전 모델 설계 및 아카이브 영상에 대한 접근에 많은 도움을 동물 행동의 통합 연구 (CISAB)의 센터에서 다니엘 반 다이크 감사하고 싶습니다. 크리스토퍼 에번스 (CISAB)는 다니엘 워너는 (시드니 대학) taxidermic 모델을 제공하는 기술 지원을 제공하고, 리처드 피터스 (호주 국립 대학교)는 초기 설계 기능을 제공했습니다.

애니메이션 디자인 객체 스캔 taxidermic의 도마뱀을 습득. 코니카 미놀타 VI - 9i와 모델을 검사합니다. 그것은 50,000 다각형의 단일 다각형 메쉬의 3D 개체 (*. lwo 및 *. OBJ 파일)을 생성합니다. 모형을 만드는 사람과 레이아웃 : Lightwave ® v8.3는 3D 애니메이션에 사용되는 두 가지 프로그램이 있습니다. Lightwave ® 모형을 만드는 사람의 설계 및이 개체를 조작합니다. Lightwave ® 레이아웃 프로그램은 애니메이션 장면을 만듭니다. Lightwave ® 모형을 만드는 사람과 레이아웃 위치, 방향 및 회전의 세 차원이 있습니다 (Y) 제목 피치 (X), 은행 (Z​​). 텍스처 취득 두가지 각도 (이마와 직교) 세 위치 (정면, 직교 복부와 지느러미), 세 신체 지역 부품 (머리, 몸, 꼬리, 그리고 팔다리)에서 12.8 메가픽셀 캐논 EOS 5D 디지털 카메라를 사용하여 라이브 도마뱀 사진 종이 흰색 시트를 통해 표준 색상 균형 흰색으로. 어도비 ® 포토샵 ® 요소 3.0 함께 사진을 융합. Lightwave ® 모형을 만드는 사람의 아틀라스 UV지도를 만듭니다. UV지도 다각형을 연결 문자열로 개체를시킵니다. 별도의 JPEG 이미지를 만들 잡아 V1.2와 아틀라스 UV지도의 JPEG 이미지를 캡처합니다. 어도비 ® 포토샵 ® 요소 3.0로 배경 레이어로 끼워 넣다. Lightwave ® 모형을 만드는 사람에서 아틀라스 UV지도에서 강조 다각형은 도마뱀의 특정 영역을 식별합니다. 자르기 영역과 배경 아틀라스 UV지도 JPEG에 비교하십시오. 모든 사진 조각은 아틀라스 UV지도 JPEG에 겹쳐있는 경우 배경을 제거하고 하나의 TIFF 파일을 만듭니다. Lightwave ® 모형을 만드는 사람으로 TIFF 파일을 가져오기 및 UV 좌표를 할당합니다. 이온 주입 Skelegons와 본즈 Lightwave ® 모형을 만드는 사람에 skelegons은 설계 박혀 있는걸되었습니다. Skelegons는 뼈에 대한 자리 표시 자로 행동. 61 뼈를 만듭니다. 머리를 시뮬레이션하는 데 사용되는 하나의 대형 skelegon와 재키 용의에서 척추의 개수를 모방하는 skelegons를 만듭니다. 아래 꼬리의 끝에 목에 가상 척추를 만듭니다. 네발로 구성 앞발을 퓨즈가 척추 칼럼을, 각각 skelegons. 모든 skelegons가 생성되면 skelegons가 뼈로 변환 예정이다 Lightwave ® 레이아웃에 전체 개체를 보냅니다. 무게 음영을 추가 독립 중량지도 골고루 운동을 배포 -100 %에서 백퍼센트하는 값을 범위가 있습니다. 팔다리, 꼬리, 그리고 긍정과 머리 (+) 무게지도 값을 지정. (-) 중량지도 값 신체에게 부정적인주세요. 보관 비디오에서 자극 캡처 오르, 피터스, 에반스, &amp; 테일러, 2002,, 아카이브 도마뱀 영상은 (초기 비디오 취득, 오르 및 에반스, 2002을 참조하십시오 자사의 인클로저에 개최되는 살아있는 도마뱀에 소니 Trinitron 모니터 소니 MiniDV 디지털 비디오 레코더 PAL 플레이어를 사용하여 표시했습니다 반 다이크와 에반스, 2007). 도마뱀의 반응은 인클로저에서 미터에 약 배치 캐논 (MV650i) 디지털 캠코더를 사용하여 기록되었다. 도마뱀 크기는 디스플레이 모니터에 펏의 대표적인 크기로 축소되었다. 애플 퀵타임 ™ v7.0에서 개별 순차 JPEG 이미지로 내보낼 수 있습니다. 자극 복제에 대한 Rotoscoping &#39;Rotoscoping&#39;는 개체 애니메이션 (Gatsey, 미들턴, 젠킨스, &amp; Shubin, 1999) 배경 시퀀스의 전경에서 프레임별로 프레임 겹쳐있는 기술입니다. 빛, 카메라, 개체 및 배경 특성이 장면에 대해 제어할 수 Lightwave ® 레이아웃에 애니메이션 보내십시오. 애플 퀵타임 플레이어 ™ 7.0을 사용하여 이미지 시퀀스 (JPEG)로 보관 디지털 비디오 및 수출에서 운동 패턴을 선택합니다. 개체 카메라보기에 있어야합니다. 배경에 이미지 시퀀스의 첫 번째 JPEG를 가져옵니다. 뼈를 사용하여 개체를 조작하고 배경 이미지를 반영하는 현재 위치에 비교하십시오. 각 프레임은 객체의 위치가 저장되는 그러한 &#39;keyframed&#39;이 될 것입니다. 다음 연속 이미지로 배경 이미지를 교체하고 새 위치로 객체를 조작. 순서가 완료될 때까지 프로세스를 반복합니다. 완성을위한 순서를 렌더링 대형 시퀀스 클립 또는 배치 처리는 렌더 팜 사령관 v2.9을 사용하여 렌더링됩니다. 렌더 팜 사령관 (RFC)은 링크까지 컴퓨터를 빠르게 렌더링에 도움이 프로세서 일련의 생산. 넷 애플 맥 G5 듀얼 프로세서 (8 스레드)가되므로 사시간하는 시간을 줄이고, 렌더링을 배포하는 데 사용되었습니다. 모든 시퀀스는 PAL - DV 표준 (; 5시 1분 압축, 수평 해상도 575 라인, 25 프레임 S - 1 720 5백76엔 픽셀 해상도)에서 유지되었다.

<ol>
	<li>Gatesy, S. M., Middleton, K. M., Jenkins, F. A., Shubin, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+preservation+of+foot+movements+in+Triassic+theropod+dinosaurs.">Three-dimensional preservation of foot movements in Triassic theropod dinosaurs.</a> Nature. 399, 141-144 (1999).</li><li>Newtek, Inc, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lightwave+[8]+Reference+Manual.">Lightwave [8] Reference Manual.</a> , (2004).</li><li>Ord, T. J., Evans, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactive+video+playback+and+opponent+assessment+in+lizards.">Interactive video playback and opponent assessment in lizards.</a> Behav. Process. 59, 55-65 (2002).</li><li>Ord, T. J., Peters, R. A., Evans, C. S., Taylor, 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=Digital+video+playback+and+visual+communication+in+lizards.">Digital video playback and visual communication in lizards.</a> Anim. Behav. 63, 879-890 (2002).</li><li>Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+considerations+and+techniques+for+constructing+video+stimuli.">Design considerations and techniques for constructing video stimuli.</a> Acta Ethol. 3, 49-54 (2000).</li><li>Van Dyk, D. A., Evans, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Familiar-unfamiliar+discrimination+based+on+visual+cues+in+the+Jacky+dragon,+Amphibolurus+muricatus.">Familiar-unfamiliar discrimination based on visual cues in the Jacky dragon, Amphibolurus muricatus.</a> Anim. Behav. 74, 33-44 (2007).</li></ol>

자금은 맥쿼리 대학의 CISAB과 심리학과 대학원 수상하고, 호주 연구위원회에 의해 지원되었다. 연구는 맥쿼리 대학 동물 윤리위원회 (2003/014)와 뉴사우스 웨일즈 국립 공원 및 야생 동물 서비스 (S11024)의 윤리적 지침에 따라 실시되었다.

컴퓨터 생성 애니메이션은 점점 동물 행동의 통신에 관한 질문을 조사하는 인기있는 도구를지고 있습니다. 애니메이션 기법을 사용함으로써, 연구자들은 신호 설계의 특정 측면에 초점을, 그리고 이전에 한정된 기술에 의한 조사하지 못했습니다 기능을 조작할 수 있습니다. 종래의 무대 동물 아직도 이론적 원리를 보여주는 발생하지만, 애니메이션 기법은 이제 더 자세한 상호 작용을 조사위한 정교한 도구를 ?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>New Item</td>
<td>Digital camera</td>
<td>Konica Minolta Holdings, Inc. </td>
<td>VI-9i</td>
<td>Used to acquire 3D mesh (done by New Dawn&#174; - Bexley North, NSW Australia)
 
</td>
</tr>
<tr>
<td>Lightwave&#174; 3D</td>
<td>Software program</td>
<td>NewTek Inc.</td>
<td>v8.3</td>
<td>To create animation</td>
</tr>
<tr>
<td>Canon</td>
<td>Digital camera</td>
<td>Canon, Inc.</td>
<td>EOS 5D</td>
<td>12.8 megapixels</td>
</tr>
<tr>
<td>Grab</td>
<td>Software program</td>
<td>Apple Computer, Inc.</td>
<td>v1.2</td>
<td>Extract texture from photographs and UV map</td>
</tr>
<tr>
<td>Adobe&#174; Photoshop&#174; Elements</td>
<td>Software program</td>
<td>Adobe Systems Inc.</td>
<td>v3.0</td>
<td>Extract texture from photographs</td>
</tr>
<tr>
<td>Render Farm Commander</td>
<td>Software program</td>
<td>Bruce Rayne&#169;</td>
<td>v2.9</td>
<td>Mass rendering across local area network

</td>
</tr>
<tr>
<td>Mac Pro</td>
<td>Computer</td>
<td>Apple Computer, Inc.</td>
<td>&nbsp;</td>
<td>Four dual processors (eight threads); Create and render animation</td>
</tr>
<tr>
<td>Sony MiniDV</td>
<td>Digital video recorder</td>
<td>Sony Corporation</td>
<td>GV-D300E</td>
<td>Playback stimuli during stimulus acquisition</td>
</tr>
<tr>
<td>Sony Trintron</td>
<td>Colour viewfinder</td>
<td>Sony Corporation</td>
<td>PVM-14M2A</td>
<td>One monitor for stimulus acquisition</td>
</tr>
<tr>
<td>Canon</td>
<td>Digital camcorder</td>
<td>Cannon Inc.</td>
<td>MV650i</td>
<td>Records behavior during stimulus acquisition</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

computer-generated animal model stimuli

동물 간의 통신은 다양하고 복잡합니다. 동물, 지진, chemosensory, 전기, 또는 시각적 신호를 청각을 사용하여 통신할 수 있습니다. 특히, 통신을위한 영상 신호 설계에 제약을 이해하는 것이 큰 관심되었습니다. 동물 상호 작용을 조사하는 전통적인 방법은 기본적인 관측 기술을 사용한, 조우가 개최하거나, 형태학의 물리적 조작. 이하 관입 방법은 거울, 스틸 이미지, 또는 모델로 원유 재생 도구를 사용 conspecifics을 시뮬레이션하기 위해 노력했다. 기술이 고급되었다로서, 비디오 재생 영상을 검토하기 위해 다른 도구 (로젠탈, 2000)로 떠오르고있다. 그러나, 한 걸음 더 나아가 이동, 지금은 컴퓨터 애니메이션의 응용 프로그램은 연구자가 특별히 conspecifics에서 사회적 반응을 이끌어내는 데 필요한 핵심 구성 요소를 격리 수 있도록하고, 상호 작용을 제어하는​​ 이러한 기능을 조작. 자, 내가 모델로 재키 용이를 사용하여 애니메이션을 만드는 방법에 대한 세부 사항을 제공하지만,이 과정은 다른 종족에 대한 적응력이있을 수 있습니다. 애니메이션 건물, 내가 객체 형태를 변경 텍스처를 추가, 뼈를 설치하고, 과장된 움직임을 방지 비교할 체중 쉐이딩을 제공하기 위해 Lightwave 3D를 사용하여 선출. 애니메이션은 다음 중요한 운동 기능을 복제하는 모터 패턴을 선택할 수와 일치합니다. 마지막 순서는 프레 젠 테이션에 대한 개별적인 클립에 렌더링합니다. 다른 적응 기술이 있지만,이 특정 메서드에 눈에과 사회 모두에 응답을 도출에 효과 입증했다하면 상호 작용을 개최.

Watch this Scientific Journal Video about Computer-Generated Animal Model Stimuli at JoVE.com

Computer-Generated Animal Model Stimuli

컴퓨터에서 생성된 모델로 재키 용이를 사용하여 자극합니다.

컴퓨터가 만든 동물 모델 자극

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Research

JoVE Journal

Biology

10.8K 조회수.  Macquarie University. 컴퓨터에서 생성된 모델로 재키 용이를 사용하여 자극합니다.

비디오: Computer-Generated Animal Model Stimuli

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

비침 투 VIVO에서 스몰 애니멀 MRI 및 MRS : 기본 실험 절차

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

연구

생물학

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 &#x2013; 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6&deg;C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5&deg;C/min, enabling further dehydration, to a temperature of -34&deg;C before being plunged into liquid nitrogen (-196&deg;C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37&deg;C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

Preimplantation 캐틀의 태아, 양, 그리고 염소 Cryopreservation

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage.  Preimplantation embryos consist predominantly ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

이전 DNA의 정제없이 식물과 동물 샘플의 Genotyping

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

마우스 모델에서 자궁 - 난관 배아 전송 및 정관

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

유전자를 사용 하 여 프로토콜 변환 연구에 대 한 적절 한 동물 모델을 식별 하기 위해 농축 분석 설정

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

기본 소 Granulosa 에스트로겐 생산의 조직 문화 모델 세포

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

소 유선 생 검 기술

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

유해 자극 감지를 위한 양적 행동 판독으로 기획자 스크런싱

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

소 난소 피질 조직 배양

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...