Name: a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster
Uploaded: 2013-09-11T15:15:00
Description: 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

Research in my laboratory is supported by the WKU Department of Biology startup funds, WKU Research Foundation RCAP-I grant # 11-8032 and by a KBRIN-AREA grant funded through a parent grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number 5P20GM103436-13. I would also like to acknowledge Dr. Tian Xu in whose laboratory I was introduced to this technique and Dr. Raymond Pagliarini who first established this technique in the Xu laboratory.

1. Induction of Benign Non-invasive Tumors
<ol>
	<li>Use stocks listed in Table 2 for the induction of benign tumors.</li>
	<li>Prepare a starter culture for the "tester" stock of the following genotype: y,w, ey-FLP1; Tub-Gal80,FRT40A; Act5C&gt;y+&gt;Gal4, UAS-GFP</li>
	<li>Prepare a starter culture for the "tested" stock of the following genotype: w; FRT40A, UAS-RasV12/CyO</li>
	<li>Collect ten female virgins from the "tester" stock and ten males from the...

<ol>
	<li>Bale, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+and+human+disease.">Hedgehog signaling and human disease.</a> Annu Rev Genomics Hum Genet. 3, 47-65 (2002).</li><li>Bier, E., Bodmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila,+an+emerging+model+for+cardiac+disease.">Drosophila, an emerging model for cardiac disease.</a> Gene. 342, 1-11 (2004).</li><li>Coombs, G. S., Covey, T. M., Virshup, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+in+development,+disease+and+translational+medicine.">Wnt signaling in development, disease and translational medicine.</a> Curr Drug Targets. 9, 513-531 (2008).</li><li>Gistelinck, M., Lambert, J. C., Callaerts, P., Dermaut, B., Dourlen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+models+of+tauopathies:+what+have+we+learned.">Drosophila models of tauopathies: what have we learned.</a> Int J Alzheimers Dis. 2012, 970980 (2012).</li><li>Marsh, J. L., Thompson, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+in+the+study+of+neurodegenerative+disease.">Drosophila in the study of neurodegenerative disease.</a> Neuron. 52, 169-178 (2006).</li><li>Reiter, L. T., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Drosophila+melanogaster+to+uncover+human+disease+gene+function+and+potential+drug+target+proteins.">Using Drosophila melanogaster to uncover human disease gene function and potential drug target proteins.</a> Expert Opin Ther Targets. 6, 387-399 (2002).</li><li>Valenta, T., Hausmann, G., Basler, K. <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+functions+of+beta-catenin.">The many faces and functions of beta-catenin.</a> EMBO J. 31, 2714-2736 (2012).</li><li>Hanahan, D., Weinberg, 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=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li><li>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=A+genetic+screen+in+Drosophila+for+metastatic+behavior.">A genetic screen in Drosophila for metastatic behavior.</a> Science. 302, 1227-1231 (2003).</li><li>Wu, J. S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+in+Drosophila.">A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila.</a> Nat Protoc. 1, 2583-2589 (2006).</li><li>Boccaccio, C., Comoglio, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+growth:+a+MET-driven+genetic+programme+for+cancer+and+stem+cells.">Invasive growth: a MET-driven genetic programme for cancer and stem cells.</a> Nat Rev Cancer. 6, 637-645 (2006).</li><li>Srivastava, A., Pastor-Pareja, J. C., Igaki, T., Pagliarini, R., 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=Basement+membrane+remodeling+is+essential+for+Drosophila+disc+eversion+and+tumor+invasion.">Basement membrane remodeling is essential for Drosophila disc eversion and tumor invasion.</a> Proc Natl Acad Sci U S A. 104, 2721-2726 (2007).</li><li>Wang, W., Eddy, R., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cofilin+pathway+in+breast+cancer+invasion+and+metastasis.">The cofilin pathway in breast cancer invasion and metastasis.</a> Nat Rev Cancer. 7, 429-440 (2007).</li><li>Brand, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+as+a+cell+and+developmental+marker+in+the+Drosophila+nervous+system.">GFP as a cell and developmental marker in the Drosophila nervous system.</a> Methods Cell Biol. 58, 165-181 (1999).</li><li>Brand, A. H., Dormand, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GAL4+system+as+a+tool+for+unravelling+the+mysteries+of+the+Drosophila+nervous+system.">The GAL4 system as a tool for unravelling the mysteries of the Drosophila nervous system.</a> Curr Opin Neurobiol. 5, 572-578 (1995).</li><li>Vidal, M., Cagan, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+models+for+cancer+research.">Drosophila models for cancer research.</a> Curr Opin Genet Dev. 16, 10-16 (2006).</li><li>Parks, A. 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=Systematic+generation+of+high-resolution+deletion+coverage+of+the+Drosophila+melanogaster+genome.">Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome.</a> Nat Genet. 36, 288-292 (2004).</li><li>Blair, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mosaic+techniques+for+studying+Drosophila+development.">Genetic mosaic techniques for studying Drosophila development.</a> Development. 130, 5065-5072 (2003).</li><li>Xu, T., 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=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117, 1223-1237 (1993).</li><li>Xu, T., Wang, W., Zhang, S., Stewart, R. A., Yu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+tumor+suppressors+in+genetic+mosaics:+the+Drosophila+lats+gene+encodes+a+putative+protein+kinase.">Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase.</a> Development. 121, 1053-1063 (1995).</li><li>Rebay, I., 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+genetic+screen+for+novel+components+of+the+Ras/Mitogen-activated+protein+kinase+signaling+pathway+that+interact+with+the+yan+gene+of+Drosophila+identifies+split+ends,+a+new+RNA+recognition+motif-containing+protein.">A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein.</a> Genetics. 154, 695-712 (2000).</li><li>Therrien, M., Morrison, D. 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>

Cancer is a complex disease with a much better understanding today than in the past. However, much still needs to be learnt and explained before we have a complete picture of the underlying mechanisms. The simple protocol presented here makes it possible to genetically induce benign and invasive tumors in a whole organism and then study the biology associated with the progression of tumors in this model. Most of the existing techniques in Drosophila and other organisms either utilize a cell culture based system ...

Progression of tumors from a benign to a metastatic state is a step wise process that is characterized by evasion of protective mechanisms present in the body 8. For example tumor cells in the body must be able to evade apoptosis and the immune system, breakthrough the specialized extracellular matrix (ECM) called Basement Membrane, and overcome any social controls imposed by the surrounding cells 8. It is through a step wise progression that the cancer cells acquire the ability to migrate and colonize distant sites in a process called metastasis. Our understanding of how the tumor cell overcomes the barriers imposed by the body is still in its infancy, however, the emerging picture from research done thus far points to a repeated use of normal developmental processes and signaling pathways by the cancer cells 11-13.
The fruit fly Drosophila melanogaster has contributed tremendously to our understanding of normal development and disease through use of sophisticated genetic techniques developed over the past several decades 14-17. Using mutagenesis and overexpression tools we have arrived at a better understanding of various oncogenes and tumor suppressor genes 18-22. However, tumor metastasis is a result of cooperation between several genetic lesions that has been studied primarily in cell culture models 23,24 as well as various xenograft models 25-27. These models though powerful have their limitations as they do not mimic entirely the conditions found in a living organism. Furthermore, transgenic models available in mice are cumbersome and not conducive to genetic analysis of invasive behavior 28,29. Several studies have attempted to understand invasion of tumor cells in Drosophila 30,31. These techniques primarily utilize transplantation of primary tumors to hosts and then rely on tracking the transplanted tumors for invasion of neighboring tissues 32,33. A powerful technique called MARCM 10 was adapted by Pagliarini and Xu to model tumor invasion in Drosophila 9. This elegant genetic modeling of tumor invasion exploited the cooperation between an activated oncogene and the loss of cell polarity. The power of this modeling lies in the fact that the invasive tumors are created in an intact organism thus circumventing the need for transplantation of tissues. To bring about the oncogenic cooperation, an activated oncogene like RasV12 is expressed in clones of cells in the larval eye-antennal disc. As a result of the MARCM technique these clones are also marked with green fluorescent protein (GFP) for easy visualization and are made homozygous for cell polarity mutants like lethal giant larvae, scribbled, and discs large. The result is GFP tagged invasive tumors in the cephalic complex. In this report I demonstrate how to induce, and visualize these invasive tumors both in the context of an intact larvae and in dissected out cephalic complex. The tumor induction presented here utilizes reagents on the second chromosome of Drosophila. In Table 2, I provide a listing of stocks on X and 3rd chromosomes that can be utilized for the same purpose. I believe that this simplified protocol will make this technique readily accessible to researchers interested in understanding the molecular basis of tumor progression.

<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

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

As a result of the protocol presented here the user will be able to induce benign tumors by overexpressing an activated oncogene in the larval eye antennal imaginal disc. The user will also be able to induce invasive tumors in the eye antennal disc by overexpressing an activated oncogene in clones of cells also mutant for a cell polarity gene. The tumors could be easily visualized with the help of a fluorescent stereomicroscope as "green fluorescent" tissue in the whole larvae or in cephalic complexes that have been diss...

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

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

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

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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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 ...

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 ...

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 ...