이 프로토콜은 제인 다드, 토마스 Jessell와 앤드류 럼즈던의 연구소에서 개발되었다. 방법은 콜라겐의 척수의 문화 explants에 콘레드 바슬러, 앤 Calof, 토마스 에들런드, 필 해밀턴, Domna Karagogeos, 아리엘 루이즈 I Altaba 및 Toshiya 야마다 등 많은 사람들이 결정. Marysia Placzek과 마크 테시어 - 라빈은 축삭지도 분자를 식별하는 수단으로 체외의 explants에서에서 축삭 성장을 사용할 수있는 기술을 개척. 버틀러 실험실에서 작품은 NIH / NINDS에서 천과 R01 NS063999의 3 월부터 기금에 의해 지원됩니다.

1 부 : Transfected COS 세포 집계의 준비 공중 방울을 사용하여 <ol><li> 종자 COS - 7 (COS) 35mm 문화 요리 세포. 그들이 제조 업체의 프로토콜에 따라 Lipofectamine2000을 사용하여 세포에 플라스미드 표현의 80 % confluency, transfect 1μg에 도달했을 때. </li><li> 방울 매달려 준비하려면, transfection 미디어를 기음과 1ml 1X의 인산 버퍼 솔루션 (PBS)로 transfected COS 세포를 헹굴. 15 분 동안 효소가없는 세포 분리 매체의 0.5ml로 세포를 처리합니다. 반응을 중지 Opti - 멤 + 1X 페니실린 / 스트렙토 마이신 / 글루타민 (P / S / G) + 10% 태아 소 혈청 (FBS)의 솔루션의 1ml을 추가합니다. </li><li> 문화 요리의 표면에서 그들을 제거하고 15ml 원뿔 튜브로 전송하는 세포를 씹다. 펠렛 세포에 2000K에서 2 분 동안 회전. Opti - 멤 + 1X P / S / G + 10% FBS의 100μl에 뜨는 및 resuspend 펠렛을 제거합니다. </li><li> 접시의 바닥 위에 뚜껑 장소 반전, 35mm 문화 접시 뚜껑의 안쪽에 여러 20μl 방울을 스팟과 세포 집계까지 몇 시간 동안 37 ° C 배양기에서 두십시오. </li></ol> 2 부 : 도설 척수 Explants의 작성 <ol><li> 어머니의 자궁에서 E11 랫 배아를 해부하다 필요한까지 L15 매체 얼음 계속. </li><li> 날카롭게 텅스텐 바늘로 즉시 forelimb 버드 아래의 지역에서, 각 배아의 줄기에서 4-6 세그먼트 섹션을 제거합니다. 플라스틱 피펫을 사용하여, 4 잘 Nunc 요리를 잘 하나에서 조직 조각을 수집하고 얼음 계속. </li><li> 모든 배아 세그먼트가 해부를하고있을 때, 1ml L15의 솔루션 + Nunc 요리 잘 순식간에 1mg dispase에 조각을 전송할 수있는 플라스틱 피펫을 사용합니다. 오분 이상없이 오래 실온에서 알을 품다. 이상 dispase있는 조직 조각을 품어하지 마십시오. </li><li> 부화하는 동안, 페트리 접시와 혼합하는 소용돌이에 L15 약 10ml에 가열 inactivated 정상 염소 혈청 (HIGS)의 0.5ml를 추가합니다. 삼분의 일에 잘 Nunc 요리의 솔루션을 1ml를 전송하고 부화가 끝나면이 솔루션에 조직 조각을 전송할 수 있습니다. 얼음 계속. 참고 : 조직 조각이 시간 동안 얼음에 &quot;나머지&quot;를 허용하는 경우 이후의 절개가 쉬울 것입니다. </li></ol> 파트 3 : 마중물 콜라겐 <ol><li> 360μl 콜라겐을 40μl 10X 최소 필수 매체를 추가 튜브를 flicking 또는 간단히 vortexing하여 신속하게 철저하게 섞는다. picofuge에 빠른 속도로 스핀 다운. 이 솔루션은 노란색 것입니다. 최대한 얼음에 보관. </li><li> (아래 참고 참조) 약간 주황색 콜라겐 솔루션을 설정하는 충분한 0.8M 나트륨 중탄산염을 (NaHCO 3) 추가합니다. 다시 말하지만, flicking에 의해 신속하게 그리고 철저하게 혼합하고, picofuge에 잠시 스핀 다운. 솔루션은 혼합 후 분홍색 남아있을 경우, 당신은 너무 NaHCO 3 추가하고 콜라겐의 새로운 튜브로 다시 시작해야합니다. </li><li> 이 시점에서 콜라겐 솔루션은 준비하는 것입니다. 그것은 얼음에 액체 유지되지만, 실온에 데려왔을 때 약 5 분 (분홍색으로 변했) 내에 고체화됩니다. </li><li> 4 잘 Nunc 요리의 각 우물에 콜라겐의 스팟 20μl. 당신의 피펫의 팁을 사용하여 작은 &quot;패드&quot;를 형성하는 콜라겐을 확산. 상온에서 설정 콜라겐을 보자. </li></ol> 참고 : NaHCO 3의 정확한 금액은 콜라겐의 각 배치에 대해 titrated해야합니다. 낮은 시작 (11μl)와 솔루션은 오렌지의 약간 그늘 때까지 다음 0.5 - 1μl stepwise를 추가합니다. &quot;오른쪽&quot;금액은 보통 콜라겐 분홍색을 돌려 것이다 NaHCO 3의 작은 금액보다 1ul 적습니다. NaHCO 3의 금액은 선형 위 또는 아래로 확장되지 않습니다. 4 부 : 도설 척수의 Explants과 콜라겐 매트릭스에서 COS 세포 집계와 함께 그들의 위치의 해부 <ol><li> 갓 둘레에 텅스텐 바늘과 고급 포셉 한 쌍의 (# 5 # 55)를 사용하여 척수를 둘러싼 mesoderm를 제거합니다. </li><li> 텅스텐 바늘로 바닥 판에 평행하게 절단하는 것은 신중하게 척수의 배면의 다섯째를 제거합니다. </li><li> 모서리가로 깨끗하게 가능한 절단하고 있는지 만드는 등의 척추 explant를 이등분하다. 필요한 경우, 모서리를 다시 잘라. 약에 가져온 유리 모세관가 장착되어 입을 피펫을 사용하여 별도의 콜라겐 패드에 각 지느러미 척추 explant을 전송합니다. 지름 0.5mm. </li><li> 지느러미 척추 explant의 측면 가장자리로 거의 같은 넓이의 정사각형으로 transfected COS 세포의 집계를 잘라. </li><li> 입 피펫을 사용하여 지느러미 척추 explant과 콜라겐 패드에이 사각형 중 하나를 전송합니다. </li><li> 입 피펫으로 입 피펫을 사용하여 초과 액체를 대기음. 기음 이상하지 마십시오. </li><li> 패드에 준비하는 콜라겐의 4μl를 적용하고, 텅스텐 바늘을 사용하여직접 조직을 건드리지 않고 explants 통해 콜라겐 이동합니다. explants을 둘러싸고있는 콜라겐의 텅스텐 바늘을 이동하여, 동양 explants 있도록 COS 세포의 집계가 등의 척추 explant의 측면 가장자리에 인접한입니다. </li><li> 콜라겐 설정한 후 explants가 콜라겐에 완전히 쌌다되는 것을 보장하기 위해 콜라겐의 4μl의 응용 프로그램을 반복합니다. </li><li> 조직 문화 후드에서 30-40시간에 대한 Opti - 멤 + 1xP/S/G 문화 0.5ml를 추가합니다. </li><li> 얼음 접시를 유지, 1xPBS에 0.​​5ml 4 % paraformaldehyde로 45 분간 explants을 수정. </li><li> 1xPBS의 차단 솔루션 + 0.1 % 트리톤 - X100 1퍼센트의 HIGS (PBTN)의 0.5ml로 두 번 씻으십시오. 4 블록 ° C 시간 중 적어도 몇, 야간 부화가 immunohistochemistry에 의해 처리 후 바람직하고 있습니다. </li></ol> 제 5 부 : Immunohistochemistry에 의해 Commissural Axons의 시각화 <ol><li> PBTN에서 차단 후, 포셉 및 48 잘 접시에 장소 4 잘 접시의 explants을 잘라 버릴거야. </li><li> 각 잘하는 기본 항체의 200μl를 추가하고 4 박 떠나 ° C. commissural axons을 시각화하기 위해 마우스 방지 Tag1 항체의 1시 6분 솔루션을 사용합니다. transfection가 성공 여부를 결정하기 위해, 또한 신호 분자가 에피토프이 태그되었는지 중 신호 분자가 테스트하거나 관련 에피토프되는 대한 일차 항체를 포함합니다. </li><li> 4 PBTN 5 X 1시간 ° C. 각 샘플을 와시 </li><li> 각 잘하는 이차 항체의 200μl를 추가하고 4 박 떠나 ° C. 안티 Tag1 항체가 기본 항체로 사용했다면, FITC 또는 Cy3 중 하나에 결합 염소 항 마우스 IgM 항체 보조를 사용합니다. 가벼운 민감한 차 항체를 사용하면 FITC 또는 Cy3로 결합하는 경우,이 시점부터는 호일로 덮여 48 잘 요리를 보관. </li><li> 각 샘플 5 4에서 X 1시간 PBTN 각 ° C. 와시 </li><li> , 3 잘 우울증 슬라이드에 explant을 전송 Vectashield 매체와 coverslip에 마운트를 초과 액체를 제거합니다. 4 ° C 또는 -20 ° C. 중 하나에서 빛이 금고에 저장 </li></ol> 6 부 : Explants 대표 이미지 성공적인 실험에서, explant와 C​​OS의 집계가 서로 인접 유지하고 콜라겐이 세트로 떨어져 표류하지 않습니다. axons의 최소한의 전면에 자라난 것이있을 것이다; 중요한 axonal 전면에 자라난가 등의 척추 explant 너무 오래 교양있는 것을 나타냅니다. explant에서 성장하고 더 blebs있을 수 없다, 조직 문화 매체와 직접 접촉으로 온다면 blebbing이 발생합니다. axons을 방향으로 신호 분자의 기능은 공촛점 현미경을 사용하여 평가됩니다. 축삭의 reorientation의 범위는 COS 세포 집계 3 가장 가까운 axons의 reorientation의 평균 각도를 측정하여 계량하실 수 있습니다. 그림 1에 표시된, COS 세포는 표현 myc - 태그 BMP7 (파란색)가 지붕 판의 explant가 commissural axons를 (그림 들어갈 수있는 방식과 유사 BMP7의 원본 (그림 1B)에서 멀리 Tag1 + commissural axons (녹색)를 방향 1A). 제어 벡터를 표현 COS 세포는 commissural axons 3,4의 방향에 영향을 미치지 않습니다. 이러한 explants 또한 모터 뉴런과 지느러미 interneurons을 장식 전사 인자 Isl1 / 2 (빨간색)에 대한 항체로 분류되었습니다. 이 결과는 최초로 표시했다는 BMP 가족 mediates의 일원 지붕 판 chemorepellent 3 활동. <img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /> 그림 1 : 제발 <a href="https://www.jove.com/files/ftp_upload/1853/1853fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다.

<ol>
	<li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The development of the rat spinal cord. , (1984).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+recombinations+in+collagen+gels.">Tissue recombinations in collagen gels.</a> Methods in molecular biology. , 325-335 (2008).</li><li>Augsburger, A., Schuchardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPs+as+mediators+of+roof+plate+repulsion+of+commissural+neurons.">BMPs as mediators of roof plate repulsion of commissural neurons.</a> Neuron. 24, 127-141 (1999).</li><li>Butler, S. J., Dodd, 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+role+for+BMP+heterodimers+in+roof+plate-mediated+repulsion+of+commissural+axons.">A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons.</a> Neuron. 38, 389-401 (2003).</li><li>Kennedy, T. E., Serafini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord.">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78, 425-435 (1994).</li><li>Charron, F., Stein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+is+an+axonal+chemoattractant+that+collaborates+with+netrin-1+in+midline+axon+guidance.">The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance.</a> Cell. 113, 11-23 (2003).</li><li>Lumsden, A. G., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323, 538-539 (1986).</li><li>Dodd, J., Morton, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+axonal+glycoprotein+expression+on+subsets+of+embryonic+spinal+neurons.">Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons.</a> Neuron. 1, 105-116 (1988).</li><li>Yamauchi, K., Phan, K. D., Butler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+type+I+receptor+complexes+have+distinct+activities+mediating+cell+fate+and+axon+guidance+decisions.">BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions.</a> Development. 135, 1119-1128 (2008).</li></ol>

이 분석을 수행하는 성공을 결정하는 중요한 요인이 처음이고, 조직이 너무 연장 기간 동안 dispase로 치료해서는 안됩니다, 이러한 치료는 조직이 매우 끈적되고 및 생존 감소가 발생하게됩니다. 둘째, 콜라겐은 완벽하게 준비하는과 최대한 얼음에 보관해야합니다. 이 설정 시작하면 그것은 처리하기 위해 점점 더 어려워 될 것입니다, 핑크 차례 즉. 세는 텅스텐 바늘은 항상 매우 날카로운 보관해...

assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons

척추 척수 1 도설 commissural axons은 축삭지도 신호를 식별하는 귀중한 모델 시스템을왔다. 여기, 우리는 시험 관내 분석, commissural axons의 방향 2 외부와 내장 신호의 효과를 연구하기 위해 광범위하게 사용되고 있습니다 &quot;reorientation 분석&quot;에 대해 설명합니다. 에 chemorepellent BMP를 포함하여,이 분석은 제인 다드, 토마스 Jessell와 앤드류 럼즈던의 실험실에서 수많은 사람들에 의해 개발되었습니다 (자세한 내용은 응답을 참조)이 분석의 버전은 주요 축삭지도 분자의 reorientation 활동을 증명하는 데 사용되었다 지붕 판 3,4 및 Netrin1 5 소닉 헤지 호그 (쉬) 척수의 바닥 판에 6 chemoattractive 활동. 척수의 지느러미 3 분의 2로부터 2-3 세그먼트로 구성된 Explants은 배아 일 (E) 11 쥐에서 해부하고 입체 콜라겐 젤 7 양식입니다. E11 등의 척추 explants은 당단백질, Tag1 8 그들 axonal 표현식에서 확인할 수 있습니다 새로 태어난 commissural 뉴런을 포함합니다. 문화 30-40시간 과정 동안 commissural 축삭의 궤도는 생체내에서 볼 비슷한 시간 코스 이들 지느러미 explants에 recapitulated 있습니다. 이 axonal 탄도는 테스트 조직이나 지느러미 explant의 측면 가장자리 중 하나를 접촉 후보 신호 분자를 표현 COS 셀 집합 중 하나를 배치하여 도전을 수 있습니다. 추가 조직의 주변에 연장 Commissural axons은 내생 지붕 판과 이소성 조직 측면에서 모두 신호의 영향 아래에서 성장합니다. commissural axons 이러한 상황에서 reoriented 아르 정도는 계량하실 수 있습니다. 이 분석을 사용하여 그것은 모두 commissural 궤도 9 직접이 신호의 필요성뿐만 아니라 commissural axons에게 3,4를 방향에 특정 신호의 자족을 확인 할 수 있습니다.

Watch this Scientific Journal Video about Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons at JoVE.com

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

이 분석은 commissural axons를 방향에 대한 신호 분자의 능력, 여기에 본 Morphogenetic의 단백질 7 (BMP7)을 평가합니다. 배아 등의 척수의 explant는 COS 세포의 secreting 후보 성장 요인의 집계에 인접한 양식입니다. explant 내에서 성장 Reoriented commissural axons은 immunohistochemistry에 의해 시각입니다.

배아 척수 Commissural Axons을 방향을 Diffusible 신호 분자의 능력을 시금

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

assaying-ability-diffusible-signaling-molecules-to-reorient-embryonic

Research

JoVE Journal

Biology

8.7K 조회수.  University of Southern California. 이 분석은 commissural axons를 방향에 대한 신호 분자의 능력, 여기에 본 Morphogenetic의 단백질 7 (BMP7)을 평가합니다. 배아 등의 척수의 explant는 COS 세포의 secreting 후보 성장 요인의 집계에 인접한 양식입니다. explant 내에서 성장 Reoriented commissural axons은 immunohistochemistry에 의해 시각입니다.

비디오: Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

신경 소마와 Axons의 Compartmentalization위한 Microfluidic 장치의 제작

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

연구

생물학

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

척수 전기 생리학

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

마우스 배아의 폐 문화, 분기의 분자 메커니즘을 평가하는 시스템

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

교양 차 마우스 배아 섬유아 세포의 신호 분자의 아데노 바이러스 - 매개 유전자 제거

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

배아 줄기 세포 생활에서 염색질 단백질 역학을 측정하는 Assays (FRAP 및 플립)를 Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2의 키나제 활동 시금 체외에서</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

위한 시스템<em&gt; 예 생체</em배아 췌장의&gt; 배양

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Blastomere Explants는 배아 개발하는 동안 세포 운명의 헌신에 대해 테스트 할

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

세포 단일 층의 기계적 자극에 의​​한 칼슘 웨이브 전파 : 소 각막 내피 세포의 예

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).