The overall goal of this procedure is to generate Conor DNA. That can be used to reproducibly stimulate intracellular innate immune DNA sensing pathways. This is accomplished by an kneeling the forward and reversal oligonucleotide, primers and subsequent blunt end ligation to form concat.
Then the concat DNA is mixed with onic lipid reagent to form transfection complexes. The transfection particles are then added to cells for in vitro, transfection, or injected into the ear pinna of a mouse. Ultimately, quantitative R-T-P-C-R is used to show the successful induction of an innate immune response by measuring the transcription of cytokines and kines.
This method can help answer key questions in innate immunity such as how cells and organisms respond to self and foreign nucleic acids. Demonstrating this procedure will be Christian, a graduate student for my laboratory. All animal studies used in this manuscript were performed under approved institutional protocols and animal care guidelines.
To begin Resus, suspend the forward and reverse primers in molecular biology grade H2O to a concentration of 10 micrograms per microliter. Then mix five microliters of the forward primer with five microliters of the reverse primer in a 1.5 milliliter micro centrifuge tube. Heat the primer mixture to the correct a kneeling temperature for 15 minutes.
Then place the mixture on the bench to cool down at room temperature while the mixture is cooling. Prepare 60%PEG 8, 000 by dissolving three grams of PEG 8, 000 in five milliliters of DDH two O.Then add 50 microliters of 60%peg 8, 010 microliters of 10 x polynucleotide kinase buffer, 27 microliters of H2O and three microliters of the polynucleotide kinase to the primer mixture. Incubate the solution for two hours at 37 degrees celsius.
Next, cool the solution to room temperature and add 11 microliters of DNA ligase buffer. Then add three microliters of DNA ligase and incubate at 37 degrees Celsius overnight. After the overnight incubation, add 300 microliters of H2O to the ligation mixture.
Then add 400 microliters of phenol chloroform and shake vigorously to mix centrifuge's tube for one minute. At 50, 000 GS in a tabletop micro centrifuge, the aqueous insolvent layers will separate leaving the aqueous layer on top and a white layer of precipitated protein between the two layers, carefully pipette the top layer into a new tube and avoid transferring the middle white layer. Then repeat the addition of 400 microliters of phenol chloroform and the centrifugation steps at least twice or until there is no precipitated protein observed after the centrifugation.
Next, add 400 microliters of chloroform to the tube centrifuge for one minute at 15, 000 Gs and again, transfer the top layer to a new tube. Add 800 microliters of 100%pre chilled ethanol to the tube and incubate at minus 20 degrees Celsius for at least one hour or overnight. Centrifuge the tube for 10 minutes at 15, 000 Gs and then aspirate the supernatant.
Wash the pellet once by adding one milliliter of 70%ethanol centrifuge again for 10 minutes at 15, 000 Gs.Then aspirate the supernatant and air dry the DNA in a fume hood resuspend, the DNA in 50 microliters of endotoxin free DDH two O.The use of endotoxin free reagents is critical to generate a DNA specific innate immune response in cells and in vivo, transfer a two microliter aliquot of the Conan DNA into a new tube and then add one microliter of DNA gel loading buffer dissolve agros in TAE buffer to make a 1%agro solution by heating on full power in a microwave oven. For two minutes, pour the gel into a casting apparatus and add four microliters of fluorescent DNA chelating dye. Next, insert a comb and leave at room temperature for approximately 20 minutes until the gel sets.
Once the gel is set, remove the comb and place the gel into an electrophoresis tank containing TAE buffer. Then load three microliters at the DNA sample or size markers into the wells. Run the gel electrophoresis for 30 to 40 minutes at 90 volts with constant current.
Visualize the DNA under ultraviolet light to observe conization. Measure the DNA concentration in the remaining sample by UV absorbence blank the spectrometer with the same D DH two O that was used to re suspend the DNA. And measure the absorbent spectrum from 200 to 300 nanometers seed cells in a tissue culture dish to be 70%confluent at time of stimulation.
Calculate the mass of DNA required for stimulation of the cells using a final concentration of one to 10 micrograms of DNA per milliliter of cell growth Medium. Add the required amount of serum free medium into a suitably sized sterile tube. Then carefully pipette the onic lipid transfection reagent into the center of the medium without touching the side of the tube.
Let it stand for five minutes. Next, add the DNA to the tube and vortex. Briefly leave at room temperature for at least 20 minutes.
Then add the transfection mixture directly to the cells. Prepare the transfection mix as described for the in vitro transfection using one microgram of DNA two microliters of lipid transfection reagent, and 18 microliters of serum free medium per mouse ear to be stimulated. Then load a sterile 20 microliter syringe containing a 30 gauge needle with the DNA transfection mix anesthetize the C 57 black six mouse using one to 2%isof fluorine.
Confirm anesthesia by loss of movement and constant respiratory rate in addition to a pinch reflex test. If necessary, monitor the animal's eyes during anesthesia and use ointment as required to avoid dryness. Using sterile technique.
Carefully inject 10 microliters of the transfection mix into the ear pinna so that a single bubble of liquid is formed in between the dermal layers of the ear. Wear a rubber thimble on your thumb, stretch the ear over it and inject at an acute angle to ensure penetration of only the first layer of skin. Reintroduce the mouse back into the cage and regularly monitor Post anesthetic recovery.
Use a heat lamp to avoid excessive cooling of the anesthetized mice. Do not leave mice unattended until they have regained sufficient consciousness to maintain sternal recumbent after 12 to 24 hours and remove ear tissue with dissection scissors. Then flash, freeze the tissue in a micro centrifuge tube by immersing in liquid nitrogen for two minutes.
For in vitro transfected samples, aspirate medium from the cells with a pipette and discard the waist. Then add one milliliter of sterile PBS to wash the cells and repeat the wash. Step two times for mouse ear tissue samples.
Grind tissue under liquid nitrogen using a sterile ceramic mortar and pestle. Then transfer the ground tissue to a micro centrifuge tube using a sterile spatula. Next, add 350 microliters of lysis buffer to either the ground mouse ear tissue, or washed transfected cells, and proceed with a commercial RNA extraction kit.
Protocol elute, RNA from the extraction kit purification column in 40 microliters of DDH two O.Quantify the RNA by UV spectroscopy as previously described. For DNA to a sterile PCR tube, add one microliter of oligo DT primer, one microliter of 10 millimolar DNTP mix and 250 micrograms of RNA. Then bring the volume up to 11 microliters with sterile D DH two o.
Incubate the PCR tube for five minutes at 65 degrees Celsius. Then add four microliters of first strand buffer, one microliter of 0.1 molar DTT point 25 microliters of reverse transcriptase and 1.75 microliters of DD H2O to the tube incubate at 50 degrees Celsius for one hour, followed by a 15 minute incubation at 72 degrees Celsius. To perform quantitative PCR use two microliters of CD NA two microliters of DDH two O five microliters of QPCR master mix, and one microliter each of the 10 micromolar forward and reverse primer stalks mix.
In a 96 well plate and analyze using a quantitative PCR machine transfection of con contemporized DNA into murn embryonic fibroblasts induces a robust transcription of C xcl 10 and IL six as measured by quantitative PCR. These results indicate that these DNA sequences are able to stimulate the innate immune DNA sensing machinery. Con categorized DNA can also be used to stimulate a cytosolic innate immune response in vivo.
This was demonstrated by injecting immune stimulatory DNA into the ear penny of mice, which also resulted in increased levels of C Xcl 10 and IL six. Following this procedure, other methods like western blotting and immuno fluorescence microscopy can be performed in order to answer additional questions, such as what are the signaling events initiated by detection of intracellular DNA.
