Name: fertilization of xenopus oocytes using the host transfer method
Uploaded: 2010-11-02T17:00:00
Description: Watch this Scientific Journal Video about Fertilization of Xenopus oocytes using the Host Transfer Method at JoVE.com

作者想感谢休斯顿实验室的关键读的手稿成员。研究是支持的美国国立卫生研究院授予DWH（GM083999）

1。手术切除的卵巢组织<ol class="lalpha"><li>准备一批新的卵母细胞培养液中（OCM;材料），根据实验的大小之间的400-800毫升。调整pH值至7.6-7.8，直到酚红变成深红色（5N氢氧化钠6个左右的小滴）。倒出少量检查在一个单独的管的pH值，因为pH电极可能污染媒体。 OCM应存放在18 ° C，并在一个星期内使用。 </li><li> 〜2L的缓冲麻醉溶液（材料）中选择3-5名女性和地方之一。我们尽量避免使用，美联储在?...

<ol>
	<li>Brun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+maturation+in+vitro:+contribution+of+the+oviduct+to+total+maturation+in+Xenopus+laevis.">Oocyte maturation in vitro: contribution of the oviduct to total maturation in Xenopus laevis.</a> Experientia. 31, 1275-1276 (1975).</li><li>Elinson, R. P., Pasceri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+UV-sensitive+targets+in+dorsoanterior+specification+of+frog+embryos.">Two UV-sensitive targets in dorsoanterior specification of frog embryos.</a> Development. 106, 511-518 (1989).</li><li>Heasman, J., Holwill, S., Wylie, C. C., C, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+of+cultured+Xenopus+oocytes+and+use+in+studies+of+maternally+inherited+molecules.">Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules.</a> Methods Cell Biol. 36, 213-230 (1991).</li><li>Holwill, S., Heasman, J., Crawley, C., Wylie, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axis+and+germ+line+deficiencies+caused+by+u.v+irradiation+of+Xenopus+oocytes+cultured+in+vitro.">Axis and germ line deficiencies caused by u.v irradiation of Xenopus oocytes cultured in vitro.</a> Development. 100, 735-743 (1987).</li><li>Houston, D. W., King, M. 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+critical+role+for+Xdazl,+a+germ+plasm-localized+RNA,+in+the+differentiation+of+primordial+germ+cells+in+Xenopus.">A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus.</a> Development. 127, 447-456 (2000).</li><li>Mir, A., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+mother+can+help:+studying+maternal+Wnt+signaling+by+anti-sense-mediated+depletion+of+maternal+mRNAs+and+the+host+transfer+technique.">How the mother can help: studying maternal Wnt signaling by anti-sense-mediated depletion of maternal mRNAs and the host transfer technique.</a> Methods Mol Biol. 469, 417-429 (2008).</li><li>Rugh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental Embryology: Techniques and procedures. , (1962).</li><li>Smith, L. D., Ecker, R. E., Subtelny, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+induction+of+physiological+maturation+in+Rana+pipiens+oocytes+removed+from+their+ovarian+follicles.">In vitro induction of physiological maturation in Rana pipiens oocytes removed from their ovarian follicles.</a> Dev Biol. 17, 627-643 (1968).</li><li>Smith, L. D., Xu, W., Varnold, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+and+oocyte+isolation.">Oogenesis and oocyte isolation.</a> Methods Cell Biol. 36, 45-60 (1991).</li><li>Woolf, T. M., Jennings, C. G., Rebagliati, M., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stability,+toxicity+and+effectiveness+of+unmodified+and+phosphorothioate+antisense+oligodeoxynucleotides+in+Xenopus+oocytes+and+embryos.">The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos.</a> Nucleic Acids Res. 18, 1763-1769 (1990).</li><li>Wylie, C., Kofron, M., Payne, C., Anderson, R., Hosobuchi, M., Joseph, E., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+beta-catenin+establishes+a+'dorsal+signal'+in+early+Xenopus+embryos.">Maternal beta-catenin establishes a 'dorsal signal' in early Xenopus embryos.</a> Development. 122, 2987-2996 (1996).</li><li>Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+maternal+VegT+in+establishing+the+primary+germ+layers+in+Xenopus+embryos.">The role of maternal VegT in establishing the primary germ layers in Xenopus embryos.</a> Cell. 94, 515-524 (1998).</li><li>Zuck, M. V., Wylie, C. C., Heasman, J., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+mRNAs+in+Xenopus+embryos:+an+antisense+approach..">Maternal mRNAs in Xenopus embryos: an antisense approach..</a> A comparative methods approach to the study of oocytes and embryos. , 341-354 (1998).</li></ol>

一个成功的转移，将导致受精和正常的卵母细胞&gt; 50％（图3）裂解。过去一般在30-60％的转让的卵母细胞存活的神经胚阶段。我们通常是75-150％实验组的卵母细胞，这将产生几种类型的分析足够的胚胎（原位RT - PCR）以及可视化的表型转移。植入大幅更多的卵母细胞似乎并不大大提高产量。此外，应至少有30％组的卵母细胞转移到保证，至少有一些通过原肠。重要的染料一般不影响这里描述的浓?...

解决方案和食谱： OCM（卵母细胞培养液中，从卫理等修改，1996年。 ） <table border="0"><tbody><tr><td> 70％的L - 15的Leibovitz的中等（含L -谷氨酰胺） </td><td> （Invitrogen公司11415-064） </td></tr><tr><td> 0.4 mg / ml的牛血清白蛋白（BSA的分数V） </td><td> （SIGMA A - 9418 - 50G） </td></tr><tr><td> 1X青霉素，链霉素</td><td> （Sigma公司的P - 0781） </td></tr><tr><td colspan="2"> 5N氢氧化钠调整pH值7.6-7.8 </td></tr><tr><td colspan="2">在18 ° C，每周新鲜，商店</td></tr></tbody></table> 10X MMR（Marc的改良林格液;扎克等人 ，1998年） <table border="0"><tbody><tr><td> 1M氯化钠</td></tr><tr><td> 20 mM的氯化钾</td></tr><tr><td> 20毫米氯化钙2 </td></tr><tr><td> 10毫米氯化镁2 </td></tr><tr><td> 150毫米的HEPES </td></tr><tr><td>调整pH值7.6-7.8 </td></tr><tr><td>保存在4 ° C </td></tr></tbody></table>麻醉的解决方案<table border="0"><tbody><tr><td> 1 g / L的3 - 氨基苯甲酸乙酯（MS222） </td><td> （Sigma公司A - 5040） </td></tr><tr><td colspan="2"> 0.7 g / L的碳酸氢钠</td></tr><tr><td colspan="2">在Amquel处理过的水溶解</td></tr><tr><td colspan="2">新鲜每周</td></tr></tbody></table>孕酮股票<table border="0"><tbody><tr><td> 100％的乙醇10毫米孕酮（Sigma公司的P - 0130） </td></tr><tr><td>为500X的股票，在100％乙醇稀释至1毫米/工作方案</td></tr><tr><td>储存在-20 ° C </td></tr></tbody></table>重要的染料（Heasman 等 ，1991年修改） <table border="0"><tbody><tr><td>蓝：0.1％耐尔蓝一</td><td> （Aldrich公司121479 - 5G）， </td></tr><tr><td>红色：0.25％中性红</td><td> （西格玛ñ - 6634）， </td></tr><tr><td>布朗：布朗俾斯麦1％ </td><td> （Sigma公司的B - 2759）， </td></tr><tr><td colspan="2">绿色（80μL蓝+ 80μL棕色）， </td></tr><tr><td colspan="2">紫红色（80μL蓝+ 80μL红色）。 </td></tr><tr><td colspan="2">第六个颜色，橙色（80μL红+ 80μL棕色），也可以使用，但往往是很难区分从褐色。 </td></tr></tbody></table>在去离子水蓝色，红色和褐色的股票于50 ml管，孵育20分钟摇摆，并在临床离心机纺。上清aliquotted到微型离心管中，并储存在-20 ° C

fertilization of xenopus oocytes using the host transfer method

往往阻碍了必要的时间和费用，产生产妇效应突变动物研究母系遗传的分子脊椎动物的早期发展的贡献。此外，许多技术如爪蟾和斑马鱼的生物过表达或抑制基因功能未能充分针对危重孕产妇的信号转导通路，如Wnt信号。在非洲爪蟾 ，在培养的卵母细胞的基因功能和操纵，随后施肥可以在一定程度上改善这些问题。手动defolliculated卵母细胞是从供体卵巢组织，注射或视为文化，然后用黄体酮刺激诱导成熟。下一步，卵母细胞引入腔体内的雌蛙排卵主机，届时他们将通过主机的输卵管移位和掌握修改和果冻大衣施肥必要。由此产生的胚胎，然后将筹集到所需的阶段和任何实验扰动的影响分析。这台主机传输方法已发现早期发展的基本机制非常有效，并没有在任何其他脊椎动物模式生物的实验可能允许广泛。

Watch this Scientific Journal Video about Fertilization of Xenopus oocytes using the Host Transfer Method at JoVE.com

Fertilization of Xenopus oocytes using the Host Transfer Method

对施肥的程序<em&gt;爪蟾卵母细胞</em由主机传输方法。

施肥爪蟾卵母细胞使用主机传输方法

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Research

JoVE Journal

Biology

Fertilization of Xenopus oocytes using the Host Transfer Method

Nuclear Transfer into Mouse Oocytes

小鼠卵母细胞的细胞核移植到

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

科研

生物学

Retrieval of Mouse Oocytes

检索小鼠卵母细胞

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

在使用分馏爪蟾卵提取物的体外核大会

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Microinjection of Xenopus Laevis Oocytes

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Visualizing RNA Localization in Xenopus Oocytes

Visualizing RNA Localization in Xenopus Oocytes

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

研究DNA损伤检验点<em&gt;爪蟾</em&gt;卵提取物

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...