 동물 감광체의 약 86 %이기 때문에 그들의 선조 세포의 80 %가 감광체 14,15,18,22,23으로 구별하고, 콘 (24)은,이 배양 물이 세포 유형에 집중 연구에 특히 적합하다. 우리는 최근 develo의이PED는 ​​따라서 유전 missexpression 연구 1 촉진함으로써이 시스템의 유용성을 넓히고,이 배양 물에서 세포의 고효율 플라스미드 형질 감염을 허용하는 간단한 기술을 특징으로한다. 이 기술의 개발은 세포 자율 방식으로 유전자 기능 연구를 허용하도록 형질의 허용 가능한 수준을 제공 할 방법 과학 문헌에서 보이드로부터 비롯. 일차 배양 신경 세포를 형질 25,26 악명 하드 때문 부분이다. 이 목적을 위해 이전에 사용 가능한 가장 일반적으로 사용되는 기술 중 일부는 3-5 % 정도의 효율을 야기하고 상당한 독성을 발휘 27-32 예컨대 리포 펙션, 칼슘 포스페이트 - 매개 된 형질 전환과 같은 화학적 형질 전환 방법을 포함. 효소 리포터 플라스미드 시스템의 사용은 신호를 증폭하여 불량한 형질 감염 효율의 문제를 피할 수있다하더라도, 그들은하지셀 고유의 효과를 판별하고, 그 결과 전체를 대표하지 않을 작은 세포군에 기초한다. 병아리에서 널리 사용되는 또 다른 방법은, RCA 단자 바이러스 감염은 세포 증식에만 적용이 망막 일차 배양 시스템 (33), 따라서 적합하지 않다. 현재 프로토콜에서 배아 병아리 눈 단계 27 (ED 5), 망막 색소 상피 (RPE)를 제거하고, 망막 컵이 플라스미드를 함유 용액으로 가득 전기 챔버에 배치에서 적출 및 사용하여 전기 천공되는 주문 제작 표준 기술 (21)을 이용하여 망막 해리와 문화를 이어 전극. 이 절차를 최적화 한 결과의 생존과 분화 특성을 손상시키지 않고 지속적으로 배양 세포 단독 감광체 인구 내의 25 %의 총 수의 22 % 정도의 형질 전환 효율을 달성 할 수 있었다문화 1. 여기에서 우리는이 기술의 성공과 재현성을 보장하기 위해이 절차의 모든 중요한 단계를 요약 한 상세한 프로토콜을 제공합니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ECM 830 Electro Square Porator</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr>
			<td>Genetrode, L-Shaped, 5 mm Gold Tip</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0115 model 512</td>
			<td>Gold tipped electrode used as anode</td>
		</tr>
		<tr>
			<td>Polyimide Tubing</td>
			<td>Vention Medical</td>
			<td>custom made</td>
			<td>Internal Diameter: 0.5mm / wall thickness: 0.15-0.2mm. Used for insulating gold tiped electrode</td>
		</tr>
		<tr>
			<td>2.5mm square box filament, 4.5mm wide</td>
			<td>Sutter Instrument Company</td>
			<td>FB245B</td>
			<td>Used to make cathode electrode</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Gibco - Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Moloney forceps</td>
			<td>Roboz</td>
			<td>RS-8254</td>
			<td>Serrated; Slight Curve; 4.5&#34; Length</td>
		</tr>
		<tr>
			<td>Dumont tweezers 5/45</td>
			<td>Roboz</td>
			<td>RS-5058</td>
			<td>Pattern #5, 45 Degree Angle; .10 X .06mm Tip Size; 109mm Length</td>
		</tr>
		<tr>
			<td>Bonn micro forceps, 1x2 teeth</td>
			<td>Roboz</td>
			<td>RS-5172</td>
			<td>Tying Platform; 1X2 Teeth, 0.12mm Teeth; 3.75&#34; Length; .3mm Tip Width</td>
		</tr>
	</tbody>
</table>

efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach

배아 병아리 망막에서 파생 된 콘 광 수용체가 풍부한 문화 망막 신경 세포, 특히 광 수용체의 생물학을 공부하는 전세계 연구자를위한 필수적인 도구가되었다. 그들은 쉽게 약물 개발을위한 높은 처리량의 기술에 적용 할 수 있으므로이 시스템의 응용 프로그램은 기초​​ 연구를 넘어. 그러나 이러한 문화 망막 광 수용체의 유전자 조작은 시스템의 유용성에 중요한 제한 포즈, 매우 어려운 것으로 입증되었습니다. 우리는 최근에 개발 및 기타 현재 사용 가능한 프로토콜 (1)에 비해 5 배 이러한 문화 망막 세포의 형질 전환의 속도를 증가 플라스미드 전기 기술 비켜 전을 검증했다. 이 방법에서 병아리 배아 눈 RPE를 제거하고, 스테이지 (27)에서 적출하고, 망막 컵 플라스미드 - 함유 용액에 배치되어 쉽게 구성된 고객이나 전기 천공을 사용하여만든 전극. 망막은 해리 배양 표준 절차를 사용하고 있습니다. 이 기술은 일반적으로 감광체 인구의 25 %의 유전자 발현을 달성 플라스미드 기반의 RNAi 기술의 사용을 통해, 예를 들면, 과발현 연구뿐만 아니라 유전자 발현의 하향 조절에 적용될 수있다. 본 공보의 비디오 포맷은 망막 일차 배양에서의 유전자 기능 연구를 가능 분야의 연구자에이 기술이 용이하게 접근 할 수 있도록한다. 우리는 또한 성공적인 결과 및 재현성이 절차의 중요한 단계에 대한 자세한 설명을 포함하고있다.

여기에서 우리는 다음 해리 세포 배양을위한 탈핵 병아리 망막 컵에 플라스미드 형질에 대한 간단한 프로토콜을 제시한다. 형질 사용자 정의 전극 (- 2 그림 1)을 쉽게 만들 수 사용 전기에 의해 달성된다. 이 프로토콜에 기재된 파라미터는 20 % 내지 27 (도 3d) (22 %의 평균) 레인지에 형질 전환 효율을 얻기 위해 최적화되었다. 위에서 언급 한 이유로, 이러한 결과는 문화 4 일...

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Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

배아 병아리 망막 세포 배양은 광 수용체 생물학의 연구에 유용한 도구를 구성한다. 우리는 이전에 배양 망막의 플라스미드 일렉트로 오보 EX에 기초한 효율적인 유전자 전달 기술을 개발했다. 이 기술은 상당히이 시스템에 대한 가능한 유전자 조작을하고, 현재 사용 가능한 프로토콜을 통해 형질 전환 효율을 증가시킨다.

차 세포 배양 연구 병아리 망막에 효율적인 유전자 전송 :<em&gt; 전 비켜</em&gt; Electroporation에 접근

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

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Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

8.1K 조회수.  The Johns Hopkins University School of Medicine.  배아 병아리 망막 세포 배양은 광 수용체 생물학의 연구에 유용한 도구를 구성한다. 우리는 이전에 배양 망막의 플라스미드 일렉트로 오보 EX에 기초한 효율적인 유전자 전달 기술을 개발했다. 이 기술은 상당히이 시스템에 대한 가능한 유전자 조작을하고, 현재 사용 가능한 프로토콜을 통해 형질 전환 효율을 증가시킨다. 

비디오: Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

최적화<em&gt; 전 비켜</em&gt; 개발의 고급 단계에 병아리 배아의 배양

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

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Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Electroporation에 의한 배아 마우스 달팽이의 Explants의 문화 유전자 전송

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

공동 문화 시스템에 신경 연결의 형성을 평가하기위한 Optogenetic 접근

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

앞으로 유전 접근은 마우스의 스트레스 저항성 유전자를 밝히기 위해서 - ES 세포에서 높은 처리량 화면

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

예 생체 병아리 소뇌 조각의 문화가 공간적으로 Electroporation에 과립 세포 전구체의 표적

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

치킨 청각 기관에 유전자 전달에 의한<em&gt; 비켜에서</em&gt; 마이크로 전기

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

소설<em&gt; 예 비켜</em&gt; 밴딩 기법 닭 배아 시스템에서 심장 내 혈류를 변경할

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

장기를위한 잠수 필터 종이 샌드위치<em&gt; 예 비켜</em초기 병아리 태아의&gt; 시간 경과 영상

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR 중재 손실 함수 분석 소 뇌과 립 세포를 사용 하 여 Utero에서 Electroporation 기반 유전자 이동

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

조류 내이 발달을 연구하는 Ovo 및 Ex Ovo 방법에서

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...