की पहचान<em> स्लीपिंग ब्यूटी</em> Linker की मध्यस्थता पीसीआर का उपयोग कर ठोस ट्यूमर में Transposon सम्मिलन
Summary
एक निष्पक्ष दृष्टिकोण का उपयोग carcinogenesis के अज्ञात ड्राइवरों की पहचान का एक तरीका वर्णित है. विधि का उपयोग करता<em> स्लीपिंग ब्यूटी</em> Transposon एक यादृच्छिक विशिष्ट ऊतकों को निर्देशित उत्परिवर्तजन के रूप में. Transposon सम्मिलन कि ट्यूमर गठन ड्राइव के जीनोमिक मानचित्रण उपन्यास ओंकोजीन और ट्यूमर शमन जीनों की पहचान
Abstract
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).
Protocol
Representative Results
Discussion
स्लीपिंग ब्यूटी transposon प्रणाली का उपयोग करते हुए एक आगे आनुवंशिक स्क्रीन परिवर्तन के कारण कैंसर की पहचान के लिए एक तरीका प्रदान करता है. उपयुक्त प्रमोटर का चयन करने के लिए किसी भी predisposing परिवर्तन करने क…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए मिनेसोटा विश्वविद्यालय में Branden Moriarity, डेविड Largaespada, और विन्सेन्ट Keng धन्यवाद करना चाहते हैं, और एडम Dupuy आयोवा विश्वविद्यालय में विकासशील प्रोटोकॉल ऊपर वर्णित में उनकी सहायता के लिए.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Sure-Seal Induction Chamber | Brain Tree Scientific | EZ-177 | |
| Cryovial | Bioexpress | C-3355-2 | |
| 10% Buffered Formalin | Sigma Aldrich | HT501128-4L | |
| Protein Precipitation solution | Qiagen | 158910 | |
| Cell lysis buffer | Qiagen | 158906 | |
| Proteinase K | Qiagen | 158920 | |
| RNase A | Qiagen | 158924 | |
| TE Buffer | Promega | V6232 | |
| BfaI | New England Biolabs | R0568S | |
| NlaIII | New England Biolabs | R0125S | |
| MinElute 96 well plates | Qiagen | 28051 | |
| QIAvac 96 Vacuum manifold | Qiagen | 19504 | |
| Multi-MicroPlate Genie, 120V | Scientific Industries, Inc. | SI-4000 | |
| T4 DNA ligase with 5x ligation Buffer | Invitrogen | 15224-041 | |
| BamHI | New England Biolabs | R0136S | |
| 25mM dNTPs | Bioexpress | C-5014-200 | |
| Platinum Taq | Invitrogen | 10966-034 | |
| FastStart Taq DNA Polymerase | Roche | 12032929001 | |
| Agarose | Promega | V3121 | |
| TAE Buffer | Promega | V4271 | |
| TE Buffer | Promega | V6232 | |
| Ethidium bromide | Promega | H5041 |
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