Name: identification of sleeping beauty transposon insertions in solid tumors using linker-mediated pcr
Uploaded: 2013-02-01T13:00:00
Description: Watch this Scientific Journal Video about Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR at JoVE.com

The authors would like to thank Branden Moriarity, David Largaespada, and Vincent Keng at the University of Minnesota, and Adam Dupuy at the University of Iowa for their assistance in developing the protocol described above.

1. Breeding and Aging of Transgenic Animals
<ol>
 <li>Select the strains of transgenic mice appropriate for your experiment. A typical experiment will use three transgenic lines; a conditional transposase mouse, a mouse harboring a concatamer of transposons, and a mouse expressing Cre Recombinase in the cells believed to be the cells of origin for the desired cancer. If the cancer being modeled has a known mutation in a large percentage of the patients it may be desirable to include this mutation at the start....

<ol>
	<li>Fearon, E. R., Vogelstein, B., 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+model+for+colorectal+tumorigenesis.">A genetic model for colorectal tumorigenesis.</a> Cell. 61, 759-767 (1990).</li><li>Wood, L. D., 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+Genomic+Landscapes+of+Human+Breast+and+Colorectal+Cancers.">The Genomic Landscapes of Human Breast and Colorectal Cancers.</a> Science. 318, 1108-1113 (2007).</li><li>Ivics, Z., Hackett, P. B., Plasterk, R. H., Izsv&#225;k, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Reconstruction+of+Sleeping+Beauty,+a+Tc1-like+Transposon+from+Fish,+and+Its+Transposition+in+Human+Cells.">Molecular Reconstruction of Sleeping Beauty, a Tc1-like Transposon from Fish, and Its Transposition in Human Cells.</a> Cell. 91, 501-510 (1997).</li><li>Starr, T. K., Largaespada, D. A. <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+using+the+Sleeping+Beauty+transposon.">Cancer gene discovery using the Sleeping Beauty transposon.</a> Cell Cycle. 4, 1744-1748 (2005).</li><li>Mueller, P. R., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Footprinting+of+a+Muscle+Specific+Enhancer+by+Ligation+Mediated+PCR.">In Vivo Footprinting of a Muscle Specific Enhancer by Ligation Mediated PCR.</a> Science. 246, 780-786 (1989).</li><li>Bergemann, T. 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=New+methods+for+finding+common+insertion+sites+and+co-occurring+common+insertion+sites+in+transposon-+and+virus-based+genetic+screens.">New methods for finding common insertion sites and co-occurring common insertion sites in transposon- and virus-based genetic screens.</a> Nucleic acids research. 40, 3822-3833 (2012).</li><li>Copeland, N. G., Jenkins, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+transposons+for+cancer+gene+discovery.">Harnessing transposons for cancer gene discovery.</a> Nature Reviews Cancer. 10, 696-706 (2010).</li><li>Collier, L. S., Carlson, C. M., Ravimohan, S., Dupuy, A. J., Largaespada, D. A. <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+solid+tumours+using+transposon-based+somatic+mutagenesis+in+the+mouse.">Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse.</a> Nature. 436, 272-276 (2005).</li><li>Starr, T. K., 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+transposon-based+genetic+screen+in+mice+identifies+genes+altered+in+colorectal+cancer.">A transposon-based genetic screen in mice identifies genes altered in colorectal cancer.</a> Science. 323, 1747-1750 (2009).</li><li>Keng, V. 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=A+conditional+transposon-based+insertional+mutagenesis+screen+for+genes+associated+with+mouse+hepatocellular+carcinoma.">A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma.</a> Nature. 27, 264-274 (2009).</li><li>Dupuy, A. J., Akagi, K., Largaespada, D. A., Copeland, N. G., Jenkins, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+mutagenesis+using+a+highly+mobile+somatic+Sleeping+Beauty+transposon+system.">Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system.</a> Nature. 436, 221-226 (2005).</li></ol>

A forward genetic screen using the Sleeping Beauty transposon system provides a method for identifying mutations that cause cancer. By selecting the appropriate promoter to control Cre Recombinase in addition to any predisposing mutations, the SB screen will identify known and novel candidate cancer genes. 
The success of an SB screen is largely dependent on the mice selected for creating the screen. For the transposon, we recommend using two different mouse strains carrying ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Sure-Seal Induction Chamber</td>
 <td>Brain Tree Scientific</td>
 <td>EZ-177</td>
 <td></td>
 </tr>
 <tr>
 <td>Cryovial</td>
 <td>Bioexpress</td>
 <td>C-3355-2</td>
 <td></td>
 </tr>
 <tr>
 <td>10% Buffered Formalin</td>
 <td>Sigma Aldrich</td>
 <td>HT501128-4L</td>
 <td></td>
 </tr>
 <tr>
 <td>Protein Precipitation solution</td>
 <td>Qiagen</td>
 <td>158910</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell lysis buffer</td>
 <td>Qiagen</td>
 <td>158906</td>
 <td></td>
 </tr>
 <tr>
 <td>Proteinase K</td>
 <td>Qiagen</td>
 <td>158920</td>
 <td></td>
 </tr>
 <tr>
 <td>RNase A</td>
 <td>Qiagen</td>
 <td>158924</td>
 <td></td>
 </tr>
 <tr>
 <td>TE Buffer</td>
 <td>Promega</td>
 <td>V6232</td>
 <td></td>
 </tr>
 <tr>
 <td>BfaI</td>
 <td>New England Biolabs</td>
 <td>R0568S</td>
 <td></td>
 </tr>
 <tr>
 <td>NlaIII</td>
 <td>New England Biolabs</td>
 <td>R0125S</td>
 <td></td>
 </tr>
 <tr>
 <td>MinElute 96 well plates</td>
 <td>Qiagen</td>
 <td>28051</td>
 <td></td>
 </tr>
 <tr>
 <td>QIAvac 96 Vacuum manifold</td>
 <td>Qiagen</td>
 <td>19504</td>
 <td></td>
 </tr>
 <tr>
 <td>Multi-MicroPlate Genie, 120V</td>
 <td>Scientific Industries, Inc.</td>
 <td>SI-4000</td>
 <td></td>
 </tr>
 <tr>
 <td>T4 DNA ligase with 5x ligation Buffer</td>
 <td>Invitrogen</td>
 <td>15224-041</td>
 <td></td>
 </tr>
 <tr>
 <td>BamHI</td>
 <td>New England Biolabs</td>
 <td>R0136S</td>
 <td></td>
 </tr>
 <tr>
 <td>25mM dNTPs</td>
 <td>Bioexpress</td>
 <td>C-5014-200</td>
 <td></td>
 </tr>
 <tr>
 <td>Platinum Taq</td>
 <td>Invitrogen</td>
 <td>10966-034</td>
 <td></td>
 </tr>
 <tr>
 <td>FastStart Taq DNA Polymerase</td>
 <td>Roche</td>
 <td>12032929001</td>
 <td></td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Promega</td>
 <td>V3121</td>
 <td></td>
 </tr>
 <tr>
 <td>TAE Buffer</td>
 <td>Promega</td>
 <td>V4271</td>
 <td></td>
 </tr>
 <tr>
 <td>TE Buffer</td>
 <td>Promega</td>
 <td>V6232</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethidium bromide</td>
 <td>Promega</td>
 <td>H5041</td>
 <td></td>
 </tr>
 </tbody>
</table>

identification of sleeping beauty transposon insertions in solid tumors using linker-mediated pcr

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

After the breeding scheme has been established, breeders should produce a litter every 19-21 days. Litter size will vary between 5 and 12 pups, depending on the age of the breeders and the genetic background. In addition, make sure that the genotype of the litter follows Mendelian genetics as a way to confirm that the breeding scheme is both correct and successful. 
For the experiment to be successful, tumor incidence in the experimental animals should be significantly greater than tumor incid...

Watch this Scientific Journal Video about Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR at JoVE.com

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

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