VCUからA·ディキンソンのスタートアップ資金がこの作品を支持した。 著者は、概略図を作成する際に、彼の芸術的才能のためのダンNacuを認識したい。

<ol>
	<li>Mossey, P. A., Little, J., Munger, R. G., Dixon, M. J., Shaw, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleft+lip+and+palate.">Cleft lip and palate.</a> Lancet. 374, 1773-1785 (2009).</li><li>Kennedy, A. E., Dickinson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Median+facial+clefts+in+Xenopus+laevis:+roles+of+retinoic+acid+signaling+and+homeobox+genes.">Median facial clefts in Xenopus laevis: roles of retinoic acid signaling and homeobox genes.</a> Dev Biol. 365, 229-240 (2012).</li><li>Kennedy, A. E., Dickinson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+Orofacial+Development+and+Median+Clefts+in+Xenopus+Laevis.">Quantitative Analysis of Orofacial Development and Median Clefts in Xenopus Laevis.</a> Anat Rec (Hoboken). , (2014).</li><li>Dickinson, A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positioning+the+extreme+anterior+in+Xenopus:+cement+gland,+primary+mouth+and+anterior+pituitary.">Positioning the extreme anterior in Xenopus: cement gland, primary mouth and anterior pituitary.</a> Semin Cell Dev Biol. 18, 525-533 (2007).</li><li>Dickinson, A. J., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+primary+mouth+in+Xenopus+laevis.">Development of the primary mouth in Xenopus laevis.</a> Dev Biol. 295, 700-713 (2006).</li><li>Dickinson, A. J., Sive, 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=The+Wnt+antagonists+Frzb-1+and+Crescent+locally+regulate+basement+membrane+dissolution+in+the+developing+primary+mouth.">The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth.</a> Development. 136, 1071-1081 (2009).</li><li>Barnett, C., 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=Syndrome+Transcription+Factor+is+critical+for+neural+crest+cell+function+in+Xenopus+laevis.">Syndrome Transcription Factor is critical for neural crest cell function in Xenopus laevis.</a> Mech Dev. 129, 324-338 (2012).</li><li>Gonzales, B., Yang, H., Henning, D., Valdez, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+characterization+of+the+Xenopus+orthologue+of+the+Treacher+Collins+syndrome+(TCOF1)+gene+product.">Cloning and functional characterization of the Xenopus orthologue of the Treacher Collins syndrome (TCOF1) gene product.</a> Gene. 359, 73-80 (2005).</li><li>Reisoli, E., De Lucchini, S., Nardi, I., Ori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+2B+receptor+signaling+is+required+for+craniofacial+morphogenesis+and+jaw+joint+formation+in+Xenopus.">Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus.</a> Development. 137, 2927-2937 (2010).</li><li>Schuff, 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=FoxN3+is+required+for+craniofacial+and+eye+development+of+Xenopus+laevis.">FoxN3 is required for craniofacial and eye development of Xenopus laevis.</a> Dev Dyn. 236, 226-239 (2007).</li><li>Slater, B. J., Liu, K. J., Kwan, M. D., Quarto, N., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+osteogenesis+and+suture+morphology+in+Xenopus+laevis:+a+unique+model+system+for+studying+craniofacial+development.">Cranial osteogenesis and suture morphology in Xenopus laevis: a unique model system for studying craniofacial development.</a> PLoS One. 4, (2009).</li><li>Vandenberg, L. N., Adams, D. S., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalized+shape+and+location+of+perturbed+craniofacial+structures+in+the+Xenopus+tadpole+reveal+an+innate+ability+to+achieve+correct+morphology.">Normalized shape and location of perturbed craniofacial structures in the Xenopus tadpole reveal an innate ability to achieve correct morphology.</a> Dev Dyn. 241, 863-878 (2012).</li><li>Bugaighis, I., Mattick, C. R., Tiddeman, B., Hobson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Facial+Morphometry+in+Children+with+Oral+Clefts.">3D Facial Morphometry in Children with Oral Clefts.</a> Cleft Palate Craniofac J. , (2013).</li><li>Farkas, L. G., Katic, M. J., Forrest, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+anatomy+of+the+face+in+Down's+syndrome:+anthropometric+proportion+indices+in+the+craniofacial+regions.">Surface anatomy of the face in Down's syndrome: anthropometric proportion indices in the craniofacial regions.</a> J Craniofac Surg. 12, 519-524 (2001).</li><li>Scheuer, H. A., Holtje, W. J., Hasund, A., Pfeifer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognosis+of+facial+growth+in+patients+with+unilateral+complete+clefts+of+the+lip,+alveolus+and+palate.">Prognosis of facial growth in patients with unilateral complete clefts of the lip, alveolus and palate.</a> J Craniomaxillofac Surg. 29, 198-204 (2001).</li><li>Parsons, K. J., Andreeva, V., James Cooper, W., Yelick, P. C., Craig Albertson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+zebrafish+jaw:+development+beyond+the+embryo.">Morphogenesis of the zebrafish jaw: development beyond the embryo.</a> Methods Cell Biol. 101, 225-248 (2011).</li><li>Farkas, L. G., Katic, M. J., Forrest, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+anatomy+of+the+face+in+Down's+syndrome:+age-related+changes+of+anthropometric+proportion+indices+in+the+craniofacial+regions.">Surface anatomy of the face in Down's syndrome: age-related changes of anthropometric proportion indices in the craniofacial regions.</a> J Craniofac Surg. 13, 368-374 (2002).</li><li>Cooper, W. J., 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=Bentho-pelagic+divergence+of+cichlid+feeding+architecture+was+prodigious+and+consistent+during+multiple+adaptive+radiations+within+African+rift-lakes.">Bentho-pelagic divergence of cichlid feeding architecture was prodigious and consistent during multiple adaptive radiations within African rift-lakes.</a> PLoS One. 5, (2010).</li><li>Klingenberg, C. P., 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=Prenatal+alcohol+exposure+alters+the+patterns+of+facial+asymmetry.">Prenatal alcohol exposure alters the patterns of facial asymmetry.</a> Alcohol. 44, 649-657 (2010).</li><li>Zhao, Y., 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=Isolated+cleft+palate+in+mice+with+a+targeted+mutation+of+the+LIM+homeobox+gene+lhx8.">Isolated cleft palate in mice with a targeted mutation of the LIM homeobox gene lhx8.</a> Proc Natl Acad Sci U S A. 96, 15002-15006 (1999).</li><li>Allam, K. A., 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+spectrum+of+median+craniofacial+dysplasia.">The spectrum of median craniofacial dysplasia.</a> Plast Reconstr Surg. , 812-821 (2011).</li><li>Sive, H. L., Grainger, R., Harlard, 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> Early development of Xenopus laevis: a laboratory manual. , (2000).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+eggs+from+Xenopus+laevis+females.">Obtaining eggs from Xenopus laevis females.</a> J Vis Exp. , (2008).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus Laevis (Daudin). , (1967).</li><li>Nieuwkoop, P. D. a. F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis. , (1994).</li><li>Abdi, H., Williams, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principal+Component+Analysis.">Principal Component Analysis.</a> WIREs Computational Statistics. 2, (2010).</li><li>Hill, T. 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> P. STATISTICS: Methods and Applications. , (2013).</li></ol>

著者らは、開示することは何もない。

アフリカツメガエルは、発達のメカニズム基礎口腔顔面開発を解剖するための有用なツールとなっています。しかし、カエルは、この領域の大きさや形状変化を記述するないプロトコルは現在ありません。ここで説明する方法では、アフリカツメガエルや他の脊椎動物における口腔顔面の表現型のより厳密な定量化を可能にすることによって口腔顔面開発の分野に大きく貢献し...

人間の先天性欠損症の最も一般的かつ破壊的な種類の中には、口腔顔面裂1として、口や顔に影響を与えるものである。不正な形式の口腔顔面構造を持つ子どもたちが生涯にわたって、複数の手術を受け、顔面disfigurements、スピーチ、聴力や食の問題に苦労しています。したがって、cranio-および口腔顔面開発に新たな研究を促進することは、ヒトにおける先天性欠損、これらのタイプの予防と治療に最も重要である。 アフリカツメガエル頭蓋顔面の開発を支配するメカニズムを解剖するための新しいツールとして浮上している（いくつかの例は、2,3,4を含む-11）。したがって、この種の頭部の発達と顔の間に大きさや形状変化を分析するための定量的な方法は3非常に強力である可能性があります。 ここでは、そのような方法を提示する。 アフリカツメガエルの研究12から適応幾何学的形態計測を持つ伝統的なサイズ測定を組み合わせる</人間の顔の形13-15を分析する研究のSUP&gt;と富。このプロトコルの目的は、研究者は、正常および異常な発達の間の異なる顔面表現型を区別するために、顔の大きさや形状を定量化できるようにすることである。この分析は、遺伝子および/または環境要因の相乗効果に起因するもののような微妙な顔面頭蓋の欠陥との間のより良好な区別を可能にする。さらに、この定量方法は、また口腔顔面欠陥の場合でもわずかな改善や救助を明らかにできた。したがって、これはそれの潜在的治療薬の分析に有用な指針になります。 ここで提示する顔の測定値と幾何形態計測の組み合わせは、大部分が1つまたは他の15-18を利用し 、現在のプロトコルよりも口腔顔面領域の大きさと形状の両方のより総合的な統計分析を可能にする。さらに、我々はの内側および外側面の両方を評価するための簡単​​な方法を提示現在の研究13,19で使用される洗練された三次元画像装置を必要とせずに、顔。 私たちは、 アフリカツメガエルでこのプロトコルは、異常な口腔顔面開発および中央値口蓋裂2,3を誘導するレチノイン酸受容体阻害剤で処理された胚をツメガエル実証する。これらの胚における口腔顔面領域の寸法及び形状の定量化は、類似の口蓋裂およびマウスモデル20,21を有するヒトに類似している顔面の変化を明らかにした。しかしながら、このプロトコルは、成長因子などの天然物質、除草剤、またはタンパク質などの口腔顔面開発に関する他の化合物の効果を評価するために利用することができる。さらに、損失または機能実験のゲインを介して遺伝子発現の摂動から生じる口腔顔面の大きさ及び形状の変化（アンチセンスまたはモルホリノCrispersを使用するには、/ Talens）もまた、このプロトコルを使用して定量することができる。最後に、我々は、このメソッド書または仕様書を開発LLY アフリカツメガエルの形態を評価する。しかしながら、容易に任意の脊椎動物の分析のために修正される。他のアプリケーションも、進化や生態の研究のために密接に関連した種を比較するため、このプロトコルを使用して含めることができます。ここで提供する例は、口腔顔面領域の分析を記述するためにこのプロトコルを利用するが、それは容易に他の領域、臓器または構造の分析のために修正することができる。 この口腔顔面定量化プロトコルは、研究コミュニティのための貴重な資源だけでなく、ビデオのデモンストレーションとして学部学生のための優れた教育ツールとなります。

<table border="0" cellpadding="0" cellspacing="0" width="818">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">Name of the reagent</td>
			<td style="width:143px;">Company</td>
			<td style="width:136px;">Catalogue number</td>
			<td style="width:236px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>fitted with AxioCamICC1 camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Inox forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile, disposable scalpel</td>
			<td>Sklar</td>
			<td>06-2015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">24-well plate</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:136px;">087721</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">Standard Disposable transfer pipettes</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:136px;">13-711-7M</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">150 mm X 15 mm Petri dishes</td>
			<td style="width:143px;">Falcon</td>
			<td style="width:136px;">351058</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Incubators</td>
			<td style="width:143px;">Ectotherm</td>
			<td style="width:136px;"></td>
			<td style="width:236px;">set to 15C or 20C&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:304px;">Modeling Clay</td>
			<td style="width:143px;">Premo, or other non-toxic modeling clay</td>
			<td style="width:136px;"></td>
			<td style="width:236px;">in black or white</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Straight teasing needle</td>
			<td style="width:143px;">Thermo Scientific</td>
			<td style="width:136px;">19010</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:304px;">Capillary Tubing (for needles)</td>
			<td>FHC</td>
			<td>30-30-1</td>
			<td style="width:236px;">Borosil 1.0mm OD x 0.5mm ID/Fiber, 100 mm each</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:304px;">Needle Puller, Model P-97&#160;</td>
			<td style="width:143px;">Sutter Instrument Co,&#160;</td>
			<td style="width:136px;">Needle Puller: P-97 Flaming/ Bown micropipette puller Filament: FB300B</td>
			<td style="width:236px;">For filaments, use Sutter 3.00mm square box filaments, 3.0mm wide.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:304px;">Pipettemen</td>
			<td style="width:143px;">Gilson</td>
			<td style="width:136px;">F144802, F123600, F123602</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">BMS-453</td>
			<td style="width:143px;">Tocris</td>
			<td style="width:136px;">3409</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:304px;">DMSO</td>
			<td style="width:143px;">American Bioanalytical</td>
			<td style="width:136px;">AB00435-01000</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Cysteine</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:136px;">52-90-4</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Paraformaldehyde powder</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:136px;">158127</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:304px;">Petri dishes</td>
			<td style="width:143px;">Falcom</td>
			<td style="width:136px;">353003, 351058</td>
			<td style="width:236px;">100 mm diameter and 150 mm in diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">100% Ethanol</td>
			<td style="width:143px;">VWR</td>
			<td>89125-170</td>
			<td style="width:236px;"></td>
		</tr>
	</tbody>
</table>

quantification of orofacial phenotypes in xenopus

アフリカツメガエルは、頭蓋顔面の開発や欠陥を支配するメカニズムを解剖するための重要なツールとなっています。口腔顔面開発を定量化する方法は、遺伝的または分子的遺伝子発現またはタンパク質機能を操作することができる物質と廃止時顔面表現のより厳密な分析を可能にする。胚の頭部の二次元画像を用いて、従来のサイズの寸法を、そのような口顔の幅、高さ及び面積 - を計測する。また、胚口開口部の真円度測定は、口の形状を説明するために使用される。これらの二次元画像の幾何学的形態計測はまた、口腔顔面領域の形状の変化をより洗練されたビューを提供するために行われる。ランドマークは、口腔顔面領域内の特定のポイントに割り当てられ、座標が作成されます。主成分分析は、その後の処置を判別主成分にランドマークの座標を低減するために使用されるグループ。これらの結果は、類似した口腔顔面形状の個人が一緒にクラスター化した散布図として表示されます。これは、統計的に両群間のランドマークの位置を比較し、判別関数分析を実行することも有用である。この分析は、ランドマークの位置の変化は、ベクトルとして見られる変換グリッドに表示される。グリッドはワーピングパターンがかなりのランドマークの位置が変更されている場所を示すために表示されるように、これらのベクターに重畳される。判別関数分析における形状の変化は、統計的尺度に基づいており、したがって、p値によって評価することができる。この分析は、唯一のステレオスコープやフリーウェアのソフトウェアを必要とし、シンプルかつアクセス可能であるため、貴重な研究と教育のリソースとなります。

ここでは、口顔の大きさおよび形状の定量分析は、未処理対照とレチノイン酸受容体阻害剤（RAR阻害剤）で処理された胚を比較することが実証された。胚をステージ24から30までこの化学阻害剤の1μM濃度（26-35 HPF）で処理し、洗い流され、ステージ42（82 HPF）で固定した。次いで、それらを処理し、プロトコルに記載のように分析した。結果は、元のデータが、以前の出版物2,3の観測?...

Watch this Scientific Journal Video about Quantification of Orofacial Phenotypes in Xenopus at JoVE.com

Quantification of Orofacial Phenotypes in Xenopus

アフリカツメガエル胚の顔面サイズおよび形状を定量化する方法が開発されている。このプロトコルでは、従来の大きさの測定は、口腔顔面開発や欠陥のより高度な分析を可能にするために、幾何学的形態計測と組み合わされる。

口腔顔面表現型の定量化で<em&gt;アフリカツメガエル</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

quantification-of-orofacial-phenotypes-in-xenopus

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Quantification of Orofacial Phenotypes in Xenopus

9.7K ビュー.  Virginia Commonwealth University.  アフリカツメガエル胚の顔面サイズおよび形状を定量化する方法が開発されている。このプロトコルでは、従来の大きさの測定は、口腔顔面開発や欠陥のより高度な分析を可能にするために、幾何学的形態計測と組み合わされる。 

ビデオ: Quantification of Orofacial Phenotypes in Xenopus

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

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 chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

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 chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

分別アフリカツメガエルの卵抽出を用いたin vitro核アセンブリで

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 ...

生物学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

アフリカツメガエルの卵母細胞で発現イオンチャネルのパッチクランプ記録

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

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

アフリカツメガエル卵母細胞のマイクロインジェクション

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

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可視化RNAの局在にアフリカツメガエル卵母細胞

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

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

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

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

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

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

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

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

の蛍光タンパク質の偏転アフリカツメガエルWntシグナルへの対応に>外胚葉

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

顔面移植<em&gt;アフリカツメガエル</em&gt;胚

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

ザ·<em&gt;アフリカツメガエル</em蛍光測定で&gt;卵母細胞のカットオープンワセリンギャップ電圧クランプ法

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

口腔顔面表現型の定量化で

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