내 실험실에서 연구는 생물학의 시작 자금의 WKU학과, WKU 연구 재단 RCAP-I는 # 11-8032을 부여하고 지원하는 국립 연구소의 종합 의료 과학 국립 연구소에서 부모 부여를 통해 자금 KBRIN 면적 그랜트 건강의 보너스 번호 5P20GM103436-13에서. 또한 그의 실험실 내가 먼저 쑤 실험실에서이 기술을 확립이 기술 박사 레이몬드 Pagliarini에 소개 된 박사 티안 쑤에 감사드립니다.

1. 양성 비 침습적 종양의 유도 <ol><li> 양성 종양의 유도를 위해 표 2에 주식을 사용합니다. </li><li> 다음과 같은 유전자형의 &quot;테스터&quot;주식에 대한 스타터 문화를 준비, 욕조 - Gal80, FRT40A, Y, EY-FLP1, w Act5C&gt; Y +&gt; 인 Gal4, UAS - GFP </li><li> 다음과 같은 유전자형의 &quot;테스트&quot;주식에 대한 스타터 문화를 준비 W; FRT40A, UAS - 라스 V12 / CYO </li><li> &quot;테스트&quot;주식에서 &quot;테스터&quot;주식 ~ 10 여성 처녀 십 명의 남성을 수집합니다. </li><li> 플라이 음식과 함께 유리 병에 모두 배치하여 &quot;테스터&quot;및 &quot;테스트&quot;주식에서 남성과 여성을 교차. </li><li> 25 ° C 배양기에서 십자가를 놓고 남성과 여성이 짝과 24-48 시간 동안 알을 낳을 수 있습니다. </li><li> 충분한 계란 증착 먼저 령 애벌레 문화가 건조되지 않도록 만드는 존재의 병을 확인합니다. <ol><li> 멸균 몇 방울 추가(문화가 건조되어있는 경우)가 촉촉한 유지하는 문화에 물을 증류. </li><li> 문화를 모니터하고 필요에 따라 단계 1.7.1를 반복을 계속합니다. </li></ol></li><li> 3 절에 설명 된대로 형광 실체 현미경을 이용하여 제 령 애벌레를 방황 관찰한다. </li></ol> 2. 침윤성 종양의 유도 <ol><li> 침습성 종양의 유도를 위해 표 2에 주식을 사용합니다. </li><li> 다음과 같은 유전자형의 위의 단계 1.2에서 스타터 문화를 사용하여, Y EY-FLP1, W, 욕조 - Gal80, FRT40A; Act5C&gt; Y +&gt; 인 Gal4, UAS - GFP </li><li> LGL 4, FRT40A, UAS - 라스 V12 / CYO, W : 다음과 같은 유전자형의 &quot;테스트&quot;주식에 대한 스타터 문화를 준비 </li><li> &quot;테스트&quot;주식에서 &quot;테스터&quot;주식 ~ 10 여성 처녀 십 명의 남성을 수집합니다. </li><li> 비행 F와 함께 유리 병에 모두 배치하여 &quot;테스터&quot;및 &quot;테스트&quot;주식에서 남성과 여성을 건너짐 우드. </li><li> 25 ° C 배양기에서 십자가를 놓고 남성과 여성이 짝과 24-48 시간 동안 알을 낳을 수 있습니다. </li><li> 충분한 계란 증착 먼저 령 애벌레 문화가 건조되지 않도록 만드는 존재의 병을 확인합니다. <ol><li> (문화가 건조되어있는 경우)가 촉촉한 유지하는 문화에 멸균 증류수를 몇 방울을 추가합니다. </li><li> 필요에 따라 건조 단계를 반복 2.7.1의 문화를 지속적으로 모니터. </li></ol></li><li> 3 절에 설명 된대로 형광 실체 현미경을 이용하여 제 령 유충을 관찰합니다. </li></ol> 3. 양성 및 침윤성 종양의 관측 <ol><li> 양성 종양의 격리 및 시각화를위한 세 번째 령 유충을 사용합니다. <ol><li> 증류수에 페인트 붓을 적시고,이 페인트 브러시를 사용하여 배양 병의 벽에서 몇 번째 령 유충을 다 쳤어요. </li><li> 1X PBS와 우울증 슬라이드에 애벌레를 배치 한 다음 젖은 페인트 브러시 스크랩을 사용하여유충의 표피에서 어떤 음식 재료를 전자. </li><li> 페트리 접시에 애벌레를 놓고 유충을 고정시키기 30 분 -20 ° C 냉장고에 둡니다. <ol><li> FLYNAP를 사용하여 유충을 고정화하는 다른 방법. </li><li> 3.1.3.2에서 유리 병에 FLYNAP에 담근 FLYNAP 지팡이를 놓고 애벌레 유리 병하자 연결 유리 병을 연결 한 후 30 ~ 40 분간 방치. </li></ol></li><li> 빛 할로 카본 오일 한 방울을 추가 커버 유리 커버 및 녹색 형광 단백질 (GFP)을 시각화 할 수있는 능력을 가진 형광 실체 현미경을 이용하여 관찰, 유리 슬라이드에 고정 된 애벌레를 놓습니다. </li><li> 양성 종양 베어링 애벌레는 두개의 복잡한 존재하는 전방 지역에서 녹색 형광을합니다. </li></ol></li><li> 침습성 종양의 관찰 위의 단계 # 2에서 설정 한 문화 일에게 (유도 후​​) 10 애벌레를 선택합니다. 침략 종양 베어링 애벌레 확장 L을 가지고 있기 때문에 10 일째 유충이 선택됩니다일반적으로 ARVAL 무대와는 pupariate 실패합니다. </li><li> 대신 세 번째 령 애벌레 사용 일 10 애벌레를 제외하고 3.1.4 단계 3.1.1를 따르십시오. <ol><li> 하루 10 침습 종양 베어링 애벌레는 두개의 복잡한 존재하는 전방 지역에서 녹색 형광을합니다. </li></ol></li><li> 형광 현미경은 디지털 카메라가 장착되어있는 경우 다음 형광 양성 및 침입 종양 베어링 애벌레의 사진을 찍을. </li></ol> 4. 양성 및 침윤성 종양의 두개의 콤플렉스 및 추가 관측의 해부 유충의 반투명 표피 어려운 종양의 침윤 정도를 관찰 할 수 있습니다. 따라서 침략의 정도는 더 유충 밖으로 두개의 복잡한을 해부 시각화됩니다. 다음 단계는 두부 복잡한 해부하기 위해 이용되어야한다. <ol><li> 젖은 페인트 붓을 사용하면 F 제 령 (양성 종양) 유충 일 (침략 종양) 10 유충을 선택각각의 문화 튜브를 롬. </li><li> 차가운 1X PBS를 포함하는 해부 접시에 잘 애벌레를 놓습니다. 유충의 표피에서 어떤 음식 입자를 긁어 페인트 브러시를 사용합니다. <ol><li> 차가운 1X PBS를 포함하는 해부 접시의 신선한 잘 깨끗한 유충을 전송합니다. </li><li> 형광을 검출 할 수있는 실체 현미경을 이용하여 GFP의 형광의 유무를 확인합니다. 비 GFP 베어링 애벌레를 폐기하십시오. </li></ol></li><li> 해부 접시에 신선한 잘에 차가운 1X PBS의 1.0 ㎖를 추가하고 포셉 한 켤레를 사용하여 양성 또는 침습성 종양 (해부 프로토콜은 종양의 두 가지 유형에 대해 동일하게 유지) 중 하나를 베어링 유충을 전송합니다. </li><li> 앞쪽 끝에서 2/3rds에 대한 포셉 한 쌍을 사용하여 유충을 누릅니다. <ol><li> 포셉의 다른 쌍을 사용하여 스마트 유충의 후방 1/3rd를 분리하고 폐기. </li><li> 유충의 앞쪽에 2/3rds에 가자. 유충 내부 압력의 내용을 떼면유충 체강 배어 나오기 것이다. </li><li> 창자, 지방 몸과 체강 밖으로 분출 한 흔적이 유충의 다른 내부 내용을 제거하는 포셉 한 쌍을 사용합니다. </li><li> 유충의 입 후크를 잡고 애벌레 표피에 밀어 포셉 한 켤레를 사용하여 완전히 유충을 반전 포셉의 다른 쌍을 사용. </li><li> 부드럽게 조심스럽게 지방을 몸, 침샘, 창자, 날개 디스크 복잡하고 어떤 기관 튜브를 제거하는 집게를 사용합니다. 두개의 복잡한 이제 반전 유충의 입으로 훅에 연결된 볼 수 있어야합니다. 뇌 반구 및 복부 신경 코드 (VNC)은 여전히​​ VNC에서 나오는 신경을 통해 유충의 표피에 연결됩니다. </li><li> 부드럽게 두부 신경과 유충의 표피 사이의 연결을 중단 집게의 쌍을 사용합니다. </li><li> VNC와 유충의 표피 사이의 신경 연결이 끊어 및 여분의 지방 기관 및 기타 조직을 제거 할 때, 두개의 공동MPLEX 명확하게 표시 될 것입니다 만 입 후크에서 유충의 표피에 첨부됩니다. </li><li> 복잡한 항체 염색과 같은 다운 스트림 응용 프로그램에 이상 시각화 고정해야하는 경우 두개의 복잡한 애벌레 표피에 붙어 남아있을 수 있습니다. </li><li> 더 다운 스트림 응용 프로그램이 다음을 수행하지 될 경우 두개의 복잡한 애벌레 표피에서 분리하고 추가 관찰과 분석을 위해 설치 미디어를 기반으로하는 글리세롤의 드롭에 배치 할 수 있습니다. 설치 매체로 VECTASHIELD를 사용합니다. </li></ol></li></ol>

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K., Wong, A. M., Rubin, 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=A+genetic+screen+for+modifiers+of+a+kinase+suppressor+of+Ras-dependent+rough+eye+phenotype+in+Drosophila.">A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in Drosophila.</a> Genetics. 156, 1231-1242 (2000).</li><li>Albini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+in+vitro+assay+for+quantitating+the+invasive+potential+of+tumor+cells.">A rapid in vitro assay for quantitating the invasive potential of tumor cells.</a> Cancer Res. 47, 3239-3245 (1987).</li><li>Zicha, D., Dunn, G. A., Brown, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> J Cell Sci. 99 (Pt 4), 769-775 (1991).</li><li>Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+in+vivo+models+of+neoplasia.">New developments in in vivo models of neoplasia.</a> Cancer Metastasis Rev. 10, 191-192 (1991).</li><li>Mueller, B. M., Romerdahl, C. A., Trent, J. M., Reisfeld, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+spontaneous+melanoma+metastasis+in+scid+mice+with+an+antibody+to+the+epidermal+growth+factor+receptor.">Suppression of spontaneous melanoma metastasis in scid mice with an antibody to the epidermal growth factor receptor.</a> Cancer Res. 51, 2193-2198 (1991).</li><li>Konantz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+xenografts+as+a+tool+for+in+vivo+studies+on+human+cancer.">Zebrafish xenografts as a tool for in vivo studies on human cancer.</a> Ann N Y Acad Sci. 1266, 124-137 (2012).</li><li>McIntyre, R. E., vander Weyden, L., Adams, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+gene+discovery+in+the+mouse.">Cancer gene discovery in the mouse.</a> Curr Opin Genet Dev. 22, 14-20 (2012).</li><li>Mattison, J., vander Weyden, L., Hubbard, T., Adams, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+gene+discovery+in+mouse+and+man.">Cancer gene discovery in mouse and man.</a> Biochim Biophys Acta. 1796, 140-161 (2009).</li><li>Miles, W. O., Dyson, N. J., Walker, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tumor+invasion+and+metastasis+in+Drosophila.">Modeling tumor invasion and metastasis in Drosophila.</a> Dis Model Mech. 4, 753-761 (2011).</li><li>Stefanatos, R. K., Vidal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+invasion+and+metastasis+in+Drosophila:+a+bold+past,+a+bright+future.">Tumor invasion and metastasis in Drosophila: a bold past, a bright future.</a> J Genet Genomics. 38, 431-438 (2011).</li><li>Beaucher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+brain+tumor+metastases+express+both+neuronal+and+glial+cell+type+markers.">Drosophila brain tumor metastases express both neuronal and glial cell type markers.</a> Dev Biol. 301, 287-297 (2007).</li><li>Beaucher, M., Hersperger, E., Page-McCaw, A., Shearn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+ability+of+Drosophila+tumors+depends+on+MMP+activity.">Metastatic ability of Drosophila tumors depends on MMP activity.</a> Dev Biol. 303, 625-634 (2007).</li><li>Joyce, J. A., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+metastasis.">Microenvironmental regulation of metastasis.</a> Nat Rev Cancer. 9, 239-252 (2009).</li><li>Igaki, T., Pagliarini, R. A., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+cell+polarity+drives+tumor+growth+and+invasion+through+JNK+activation+in+Drosophila.">Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila.</a> Curr Biol. 16, 1139-1146 (2006).</li><li>Uhlirova, M., Jasper, H., Bohmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-cell-autonomous+induction+of+tissue+overgrowth+by+JNK/Ras+cooperation+in+a+Drosophila+tumor+model.">Non-cell-autonomous induction of tissue overgrowth by JNK/Ras cooperation in a Drosophila tumor model.</a> Proc Natl Acad Sci U S A. 102, 13123-13128 (2005).</li><li>Uhlirova, M., Bohmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JNK-+and+Fos-regulated+Mmp1+expression+cooperates+with+Ras+to+induce+invasive+tumors+in+Drosophila.">JNK- and Fos-regulated Mmp1 expression cooperates with Ras to induce invasive tumors in Drosophila.</a> EMBO J. 25, 5294-5304 (2006).</li><li>Menut, L., 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=A+mosaic+genetic+screen+for+Drosophila+neoplastic+tumor+suppressor+genes+based+on+defective+pupation.">A mosaic genetic screen for Drosophila neoplastic tumor suppressor genes based on defective pupation.</a> Genetics. 177, 1667-1677 (2007).</li><li>Wu, M., Pastor-Pareja, J. C., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+Ras(V12)+and+scribbled+clones+induces+tumour+growth+and+invasion.">Interaction between Ras(V12) and scribbled clones induces tumour growth and invasion.</a> Nature. 463, 545-548 (2010).</li></ol>

I는 공개 아무것도 없어.

암은 과거에 비해 훨씬 더 잘 이해 오늘날 복잡한 질병입니다. 그러나, 많은 여전히​​ 배우고 우리가 기본 메커니즘의 완전한 그림이 전에 설명해야합니다. 여기에 제시된 간단한 프로토콜은 가능한 유전자 전체 유기체의 양성 및 침입 종양을 유발하고이 모델에서 종양의 진행과 관련된 생물학을 연구 할 수 있습니다. 초파리 및 다른 생물체에서 기존 기술의 대부분은 종양 발생 및 전이...

전이 된 상태로 양성의 종양의 진행 상황은 본체 (8)에 존재하는 보호 메커니즘을 회피 특징 단계 현명한 방법입니다. 예를 들어 몸에있는 종양 세포는 세포 사멸과 면역 체계를 회피 지하실 멤브레인라고하는 특수 세포 외 기질 (ECM)을 돌파구와 주변 세포 (8)에 의해 부과 된 사회 통제를 극복 할 수 있어야합니다. 이는 암세포가 전이 불리는 프로세스에서 먼 위치를 이주 및 정착 할 수있는 능력을 습득하는 것이 현명 단계 진행을 통해서이다. 종양 세포는 몸에 의해 부과 된 장벽을 극복하는 방법에 대한 우리의 이해는하지만, 연구의 새로운 그림이 암 세포 11 ~ 13로 정상 발달 과정 및 신호 전달 경로의 반복 사용으로 지금까지의 포인트를 수행, 아직 초기 단계에있다. 열매는 초파리 melanogaster의 우리 싶게에 엄청난 기여하고있다 비행지난 몇 년간 14 ~ 17에 걸쳐 개발 정교한 유전자 기술의 사용을 통해 정상적인 발달과 질병의 erstanding. 우리는 다양한 종양 유전자와 종양 억제 유전자 18-22의 더 나은 이해에 도착했습니다 돌연변이 유발 및 과발현 도구를 사용하여. 그러나, 종양 전이가 세포 배양 모델 (23, 24)뿐만 아니라, 다양한 이종 이식 모델에서 25-27 주로 연구되어 여러 유전자 ​​병변 간의 협력의 결과이다. 그들은 완전히 살아있는 유기체에서 발견되는 조건을 모방하지 않는 한이 모델 강력한하지만 자신의 제한이 있습니다. 또한, 마우스에서 사용할 수있는 형질 전환 모델은 복잡하고 침략적인 행동 (28, 29)의 유전자 분석에 도움이되지 않습니다. 몇몇 연구는 초파리 (30, 31)에서 종양 세포의 침윤을 이해하려고했습니다. 이러한 기술은 주로 호스트 일차 종양 이식을 활용하고 TRA 추적에 의존이웃 조직 (32, 33)의 침략 종양을 nsplanted. MARCM 10라는 강력한 기술은 초파리 9 종양의 침략을 모델로 Pagliarini와 쑤으로 적응했다. 종양의 침윤이 우아한 유전 모델링은 활성화 된 종양 유전자 및 세포 극성의 손실 사이의 협력을 악용. 이러한 모델링의 전력은 침습 종양 따라서 조직의 이식을위한 필요성을 우회 본래 유기체에서 생성된다는 사실에있다. 종양의 협력에 대해 가지고, 라스 V12 등 활성화 된 종양 유전자는 애벌레 눈 더듬이 디스크에있는 세포의 클론으로 표현된다. MARCM 기술의 결과로 이들 클론은 쉽게 시각화를 위해 녹색 형광 단백질 (GFP)로 표시되어 치명적인 거대한 애벌레 남겼, 대형 디스크와 같은 세포 극성 돌연변이에 대한 동형 접합 만들어집니다. 결과는 GFP가 침습성 종양 태그입니다 두개의 복잡한. 이 보고서 I유도하는 방법을 설명하고, 그대로 애벌레의 맥락에서 밖으로 해부 두개의 단지에 두 가지 침습적 종양을 시각화. 여기에 제시된 종양 유도 초파리의 두 번째 염색체에 시약을 사용한다. 표 2에서, 내가 같은 목적을 위해 이용 될 수있는 X와 3 번째 염색체에 주식의 목록을 제공합니다. 나는이 간단한 프로토콜은 종양의 진행의 분자 기초를 이해에 관심이 연구자들에게이 기술을 쉽게 액세스 할 수 있도록 것이라고 믿는다.

<table border="1">
	<tbody>
 <tr>
 <td>10X PBS (phosphate buffered saline) pH 7.2 stock solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Chilled 1X PBS pH7.2 working solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td>Make fresh and refrigerate, can be used up to a week</td>
 </tr>
 <tr>
 <td>Flynap</td>
 <td>Carolina Biologicals</td>
 <td></td>
 <td>Fly anesthesia needed to anesthetize larvae</td>
 </tr>
 <tr>
 <td>Fixative</td>
 <td>0.1M PIPES, pH 7.2, 4% Paraformaldehyde</td>
 <td></td>
 <td>Needed to fix the dissected cephalic complex</td>
 </tr>
 <tr>
 <td>Ice Bucket</td>
 <td>Several</td>
 <td></td>
 <td>Maintain solutions on ice. Also, dissect cephalic complex in chilled 1X PBS and then place on ice in an Eppendorf tube</td>
 </tr>
 <tr>
 <td>1.7ml Eppendorf tube</td>
 <td>Various</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Glass slides, cover glass</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vectashield Mounting Media or any other mounting media</td>
 <td>Vector Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon 200 or 700 Oil</td>
 <td>Polysciences Inc. or Halocarbon.com</td>
 <td></td>
 <td>Halocarbon 200 is used to mount the larvae for visualization on a fluorescence stereoscope</td>
 </tr>
 <tr>
 <td>Sally Hansen "Hard as Nails" nail polish</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td>Needed to seal the edges of Coverslip</td>
 </tr>
 <tr>
 <td>A Leica MZ16.5 fluorescence stereomicroscope or any other fluorescence stereomicroscope</td>
 <td>Leica and others</td>
 <td></td>
 <td>Needed to observe the GFP fluorescence in larvae</td>
 </tr>
 <tr>
 <td>Dumont #5 forceps</td>
 <td>Fine Science Tools</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pyrex 9 well spot plate or any other dissection dish</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paint Brush</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Materials needed to perform the experimental protocol presented in this article.</td>
 </tr>
 </tbody>
 </table>

a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster

초파리는 복잡한 질병 1-7에 대한 우리의 이해에 상당히 기여하고 있습니다 지난 몇 년과 오늘 정상 개발 및 질병의 유전 적 기초에 대한 우리의 이해를 조명하고있다. 전이 된 상태로 양성의 종양의 진행 상황은 복잡한 이벤트 8하고 우리가 더 나은이 질병 (9)의 유전 적 기초를 이해하는 데 도움이 초파리 모델로하고있다. 여기에 유 전적으로 유도 관찰하고 초파리 유충에 종양의 진행을 분석하는 간단한 프로토콜을 제시한다. 종양 유도 기술은 MARCM 시스템 (10)을 기반으로 활성화 된 종양 유전자, 라스 V12 세포 극성 유전자의 손실 (남겼, 디스크 크고 치명적인 거대한 애벌레) 침략 종양 9를 생성 사이의 협력을 활용한다. 나는이 종양은 그대로 유충으로 시각화 할 수있는 방법을 설명하고그럼 어떻게 이러한 추가 분석을 위해 해부 할 수 있습니다. 여기에 제시된 단순화 된 프로토콜은 가능한이 기술은 종양의 침공에있는 유전자의 역할을 이해에 관심있는 연구자에 의해 이용 될 수 있도록해야한다.

프로토콜의 결과는 여기에 제시된 바와 같이, 사용자는 유충의 눈 더듬이, 상상 디스크에 활성화 된 종양 유전자를 과발현에 의해 양성 종양을 유도 할 수있을 것입니다. 사용자는 또한 세포 극성 유전자의 돌연변이 세포의 복제에 활성화 된 종양 유전자를 과발현에 의해 눈 더듬이 디스크에 침략 종양을 유도 할 수있을 것입니다. 종양은 쉽게 전체 유충이나 애벌레 체강 (그림 1) 중 ?...

Watch this Scientific Journal Video about A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster at JoVE.com

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

RAS V12 및 남겼, 종양 세포는 또한 침략적인 행동을 표시 초파리에있는 종양의 성장 결과와 같은 세포 극성 유전자의 돌연변이와 같은 활성화 된 발암 유전자 간의 협력. 여기에 양성 및 침입 종양의 유도 및 관찰을위한 간단한 프로토콜을 제시한다.

의 두부 컴플렉스 유전 유도하고 양성 및 침윤성 종양의 시각화를위한 프로토콜<em&gt; 초파리</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

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A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

9.4K 조회수.  Western Kentucky University.  RAS V12 및 남겼, 종양 세포는 또한 침략적인 행동을 표시 초파리에있는 종양의 성장 결과와 같은 세포 극성 유전자의 돌연변이와 같은 활성화 된 발암 유전자 간의 협력. 여기에 양성 및 침입 종양의 유도 및 관찰을위한 간단한 프로토콜을 제시한다. 

비디오: A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

면역 결함 마우스의 확립과 인간 Retinoblastoma 종양의 전파

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

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Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

유아의 뇌 신진 대사와 혈류 역학의 비 침습적 광학 측정

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

전립선 발암 및 양성 전립선 비대증의 평가를위한 신장 캡슐 Xenografting 및 피하 펠렛 주입

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

면역 본래대로 사람 MUC1 유전자 변형 마우스의 침략 과도 셀 방광 암의 유도 : 면역 요법 개발을위한 모델

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

마우스에서 가속 죽상 경화증의 유도: "와이어 부상" 모델

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

털이 마우스의 귓불에서 생체 내에 현미경 및 혈전 유도

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

왼쪽된 심 실 비 대의 유도 대 한 생쥐에서 최소 침 습 가로 대동맥 수축의 기술

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

비-침략 적 쥐에서 Dorsiflexor 근육 기능 평가

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

근접 결찰 분석분석서를 활용한 인간 췌장 베타 세포에서 내인성 미토파기 복합체의 시각화

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

재건 미세 수술에서 허혈 재관류 손상의 연구를위한 전 임상 쥐 모델

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...