VCU에서 A. 디킨슨 시동 돈이 작업을 지원했다. 저자는 개략적 인 그림을 만드는 그의 예술적 재능 댄 Nacu을 인정하고 싶습니다.

<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>

저자가 공개하는 게 없다.

아프리카 발톱 개구리는 laevis의 발달 기전 구강 안면 개발을 해부하는 데 유용한 도구가되었다; 그러나, 개구리에서이 지역의 크기와 모양 변화를 기술 한 프로토콜은 현재 존재하지 않는다. 여기에 설명 된 방법은 제노 푸스 다른 척추 동물에 구강 안면 표현형보다 엄격한 정량 허용함으로써 구강 안면 개발 분야에 크게 기여할 것이다. 제대로이 프로토콜을 ?...

인간의 출생 결함의 가장 흔하고 치명적인 유형 중 같은 구강 안면 쪼개진 조각 1로 입과 얼굴에 영향을 미치는 것들이다. 잘못된 구강 안면 구조와 아이들은 평생 동안 여러 수술을 받아야 얼굴 disfigurements, 언어, 청각 먹는 문제를 투쟁. 따라서, cranio- 및 구강 안면 개발의 새로운 연구를 촉진하는 것은 인간의 출생 결함의 이러한 유형의 예방 및 치료에 중요합니다. 아프리카 발톱 개구리 laevis의는 두개 안면 개발을 관리하는 메커니즘을 해부를위한 새로운 도구로 떠오르고있다 (몇 가지 예 2,3,4 포함 -11). 따라서,이 종류의 헤드의 개발 및 얼굴 중 크기 및 형상 변화를 정량적으로 분석하는 방법은 세 매우 강력 할 수있다. 여기서, 우리는 이러한 방법을 제시한다; Xenopus의 연구 (12)에서 적응 기하학적 morphometrics 전통적인 크기 측정을 결합 </인간의 얼굴 형태 13-15을 분석하는 연구의 SUP&gt;를 찾을 수있다. 이 프로토콜의 목표는 연구자가 정상 및 비정상 개발하는 동안 다른 구강 안면 표현형을 구별하는 얼굴 크기와 모양을 정량화 할 수 있도록하는 것입니다. 이 분석은 유전자 및 / 또는 환경 적 요인의 시너지 효과에서 발생하는 것과 같은 미묘한 두개 안면 결함 사이의 더 나은 차별화 수 있습니다. 또한, 이러한 정량화 방법은 또한 구강 안면 결함에도 약간 개선 또는 구조를 밝힐 수있다. 이것은 따라서 잠재적 인 치료제 분석에 유용한 가이드한다. 여기 제시 안면 측정 및 기하학적 morphometrics의 조합은 대체로 하나 또는 다른 15-18 활용 크기 및 현재의 프로토콜보다 구강 안면 영역의 형상 모두 포괄적 통계 분석을 허용한다. 또한, 우리는 내측과 외측의 양쪽면을 평가할 수있는 간단한 방법을 제시현재 연구 13, 19에서 사용 된 복잡한 3 차원 화상 장비 없이도 얼굴. 우리는 아프리카 발톱 개구리에이 프로토콜은 비정상적인 구강 안면 개발 및 중간 구개열 2,3를 유도하는 레티노 산 수용체 억제제로 치료 배아를 laevis의 보여줍니다. 이러한 배아의 구강 안면 영역의 크기와 모양의 정량화 비슷한 구개 쪼개진 조각 및 마우스 모델 (20, 21)와 인간과 유사하다 midface의 변화를 공개했다. 그러나이 프로토콜은 예컨대 성장 인자와 같은 천연 물질, 제초제, 또는 단백질과 같은 구강 안면 발달에 다른 화합물의 효과를 평가하는데 이용 될 수있다. 또한, 손실 또는 함수 실험의 이득을 통해 유전자 발현을 교란으로부터 발생하는 구강 안면 크기 및 형태의 변화 (안티센스 morpholinos 또는 Crispers를 사용하여이 / Talens)도이 프로토콜을 이용하여 정량화 할 수있다. 마지막으로, 우리는이 방법의 사양보다 개발에서야 Xenopus의 형태를 평가하기 위해; 그러나, 용이하게 임의의 척추 동물의 분석을 위해 수정된다. 다른 응용 프로그램도 진화 또는 생태 연구에 밀접하게 관련 종을 비교하는이 프로토콜을 사용하여 포함 할 수있다. 우리가 여기에서 제공하는 예는 구강 안면 영역의 분석을 설명하는이 프로토콜을 사용하지만, 쉽게 다른 지역, 기관, 또는 구조의 분석을 위해 수정할 수 있습니다. 이 구강 안면 정량화 프로토콜은 연구 지역 사회를위한 귀중한 자원뿐만 아니라, 비디오 데모와 같은 학부 학생들을위한 훌륭한 교육 도구가 될 것이다.

<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

Xenopus의 개발은 두개 안면 결함을 관리하는 메커니즘을 해부위한 중요한 도구가되었다. 구강 안면 현상을 정량화하는 방법은 유전자 또는 분자 유전자 발현 또는 단백질의 기능을 조작 할 수있는 물질과 폐기시 구강 안면 표현형보다 엄격한 분석을 허용 할 것이다. 배아 머리의 두 차원 이미지를 사용하여, 기존 사이즈의 치수를-같은 구강 안면 폭, 높이 등 및 지역 - 측정한다. 또한, 배아 입 개구의 진원도 측정 입의 형상을 설명하는 데 사용된다. 이러한 이차원 영상의 기하학적 morphometrics 또한 구강 안면 영역의 형상의 변화에​​보다 정교한 뷰를 제공하기 위해 수행된다. 랜드 마크는 구강 안면 영역의 특정 지점에 할당 및 좌표가 생성됩니다. 주성분 분석은 치료를 판별 원리 성분 표식 좌표를 줄이기 위해 사용되는그룹. 이러한 결과는 비슷한 구강 안면 형태와 개인이 함께 클러스터링하는 산포도로 표시됩니다. 또한 통계적 두 치료군 간의 랜드 마크의 위치를​​ 비교 판별 함수 분석을 수행하는 것이 유용하다. 이 분석은 랜드 마크의 위치 변화가 벡터로 볼 수 있습니다 변환 그리드에 표시됩니다. 그리드는 왜곡 패턴이 중요한 랜드 마크 위치가 변경된 위치를 표시하는 표시되도록이 벡터에 놓이게됩니다. 판별 함수 분석 형상 변화는 통계적 측정치에 기초하며, 따라서 p- 값에 의해 평가 될 수있다. 이 분석은 입체경 프리웨어 소프트웨어를 필요로하는, 단순하며, 따라서 귀중한 연구 및 교육 자료가 될 것이다.

여기서, 구강 안면 크기 및 형상의 정량 분석​​ 미처리 컨트롤 레티노 산 수용체 억제제 (RAR 억제제)로 처리 배아를 비교하는 것으로 입증되었다. 배아, 30 (26 ~ 35 HPF)로 단계 24에서이 화학 물질 억제제의 1 μM 농도로 처리 세척하고, 단계 (42) (82 HPF)로 고정되었다. 그런 다음 처리 프로토콜에 기술 된 바와 같이 분석 하였다. 결과는 원본 데이터,하지만 이전의 출판물 2,3 관측과 일치한다. ?...

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

Quantification of Orofacial Phenotypes in Xenopus

아프리카 발톱 개구리 laevis의 배아의 구강 안면 크기 및 형상을 정량화하는 방법이 개발되었다. 이 프로토콜에서, 기존의 크기 측정은 구강 안면 현상 결함의보다 정교한 분석을 허용하기 위해 기하학적 morphometrics과 결합된다.

구강 안면 표현형의 정량화에<em&gt; Xenopus의</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

Research

JoVE Journal

Biology

Quantification of Orofacial Phenotypes in Xenopus

9.7K 조회수.  Virginia Commonwealth University.  아프리카 발톱 개구리 laevis의 배아의 구강 안면 크기 및 형상을 정량화하는 방법이 개발되었다. 이 프로토콜에서, 기존의 크기 측정은 구강 안면 현상 결함의보다 정교한 분석을 허용하기 위해 기하학적 morphometrics과 결합된다. 

비디오: Quantification of Orofacial Phenotypes in Xenopus

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.

Xenopus Laevis의 Oocytes의 Microinjection

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

연구

생물학

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.

머릿속 RNA 현지화에서 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 ...

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.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 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 ...

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.

에 Postembryonic Phenotypes을 식별하기 위해 RNAi 심사 C. elegans</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.

의 얼굴 이식<em&gt;는 Xenopus laevis의</em&gt; 태아

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.

<em&gt; Xenopus의</em형광 측정으로&gt; 난자 절개 바셀린 갭 전압 클램프 기술

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

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &amp;#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

β-catenin의 저하의 재구성<em&gt; Xenopus의</em&gt; 계란 추출

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Blood Perfusion in African Clawed Frogs for transcriptomics and proteomics(전사체학 및 단백질체학을 위한 아프리카 발톱 개구리의 혈액 관류)

Effective Rapid Blood Perfusion in Xenopus

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol in Xenopus, aimed at a consistent and drastic reduction of blood within all tissues, is defined. Perfusion is carried out by inserting a needle directly into the ventricle of the heart and pumping heparinized phosphate-buffered saline (PBS) through the vascular system. The procedure can be completed in approximately 10 min per animal. The blood is dominated by a few highly abundant proteins and cell types, creating numerous issues as these proteins mask most other molecules and cell types of interest. The reproducible characterization of adult Xenopus tissues with quantitative proteomics and single-cell transcriptomics will benefit from applying this protocol prior to organ sampling. The protocols for tissue sampling are defined in companion papers. These procedures are aimed at the standardization of practices across Xenopus of different sex, age, and health status, specifically X. laevis and X. tropicalis.

저자 스포트라이트: Xenopus의 효과적인 빠른 혈액 관류  

Presented here is an effective rapid blood perfusion protocol to prepare tissue samples from African clawed frogs for transcriptomics and proteomics studies.

Xenopus의 효과적인 급속 관류

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol ...