This procedure shows how to identify novel oncogenes and tumor suppressor genes by mapping transpose on insertions in the mouse that drive tumor formation. This is accomplished by first generating and harvesting tumors from the transgenic mouse model. The next steps of the procedure are to isolate the tumor DNA, perform linker mediated PCR, then sequence the amplified DNA samples and analyze the transpose on insertions.
Ultimately, the procedure identifies candidate tumor driver genes. The main advantage of using linker mediated PCR in conjunction with an unbiased for genetic screen using sleeping beauty transposons, is that we can easily identify new genetic drivers of cancer. Always begin the dissection by spraying the exterior of the sacrificed mouse with 70%ethanol.
The collected tissues and tumors should be divided into four sections, 50 to 100 milligrams in size. Fix one section in 10%buffered formin for 18 hours, followed by 70%ethanol. This section can be used for h and e staining and or immunohistochemistry.
Snap freeze the remaining sections in liquid nitrogen. These can be stored in a freezer and used for D-N-A-R-N-A and protein isolation to collect DNA from a frozen tumor sample. First finely mince the tissue with a razor blade.
Next, digest the tissue in a 1.5 milliliter tube with one milliliter of cell lysis buffer, and five microliters of 20 milligrams per milliliter. Protease K solution vortex the mixture thoroughly and proceed with a standard isopropanol based DNA extraction. Begin this protocol by annealing the linkers in a 1.5 milliliter tube.
Mix 50 microliters of each linker solution with two microliters of concentrated sodium chloride. Then place the tube in a 95 degree Celsius heat block. After five minutes, turn off the heat and let it slowly cool to room temperature, which can take an hour or more after cooling.
The anal linkers may be stored in the freezer until needed. Prepare 2 96 well plates by Ali quoting one microgram of DNA from each sample into corresponding wells in both 96 well plates. For example, place one microgram of DNA from sample one in well A one of both well plates.
The DNA in one of the 96 well plates will be digested using the right side enzyme NLA three and digest the DNA samples in the other 96 well plate using the left side enzyme BFA one. Allow the DNA to digest overnight at 37 degrees Celsius. The next day, finish the digestion by heat, inactivating the enzymes for 20 minutes.
Now clean up the digestion reactions using a mini loot 96 well plate. Using the multi-channel pipette, transfer each sample to the mini loot. 96 well plate.
Place the mini loot 96 well plate in a vacuum manifold. Apply a vacuum to the plate until the wells look dry. Typically, 15 minutes Throughout this protocol.
Great care needs to be taken not to contaminate your samples. When working with 96, well plates keep wells covered with caps as much as possible and avoid passing your pipette over open wells because any drop of unwanted liquid can ruin the sample. Remove the mini loot plate from the vacuum manifold and block the bottom of plate with paper towel until it is completely dry.
Resuspend the DNA in the mini loot. 96 well plate by adding 30 microliters of double distilled water to each. Well place the mini loot plate on an orbital shaker set to high for two minutes.
Next, transfer the mini loot plate contents to a clean 96 well plate. To perform the linker ligation reactions. Prepare the ligation reaction by mixing a sufficient amount of ligation buffer, a kneeled linkers from step 3.2 T four DNA ligase and water in a reagent reservoir.
Use a multi-channel pipette to transfer 10 microliters of digested DNA from the previous step into a new 96 well plate. Then use a multi-channel pipette to Eloqua 10 microliters of the ligation reaction to all wells of the 96 well plate. After all, the wells have been loaded with DNA and ligation reagents.
Incubate the plate for four hours to overnight at 16 degrees Celsius. The next morning. End the ligation by inactivating the T four Ligase at 65 degrees Celsius for 10 minutes.
As before, clean up the ligation reaction using mini loot plates and a vacuum manifold to dry the plate and then blot the bottom of plate until it is completely dry. Resuspend the DNA in 30 microliters of double distilled water. Place on an orbital shaker for two minutes and then transfer the mini loot plate contents to a clean plate.
Next, perform a second DNA digestion with bam H one to destroy the remaining concat transposons. Digest the DNA at 37 degrees Celsius for three to six hours or overnight, and then the digestion is cleaned up Using mini loot plates following the same procedure as before. Perform a 30 cycle PCR using a primer specific for the transpose on and a primer specific for the linker.
The reaction should contain three microliters of the digested DNA with linkers. Next, clean up the PCR reaction using mini loot. 96 well plates and a vacuum manifold as performed previously.
Resuspend the DNA in 30 microliters of water and transfer the suspensions to a clean 96. Well played proceed by diluting two microliters of the PCR reaction products into 148 microliters of double distilled water using just two microliters of the diluted products. Perform 30 cycle PCRs using nested versions of the primers in 100 microliter volumes.
These primers are for the Illumina GA two X machine. Each sample will have a unique 12 base pair barcode from the PCR run, 45 microliters of each product on a 1%gel with AUM bromide. There should be a smear of PCR products representing amplicons of the many different transposon insertions in the tumor.
Use the previously described vacuum wash method to clean up the remaining 50 microliters of PCR, then resuspend the DNA in 50 microliters of water and perform the vacuum wash again. Now Resus, suspend the DNA in 30 microliters of TE and transfer it to a clean 96 well plate. Determine the concentration of DNA in each sample.
Then combine equal amounts of DNA from each sample in a single tube. It is possible to sequence several hundred DNA samples in a single lane of an Illumina run as long as each sample was generated using a distinct barcoded primer using the protocol. A successful L-M-P-C-R should amplify hundreds of transpose on insertions in a single tumor after running half of the secondary PCR product on a 1%gel, a smear of DNA represents this amplification.
If LM PCR is unsuccessful, there will be an absence of the smear of DNA. Instead, you will see definitive bands of DNA sequencing one to 200 tumors in a single lane of the Illumina GA two X platform should produce over 30 million sequences of 100 base pairs each While attempting this procedure. It's important to remember to work slowly and carefully to ensure that no contamination occurs following this procedure.
Analysis of newly discovered genetic drivers of cancer can be performed in order to answer additional questions like, which of these drivers could be targeted for therapy?
