저자는 그림 1과 도움과 토론 연구소의 다른 회원 배아 헤드를 도시에 아이린 최 감사드립니다. 이 작품은 NIH 보조금 DE012728 (TW)에 의해 지원됩니다

1. Dispase II 준비 <ol><li> 준비 신선한 Hepes 버퍼 식염 (HBS) (50 밀리미터 Hepes / 코 산도 7.4, 150 MM NaCl).에게 16.8 ML Ultrapure H 2 O.으로 2 ML 0.5 M Hepes / 코 산도 7.4 및 1.2 ML 2.5 M NaCl을 추가 </li><li> 10 MG / ML Dispase II하십시오. 0.2 g Dispase II를 (로슈, 고양이 # 4942078001) 무게 20 ML HBS에 용해. 장기 저장을 위해 -20 ° C에서 각각 Eppendorf 튜브 및 저장소에 1.0 ML의 aliquots를 준비합니다. </li><li> 사용하기 전에 PBS에서 10 MG / ML Dispase II 1시 10분을 희석. 얼음에 희석 Dispase II를 놔. </li></ol> 2. 임신 여성에서 CD-1 E10.5의 태아를 해부 모든 동물 실험은 콜로라도 덴버의 대학 (UCD) 동물 관리 및 사용위원회의 승인을 프로토콜에 따라 수행되었다. CD-1 (ICR) 마우스는 할란 연구소 (인디애나 폴리스, IN)에서 얻은되었습니다. <ol><li> 70 % 에탄올로 분해 공간 및 도구를 청소하십시오. </li><li> approv을 사용하여 임신 CD-1 여성을 안락사시켜야에드 프로토콜. </li><li> 70 % 에탄올로 복부를 뿌려서하면 다음 복부를 엽니 다. 자궁에서 mesometrium을 멀리 찢어 자궁과 다른 끌어 포셉 한 세트를 사용합니다. 몸에서 자궁을 제거합니다. 복부의 다른 쪽 자궁 뿔에 대해 반복합니다. </li><li> 간단히 피를 씻어 10 cm 배양 접시에 얼음 차가운 PBS로 자궁을 씻어. </li><li> 얼음 차가운 PBS로 새 페트리 접시에 자궁을 전송합니다. 주입 사이트 사이에서 절단하여 한 배아 조각으로 분리합니다. </li><li> stereomicroscope 아래 얼음처럼 차가운 PBS에 하나 배아를 전송합니다. 난황 주머니에 배아를 노출 할 수있는 컷 사이트 중 하나에서 시작 자궁의 근육 벽을 찢어 고급 집게를 사용하십시오. 난황 주머니와 양막 보지. 얼음에 PBS로 6 cm 페트리 접시에 해부하는 배아를 전송합니다. 모든 배아는 이런 방식으로 수집 될 때까지 진행합니다. </li></ol> 3. Dispase II 치료 <ol><li> 6 cm 배양 접시에있는 PBS 모든 배아를 씻으십시오. 교체PBS 10 ML 희석 Dispase II와 PBS. 25 분에 37 ° C에서 페트리 접시 및 배양을 커버 - 세균 배양기 나 따뜻한 방은 충분해야합니다 - 우리는이 단계의 CO 2 조직 문화 인큐베이터를 사용하지 않았습니다. </li><li> 페트리 접시를 타고 stereomicroscope 아래의 배아를 관찰합니다. 외배엽이 느슨되고있는 것,이 조직 계층과 기본 mesenchyme 사이에 명확한 차이가 typified. 그렇지 않은 경우, 37에서 추가로 5 분 ° C.에 품다 그런 다음 얼음에 페트리 접시를 넣어. </li></ol> 4. 그대로 얼굴 프로미넌스 prominences을 해부 <ol><li> (VWR, 고양이 # 14672-380) 일회용 유리 피펫을 사용하여 stereomicroscope 미만 Dispase II는 6 cm 배양 접시에 얼음처럼 차가운 PBS로 배아를 취급 전송합니다. </li><li> 배아를 개최 할 포셉 한 쌍의를 사용하십시오. 머리에 부착 된 얼굴 프로미넌스 prominences의 경계를 (그림 1의 경계를 표시자를주의 깊게 고급 집게의 또 다른 한 쌍의를 사용하여얼굴 프로미넌스 prominences). 하나 MnP의 측면, 다음 MxP, 그리고 마지막으로 FNP에서 시작합니다. 그대로 밖으로 얼굴 프로미넌스 prominences (그림 2A)를 해부하기 위해 반대쪽으로 동일한 작업을 수행. </li></ol> 5. Mesenchyme에서​​ 별도의 얼굴 외배엽 <ol><li> 3 배 작동 거리와 stereomicroscope 아래 얼굴 프로미넌스 prominences을 관찰합니다. 부드럽게 위아래로 5 배 얼굴 프로미넌스 prominences을 피펫하는 긴 유리 파스퇴르 피펫을 사용합니다. 이 후, 외배엽으로 흩어져 용이합니다. </li><li> 얼굴 프로미넌스 prominences을 보유 할 포셉 한 쌍의를 사용하십시오. 아주 천천히 외배엽에서 껍질에 포셉의 다른 쌍을 사용합니다. 그림 2B는 얼굴 외배엽의 분리 시트를 보여줍니다. </li><li> 얼음에 PBS와 1.5 ML Eppendorf 튜브에 얼굴 외배엽를 전송하는 새로운 긴 유리 파스퇴르 피펫을 사용합니다. 얼음에 PBS와 함께 또 다른 1.5 ML Eppendorf 튜브에 mesenchyme를 전송합니다. </li><li> 각각의 해부를위한 새로운 얼음 차가운 PBS를 사용합니다. 샘플을 수집합니다. </li><li> t을 씻으십시오PBS로 그는 샘플. 4 ° C, 3 분 500 XG에서 원심 분리기. </li><li> 긴 유리 파스퇴르 피펫을 사용하여 PBS를 대기음. 샘플 칩 분석을 위해 사용됩니다 경우, 6.1 단계로 진행합니다. RNA 추출의 경우, 6.2 단계로 진행합니다. 단백질 추출의 경우, 6.3 단계로 진행합니다. </li></ol> 6. 또한 실험에 대한 공정 샘플 <ol><li> 칩 분석을위한 샘플 <ol><li> crosslink에 1 %의 포름 알데히드와 함께 1 ML PBS를 추가합니다. 샘플을 균질화. </li><li> 회전 10 분 동안 실온에서 알을 품다. </li><li> unreacted 포름 알데히드을 해소하기 위해 각 튜브에 1.25 M 글리신의 110 μl를 추가합니다. </li><li> 섞어서 5 분 동안 실온에서 알을 품다. </li><li> 3 분에 대해 4 ° C에서 500 XG에서 봐. 표면에 뜨는 제거합니다. </li><li> 조직을 씻어 한 ML 차가운 PBS를 추가합니다. </li><li> 3 분에 대해 4 ° C에서 500 XG에서 봐. </li><li> PBS를 제거하고 PBS 한 번 씻어 반복합니다. </li><li> 표면에 뜨는 제거, 액체 질소의 조직을 고정 스냅인을 종료합니다. SA를 저장-80 ° C에서 후속 풀링 및 처리를 위해 mples. </li></ol></li><li> RNA 추출을위한 샘플 <ol><li> RNAlater (Ambion)를 포함하는 튜브의 얼굴 외배엽과 mesenchyme를 놓습니다. </li><li> ° C는 조직의 철저한 침투를 허용 할 4에 박 RNAlater 솔루션에 샘플을 품다. </li><li> 이후 풀링 및 처리를 위해 -20 ° C에서 저장합니다. </li></ol></li><li> 단백질 추출을위한 샘플 </li></ol> 후속 처리를 위해 샘플에 용해 버퍼를 추가합니다. 또는 액체 질소의 조직을 동결 이런. -80 ° C에서 후속 풀링 및 처리를 위해 샘플을 저장합니다. 참고 사항 <ol><li> 시간 제약. 을 통해 해부의 시작부터 제 6의 시작 부분에 4-6 시간을 계획합니다. </li><li> 때 실력, 그것은 섹션 4와 5 (명성의 해부 및 외배엽 분리)를 수행하기 위해 배아 당 ~ 10-15 분 정도 소요됩니다. </li><li> 샘플 RNA 분리, 모든 solutio에있는 경우N 먼저 Diethylpyrocarbonate로 치료해야합니다. 또한 포셉과 멀리 RNase있는 작업 공간을 청소. </li><li> Dispase II 처리 및 dissections를 제외한 모든 시간에 얼음에 샘플을 보관. 해부에 사용 된 PBS 감기에 있는지 확인하기 위해 각 절개에 대한 새로운 얼음 차가운 PBS로 변경합니다. </li><li> 샤프 포셉 정확한 절개를 얻을 중요합니다. 모든 오염을 방지하기 위해 완전히 포셉를 청소합니다. </li><li> 다른 프로미넌스 prominences에서 샘플을 수집하기 위해 Dispase II 치료 후 각각 부각을 해부. 그런 다음 외배엽과 mesenchyme 구분합니다. </li><li> Dispase II 치료 후 얼굴 외배엽이 흩어져 쉽게합니다. 다른 외배엽에서 오염에 특별한주의를 기울이십시오. 모든 잠재적으로 오염 된 조직을 폐기하십시오. </li><li> 일단 샘플 제 6 항에서 해당 지점에 도달 할 때까지 멈추지 말고, 시작합니다. 얼굴 외배엽의 분리를 완료하고 최대한 빨리 할 수​​있는 한 모든 배아에 대한 mesenchyme. </li><li> 전자Dispase II의 nzyme 활동은 일괄 및 브랜드 사이에 다를 수 있습니다. 이것은 다른 브랜드와 Dispase II의 배치에 대한 치료 시간을 조정할 필요가 있습니다. </li></ol>

<ol>
	<li>Wilkie, A. O., Morriss-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+craniofacial+development+and+malformation.">Genetics of craniofacial development and malformation.</a> Nat. Rev. Genet. 2, 458-468 (2001).</li><li>Gritli-Linde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2+-+The+Etiopathogenesis+of+cleft+lip+and+cleft+palate:+usefulness+and+caveats+of+mouse+models.">Chapter 2 - The Etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models.</a> Current Topics in Developmental Biology. 84, 37-138 (2008).</li><li>Schutte, B. C., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+faces+and+factors+of+orofacial+clefts.">The many faces and factors of orofacial clefts.</a> Human Molecular Genetics. 8, 1-7 (1999).</li><li>Chai, Y., Maxson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+craniofacial+morphogenesis.">Recent advances in craniofacial morphogenesis.</a> Dev. Dyn. 235, 2353-2375 (2006).</li><li>Jiang, R., Bush, J. O., Lidral, A. C. <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+upper+lip:+Morphogenetic+and+molecular+mechanisms.">Development of the upper lip: Morphogenetic and molecular mechanisms.</a> Dev. Dyn. 235, 1152-1166 (2006).</li><li>Reid, B. S., Yang, H., Melvin, V. S., Taketo, M. M., Williams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectodermal+WNT/&#946;-catenin+signaling+shapes+the+mouse+face.">Ectodermal WNT/&#946;-catenin signaling shapes the mouse face.</a> Developmental Biology. 349, 261-269 (2011).</li><li>Feng, W., 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=Spatial+and+temporal+analysis+of+gene+expression+during+growth+and+fusion+of+the+mouse+facial+prominences.">Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.</a> PLoS ONE. 4, e8066 (2009).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat. Genet. 37 (11), 1181-1186 (2005).</li><li>Nagai, T., Aruga, J., Takada, S., 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+expression+of+the+mouse+Zic1,+Zic2,+and+Zic3+gene+suggests+an+essential+role+for+Zic+genes+in+body+pattern+formation.">The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.</a> Developmental Biology. 182, 299-313 (1997).</li><li>Britsch, S., 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+transcription+factor+Sox10+is+a+key+regulator+of+peripheral+glial+development.">The transcription factor Sox10 is a key regulator of peripheral glial development.</a> Genes Dev. 15 (1), 66-78 (2001).</li></ol>

저자는 경쟁 이익이 문서의 내용과 관련된 기타 충돌이 없습니다.

이 프로토콜은 초기 Dispase II 치료 단계에 따라 배아 마우스 얼굴 외배엽과 mesenchyme을 분리 할 수​​있는 간단한 방법을 제공합니다. 이전 연구에서, 우리는 이전 Dispase II 치료에 얼굴 프로미넌스 prominences의 절개를 수행했지만, 우리는 지속적으로 mesenchyme는 &quot;끈적&quot;과 조작이 더 어려워지고있는 것으로 나타났습니다. 새로운 프로토콜은이 문제를 방지합니다. 조합 Dispase II 아래로 pipetting과...

<table>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <td>Dispase II (neutral protease, grade II)</td>
 <td>Roche</td>
 <td>4942078001</td>
 <td>Make 10 mg/ml Dispase II stock solution in HBS. Aliquot and store at -20 &deg;C . </td>
 </tr>
 <td>RNAlater</td>
 <td>Ambion</td>
 <td>AM7021</td>
 <td>&nbsp;</td>
 </tr>
</table>

separation of mouse embryonic facial ectoderm and mesenchyme

Orofacial 갈라진 틈은 1,000 신생아 세계 1,2에서 1.5에 영향을 미치는 가장 자주 craniofacial 결함입니다. Orofacial clefting는 이상 얼굴의 개발 3로 인해 발생합니다. 얼굴의 인간과 마우스, 초기 성장 및 패턴에서 조직의 몇몇 작은 꽃 봉오리, 4,5 얼굴 프로미넌스 prominences에 의존하고 있습니다. 이점 정면 코 프로세스 (FNP), 턱의 프로미넌스 prominences (MxP)와 mandibular의 프로미넌스 prominences (MDP) : 얼굴은 여섯 가지 주요 프로미넌스 prominences에서 파생됩니다. 이러한 프로미넌스 prominences는 놓인 상피으로 쌌다 아르 mesenchyme의 swellings로 구성되어 있습니다. 여러 종의 연구는 얼굴 외배엽과 mesenchyme 사이의 누화 신호를하면 얼굴 6 형성에 중요합니다 것으로 나타났습니다. 그러나,이 신호를 릴레이에 참여 유전자에 관한 기계론의 내용은 부족합니다. 하나의 유전자 표현의 포괄적 인 이해를 얻을 수있는 방법 구속력이 전사 인자 및 develo과 관련된 염색질 마크핑 얼굴 외배엽과 mesenchyme은 분리 조직 구획을 분리하고 특성화하는 것입니다. 여기 배아 일 (E) 10.5 프로미넌스 prominences의 융합을 앞에 마우스 얼굴 형성에 중요한 발달 단계에서 얼굴 외배엽과 mesenchyme를 분리하기위한 방법을 제시한다. 우리의 방법은 우리가 이전에 얼굴 프로미넌스 prominences에게 7 해부에 사용했던 방법에서 구성된다. 이 이전 연구에서 우리는 근친 C57BL을 고용 한 /이 변형 등 6 마우스 유전학, 유전체학 및 얼굴 형태 8에 대한 표준이되었습니다. 여기하지만 가능한 조직의 더 한정된 수량으로 인해, 우리는 축산에 대해보다 강력한를 구입 저렴 outbred CD-1 변형을 활용, 모든 근친보다 쓰레기 당 더 많은 배아 (12-18)를 생산 돌봐 있습니다 마우스 변형 8. 배아 분리 후, 중성 단백 분해 효소 Dispase II는 전체 배아을 치료하는 데 사용되었습니다. 그런 다음 얼굴 프로미넌스 prominences가 해부했다에드 아웃, 그리고 얼굴 외배엽은 mesenchyme에서​​ 분리되었다. 이 방법은 얼굴 외배엽과 mesenchyme 모두 그대로 유지합니다. 이 방법을 사용하여 얻은 샘플은 단백질 감지, 염색질 immunoprecipitation (칩) 분석, microarray 연구 및 RNA-seq 등의 기술을 사용할 수 있습니다.

Dispase II 치료 후, 배아의 외배엽이 늘어 경향이있다. 얼굴 프로미넌스 prominences는 절개 (그림 2A) 후 그대로 있어야합니다. 격리 된 얼굴 외배엽는 명확하고 mesenchyme 조직 (그림 2B)은 무료입니다. 프로토콜의 효능을 결정하기 위해 우리는 forebrain에 대한 assayed 교차 오염을 감지 할 수 Zic3 9 ectodermal 특정 cdh1 5 역 transcriptase PCR을 사용하여 중간 엽 특정 sox10 <sup...

Watch this Scientific Journal Video about Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme at JoVE.com

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

태아 얼굴 외배엽과 mesenchyme의 분리를위한 프로토콜이 설명되어 있습니다. 우리는 먼저 전체 배아를 취급하는 전체 얼굴 프로미넌스 prominences을 해부하기 위해 Dispase II를 사용하고 얼굴 외배엽과 mesenchyme 구분합니다.

마우스 배아 얼굴 외배엽과 Mesenchyme의 분리

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

separation-of-mouse-embryonic-facial-ectoderm-and-mesenchyme

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10.7K 조회수.  University of Colorado Denver Anschutz Medical Campus. 태아 얼굴 외배엽과 mesenchyme의 분리를위한 프로토콜이 설명되어 있습니다. 우리는 먼저 전체 배아를 취급하는 전체 얼굴 프로미넌스 prominences을 해부하기 위해 Dispase II를 사용하고 얼굴 외배엽과 mesenchyme 구분합니다.

비디오: Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

단백질의 전기 영동 분리

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

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Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

절연 및 마우스 배아 새싹 세포의 유도

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

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 ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

배아 마우스 신장의 Nephrogenic 지대의 세포의 분리 및 문화

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

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

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

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

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

의 형광 단백질의 편광 Translocation XenopusWnt 신호에 응답> Ectoderm

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

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Craniofacial Mesenchyme의 Electroporation

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

모터 뉴런에 마우스 배아 줄기 세포의 효율적인 분화

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Culturing 인간의 배아와 유도 Pluripotent 줄기 세포에 적합 마우스 배아 Fibroblast 세포의 준비

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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 ...