We are grateful to Gurdon laboratory members for useful discussion. K.M. is a Research Fellow at Wolfson College and is supported by the Herchel Smith Postdoctoral Fellowship and the Great Britain Sasakawa Foundation. Gurdon laboratory is supported by grants from the Wellcome Trust (RG69899) and MRC to J.B.G.

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The authors have nothing to disclose.

우리는 여기에 세부 산모의 요인을 고갈하거나 수정하기 전에 외생 적 요인을 과발현하는 새로운 방법. 이 시스템은 두 microinjections 필요하지만, 대신에 호스트 전송 방법에 사용 7,17 개구리 수술을 건너 뛴다. 그것은 동물 보호의 관점에서 개구리 수술 단계를 제거하는 최적이다. 또한, 우리는 우리가 실험의 성공에 영향을 미치는 하나의 생물학적 요인을 제거 할 수 있다는 것을 의미, 전송 실험에 대한 호스트 여성 개구리의 품질을 고려 할 필요가 없습니다. 따라서이 시스템에 PMSG 감작 개구리에서 얻은 난자의 질이 성공적으로 실험을위한 핵심 주요 생물학적 요인이다. 난자의 품질은 난소 또는 후 defolliculation의 수집 후 판단 할 수있다. 어떤 이상이 이러한 단계에서 관찰되는 경우, 새로운 개구리에서 다른 난소를 수집하는 최적이다. 당신이 난자의 품질을 확인하는 또 다른 점은 난자의 성숙 후입니다. progesteron의 경우 80 % 미만전자 처리 된 난자가 성숙의 징후를 보여, 당신은 더 분석을위한 배아의 충분한 수를 얻을 수없는 경우가 있습니다. 효소 defolliculation 대​​신 수동 defolliculation의 사용은 또한 defolliculation 필요한 시간을 삭감하는 본 시스템의 또 다른 장점이다. 그러나, 수동 defolliculation 효소 처리가보다 나은 개발을 줄 수 있다는 것을 여전히 가능하다. 도 3에 도시 된 바와 같이, 시험 관내에서의 거의 10 %가 수영 올챙이 스테이지에 도달 할 수 난자 성숙. 이 자료는 8 및 새로운 주입 실험에서보고 된 13 개의 독립적 인 실험에서 수집됩니다. 호스트 전사 방법은 전사 난자 30-60 %의 neurula 스테이지 (17)에 도달 할 수 있기 때문에 의미있는 데이터를 얻기위한 각 난자 75-150 치료를 필요로한다. 우리의 방법은 일반적으로 절단 배아의 약 40-60%가 도달 할 수 있기 때문에 각 실험군에서 200 ~ 300 난자로 시작근육 응답 무대. 이러한 데이터는 두 가지 방법이 합리적인 개발 속도를 지원하는 것이 좋습니다. 우리는 지금까지이 방법은 변형을 통해 개발을 지원하는 것을 나타내는,이 기술을 사용하여 제어 mRNA의 주입 및 제어 안티센스 올리고 주입 된 난자에서 10 살아있는 개구리를 얻었다. 우리 난자 조작-ICSI 방법 곧 수정 후, 초기 배아 발달 동안 모체 인자의 역할을 테스트 할 수있는 기회를 제공한다. 또한, 수정 전에 염색질 수정 인자의 과발현은 가능한 개발에 필요한 산모의 염색질 상태를 이해하기위한 수정하기 전에 산모의 염색질을 개조 할 수 있습니다. 이 전략은 또한 이들도 수정하기 전에 표현 될 수 있기 때문에 전사 활성제와 같은 이펙터 클레아 제 (TALEN) 18, 19 및 CRISPR / CAS9 (20, 21)로 현재 유전자 편집 시스템과 잘 작동 할 수 있습니다. 따라서, 우리의 새로운 접근 방식은 potenti가알은 미래 많은 응용에 사용될 수있다.

아프리카 발톱 개구리 laevis의 개발 1 연구 널리 사용되는 강력한 모델 생물이다. 제노 푸스 달걀 풍부한 (포유 동물 대응 물에 비해 약 0.1 mm 인에 약 1.2-1.4 mm) 비정상적으로 큰 때문이다. 계란 (4,000-8,000 세포 단계 8-8.5에서 발생, MBT) 중간 포배 전이 될 때까지 배아 발달을 구동하기에 충분하다 모계 합성 및 저장 구성 요소가 포함되어 있습니다. MBT이어서 발전 지시 배아 유전자 산물을 생산 접합체 게놈 활성화를 수반한다. 많은 연구 개발을위한 중요한 산모 요인을 파악하는 것을 목표로. 많은 연구는 단백질의 경우 모계 저하 스테이지 낭배 2-4에서 관찰 될 수있는 수정 된 배아에 모르 폴리 노 올리고 뉴클레오티드를 포함 안티센스 올리고 뉴클레오티드 (올리고)의 주입에 의존한다. 대안 적으로,이 수정 된 mRNA를 주입하여 EMbryos 유전자 기능을 방해하거나 과발현 단백질의 운명을 추적. 그러나, 하나의 세포 단계 배아 내로 주입 정상적으로 MBT 전에 초기 배아 단계에서 모체 단백질의 발현 수준에 영향을 미치지 않는다. 와일리 Heasman 및 5는이 문제를 극복하기 위해 호스트 전송 방법을 확립. 이들 방법에서, 수동 defolliculated 난자 안티센스 올리고으로 주입되고, 호스트 (6)에 전송 암컷. 초기 배아 발달의 하향 조절 단백질의 역할을 조사 할 수 있도록 단백질을 하향 조절되기 전에 수정된다. 7에서 평가로서이 모체 공핍있어서, 모체 단백질의 몇몇 독특한 역할 발달 식별되었다. 수정 이전의 mRNA의 모성 결핍 또는 과잉 발현이 매뉴얼 defolliculation 및 호스트 전송없이 달성되는이 보고서, 우리는 세부 사항 우리의 최근에 개발 된 방법에서,8. 수동 defolliculation 시간과 호스트의 전송은 종종 많은 따라서 임산부 공핍 방법의 빈번한 사용을 방해하는, 숙련 된 기술과 동물의 수술 별 라이선스를 필요로 요구한다. Defolliculation 및 호스트 전송은 각각 효소 defolliculation 9, 10 및 세포질 내 정자 주입 (ICSI) 11 ~ 13로 치환된다. 계란 ICSI 원래 형질 개구리 (12)를 제조 하였다. 정자와 배아 핵은 체외 성숙 양서류의 난자 11,14,15에 이식했다. 여기, 우리가 있었던 체외 성숙 난자에 정자를 주입하는 단계별 방법을 보여 안티센스 올리고 또는 ​​mRNA의 사전을 주입.

<table border="0" cellpadding="0" cellspacing="0" width="589">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:87px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pregnant Mare Serum Gonadotropin (PMSG)&#160;</td>
			<td style="width:87px;">MSD Animal Health</td>
			<td style="width:119px;">Vm 01708/4309</td>
			<td>PMSG-Intervet 5000iu powder and solvent for solution for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate salt (MS222)</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>For anesthesia</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Liberase TM Research Grade</td>
			<td style="width:87px;">Roche</td>
			<td style="width:119px;">05 401 127 001</td>
			<td>For oocyte defolliculation. Store at -20oC</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:216px;">Drummond Nanoject</td>
			<td style="width:87px;">Drummond Scientific Company</td>
			<td style="width:119px;">3-000-205/206</td>
			<td>For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">glass capillary&#160;</td>
			<td style="width:87px;">Alpha laboratories</td>
			<td style="width:119px;">7&#8221; Drummond #3-000-203-G/XL</td>
			<td>For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette Puller&#160;</td>
			<td style="width:87px;">SUTTER Instrument</td>
			<td style="width:119px;">Model D-97</td>
			<td>For microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraPure Agarose</td>
			<td style="width:87px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td>For invitro maturation and ICSI</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">14 ml ultra-clear centrifuge tube&#160;</td>
			<td style="width:87px;">Beckman Coulter United Kingdom Ltd</td>
			<td style="width:119px;">344060</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">OptiPrep Density Gradient Medium (Iodixanol)</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D1556</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digitonin</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D141</td>
			<td>Cell permeabilization reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaker</td>
			<td style="width:87px;">Hybaid&#160;</td>
			<td style="width:119px;">HB-SHK-1</td>
			<td>For oocyte defolliculation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope (Stereo zoom microscope)</td>
			<td style="width:87px;">ZEISS</td>
			<td style="width:119px;">Semi SV6</td>
			<td>For oocyte collection and microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 &#956;m pore filter</td>
			<td style="width:87px;">CellTrics</td>
			<td style="width:119px;">04-0042-2317</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultracentrifuge</td>
			<td style="width:87px;">Beckman Coulter</td>
			<td style="width:119px;">Optima L-100XP</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50 ml centrifuge tube</td>
			<td style="width:87px;">Cellstar Greiner Bio-One</td>
			<td style="width:119px;">227261 or 210261</td>
			<td>Oocyte collection and defolliculation reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml centrifuge tube</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">352097</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">90 mm Petri dish&#160;</td>
			<td style="width:87px;">Thermo Scientific</td>
			<td style="width:119px;">101VR20/C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easy-Grip Cell Culture Dish, 60x15 mm</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">353004</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easy-Grip Cell Culture Dish, 35x15 mm</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">351008</td>
			<td></td>
		</tr>
	</tbody>
</table>

manipulation and in vitro maturation of xenopus laevis oocytes, followed by intracytoplasmic sperm injection, to study embryonic development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

체외 성숙 난자에 사용 ICSI 배아의 배아 개발 (그림 3A)를 조사 하였다. MII 단계에 GV 난자의 성숙 속도는 가변적 크게 난자의 품질에 따라 달라집니다. 좋은 실험에서, GV 난자의 거의 100 %가 마침내 MII 단계에서 계란되고, 난자의 성숙의 프로게스테론과 쇼 징후에 응답합니다. 모든 성숙 된 난자는 ICSI를 실시하고 주입 절단 계란의 약 25 % (그림 3A, N = 제어 (13)는 올리고 주입 난 모세포를 스크램블 N = 더 올리고 주입 난 모세포 7) 하였다. 약 60 % 또는 제어 올리고 주입 난 모세포 또는 비 - 주입 난 모세포로부터 생성 된 절단 배아의 80 %는 각각, 포배 / 스테이지 낭배 도달. 포배 / 낭배 배아의 82 %를 n에 좋은 품질의했다 반면 포배 / 낭 배기 배아 중 올리고 주입 시료에서 배아의 약 절반 (이상 분열 및 세포 사멸의 거의 흔적) 좋은 품질했다에 주입 된 샘플 (도 3A). 비 주입 시료에서 60 % 및 절단 배아의 29 %는 (그림 3A)를 한 동안 마지막으로, 41 % 및 올리고 주입 시료에서 절단 배아의 11 %는 각각 근육 반응과 수영 올챙이 단계에 도달했다. 이 ICSI 배아는 정상 및 비정상 배아 (그림 3B)의 혼합물이다. 그들 중 일부는 변형을 거쳐 개구리 8 성숙 개발한다. 이러한 결과는 IVM과 ICSI 다음 GV 난자에 안티센스 올리고 뉴클레오티드의 주입, 효율적인 초기 배아 발달을 허용하는 것이 좋습니다. 안티센스 올리고 자체의 주입 배아 개발을 감소하지만, 우리는 여전히 성장 발달 검사로 많은 실험을 위해 충분한 배아를 획득 할 수 있으며, RT-PCR, 등등 웨스턴 블롯합니다. <img alt="그림 1" src="/files/ftp_upload/52496/52496fig1highres.jpg" /> 무화과<html> 우레 1 : 아프리카 발톱 개구리의 전형적인 예는 체외 성숙 실험을위한 좋은 품질 또는 나쁜 품질의 난자를 laevis의. (A) 나쁜 품질 난자의 예. 예를 들어, 곳곳에 색소 침착을 보여주는 난자 후속 실험에 사용되지 않습니다. 각 Xenopus의 난자는 직경 약 1.2-1.4 mm이다 laevis의. (B) 부분 defolliculated 난자. 난자의 우측 절반은 혈관의 존재에 의해 식별 될 수있는 모낭 세포에 의해 덮여있다. 화살표는 모낭 세포의 자유와 주사 바늘이 주입된다되는 영역을 도시한다. (C)의 동일한 크기이며 동물 반구 및 식물성 반구 사이에 명확한 명암 고르게 착색 동물 반구 표시 양질 난자의 예. 2496 / 52496fig2highres.jpg &quot;/&gt; 그림 2 :. Xenopus의 전과 후 성숙 난자를 laevis의 난자 성숙 후, 분명 흰색 반점 동물 반구 모낭 세포의 상단에 나타납니다 박리된다. 각 아프리카 발톱 개구리 laevis의 난자는 직경이 약 1mm이다. <img alt="그림 3" src="/files/ftp_upload/52496/52496fig3highres.jpg" /> 그림 3 : 체외 성숙 난자에에서 생산 ICSI 배아의 개발. 각 단계에 ICSI 배아의 (A) 개발이 요약되어있다. 컨트롤이 IVM과 ICSI 다음 안티센스 올리고 뉴클레오티드 (제어 올리고 주입) 또는 주입 (비 주입)을하지 않고, 스크램블와 난 모세포가 주입되었다. 각 단계에서 배아의 해당 번호는 바 위에 표시됩니다. ± SEM이 표시됩니다 의미. N = 4-13 독립적 인 실험 / 다른 Females. (B) ICSI 배아 생존의 예. 이러한 tailbud 단계 배아 약 200 난자 출발 물질로서 사용 된 하나의 실험 제작 하였다.

Watch this Scientific Journal Video about Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development at JoVE.com

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

조작 및<em&gt; 체외</em의&gt; 성숙<em&gt; 아프리카 발톱 개구리 laevis의</em세포질 내 정자 주입에 이어&gt; 난 모세포, 배아 발달을 연구하는

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

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JoVE Journal

Developmental Biology

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

22.1K 조회수.  University of Cambridge. We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

비디오: Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

형광 단백질의 발현에<em&gt; 창고 기</em&gt; 미 수정 난자에 mRNA의 주입에 의한

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

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발생학

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 ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

공 촛점 분석을 사용하여<em&gt; 아프리카 발톱 개구리 laevis의</em&gt;의 Wnt 및 쉬 Morphogen 그라디언트의 변조기를 조사하기 위해

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

줄기 세포처럼<em&gt; Xenopus의</em&gt; 배아의 Explants는 초기 신경 발달의 특징을 연구하는<em&gt; 체외</em&gt; 및<em&gt; 생체</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

개발에 미세 주입을 대상으로 기술<em&gt; Xenopus의</em&gt; 신장

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

사용 모성 유전자 제품의 기능 조작<em&gt; 체외</em제브라 피쉬&gt; 난자의 성숙

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

의 배아 혈관 신생에 대한 시각화 및 정량 분석<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Organotypic 문화 메서드 배아 치킨 조직 개발 연구를

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

제노푸스 laevis 배아에서 눈부안으로 DNA의 미세 주입 및 GFP의 이미징 그대로 광학 축색 아버를 표현, 살아있는 제노푸스 올챙이

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

제노푸스 라에비스의 신경 전구체 특성화를 위한 형광 칼슘 이미징 및 후속 시토 혼성화

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...