The overall goal of this procedure is to isolate E 8.5 mouse embryos for use in chromatin, immunoprecipitation, or chip assays. This is accomplished by first isolating the mouse embryos and preparing single cell suspensions. The second step of the procedure is to chemically cross-link DNA bound proteins to the genomic DNA, followed by sonication to fragment the DNA.
The third step of the procedure is to immuno precipitate protein chromatin complexes and isolate purified DNA. The final step of the procedure is to analyze the purified immuno precipitated DNA by conventional PCR or by quantitative real-time PCR. Ultimately, results can be obtained that show protein interactions with regulatory sequences of differentiation specific genes during the initiation of the differentiation program in developing tissues and organs.
This semester can help answer good question about the initiation of differentiation program during embryogenesis through the identification of a regulatory factors that interact with the regulatory sequences of differentiation specific gene as they become transcriptionally competent. To begin this procedure, uterine horns are removed from a mouse sacrificed at embryonic day 8.5. According to an approved protocol, the tissue is placed into a Petri dish containing fresh section media using scissors.
Cut each implantation site individually and place them in a Petri dish containing fresh dissection media to avoid excess blood. With the eight of forceps, remove the uterine layer from each implantation site to expose the de sidu isolate individual embryos using forceps. At this stage in development, the embryos are surrounded by the parietal yolk sack, which contacts the deum tissue, the visceral yolk sack, and the amnion.
The amnion is a transparent membrane that is in direct contact with the embryo. The visceral yolk sack is located between the amnion and the parietal yolk sack and is readily distinguished in the 8.5 to eat 9.5 embryos by the presence of prominent blood vessels that nurture the embryo. Remove each embryo from its surrounding membranes and transfer individually into a new plate containing dissection media to remove excess blood.
Then transfer each embryo to a 1.5 milliliter einor tube containing 200 microliters of dissection media. Homogenization of the isolated embryo begins with the addition of 200 microliters of collagenase. Type two solution to each einor tube containing the embryos.
Gently shake samples in a 37 degree Celsius shaker at 100 RPM for 20 minutes. Then resuspend the samples by petting to disrupt clumps of cells. Apply the cell suspension on the top of a 40 micron mesh size sterile cell strainer placed over a 1.5 milliliter eend rph tube.
Immediately apply 600 microliters of room temperature one X-D-P-B-S to the top of the cell strainer to complete the separation. Once the suspension has been strained, discard the cell strainer centrifuge the samples at 4, 000 G for five minutes at four degrees Celsius. Following centrifugation, discard the supernatants reus.
Suspend each pellet in one milliliter of room temperature one X-D-P-B-S. Then centrifuge the samples at 4, 000 G for one minute at room temperature. After discarding the snat for a second time, resuspend each palate in 200 microliters of room temperature dissection media cross-link the chromatin by adding 5.6 microliters of 37%formaldehyde to the 200 microliter samples for a final concentration of 1%formaldehyde.
Incubate samples for 10 minutes at room temperature following incubation, centrifuge the samples at 4, 000 G for three minutes at four degrees Celsius and discard the supernatant. Resuspend the cells with cold one X-D-P-B-S containing 80 microliters per milliliter protease inhibitor cocktail or PIC. Then centrifuge the samples at 4, 000 G for three minutes at four degrees Celsius.
After discarding the supernatant, the samples can be frozen at minus 80 degrees Celsius and are stable for several months prior to sonication thaw the frozen cell pellet on ice or placed the pellet from the previous step on ice. Re suspend the cell palette in 100 microliters of room temperature SDS lysis buffer after resus suspension. An additional 100 microliters of room temperature SDS lysis buffer is added and the sample is resuspended by pipetting and inversion.
Incubate the cell suspension on ice for 10 minutes. Following incubation, set up the ator to shear the cross-link DNA to between 200 and 500 base pairs. In this demonstration, a DIO geno bio rupture UCD 200 is used.
Sonicate the sample surrounded by an ice slurry. Do not allow it to warm above four degrees Celsius or to freeze centrifuge. The sonicate sample at 14, 000 G for 10 minutes at four degrees Celsius.
Transfer the supernatant into a fresh eend DPH tube pre-cool on ice. Divide the sonicated sample into a maximum of five aliquots. Store one aliquot at minus 20 degrees Celsius to reserve his input for the cross-link reversal.
Step dilute the other aliquots tenfold in chip dilution buffer containing freshly added PIC at one microliter per 800 microliters of buffer. First, determine which aero speeds will be used for pre clearing based on the antibody to be used for the chip assay protein. A beads are used here because the antibody to be used for chip is rabid.
Pre-clear the diluted superin by adding 75 microliters of a 50%of salmon sperm, DNA and protein. A aero speeds incubated four degrees Celsius with rotation for one hour following incubation centrifuge the samples at 2, 500 G for one minute at four degrees Celsius. Transfer the supines to a new pre-cool eend DPH tube.
Add four micrograms of antibody per sample to the pre-cleared Eloqua and incubate overnight at four degrees Celsius with rotation. Begin by adding 60 microliters of the salmon sperm, DNA protein, a aeros bead suspension into each sample and incubate at four degrees Celsius with rotation for one hour. Centrifuge the samples at 1000 G for one minute at four degrees Celsius.
Following centrifugation. Carefully remove the supines and discard keeping the DNA protein bead complex on ice. Resus suspend and wash the pallets as described in the written protocol with buffers one through four.
Each wash is comprised of a five minute incubation period with rotation followed by centrifugation at 1000 G for one minute and removal of the supernatant after washing. Elute the chromatin antibody complex by Resus Suspending the washed chromatin protein bead complex in 250 microliters of freshly prepared elution buffer vigorously Vortex each sample for five seconds and then incubate the samples at room temperature for 15 minutes with rotation following centrifugation at 2, 500 G for one minute. At room temperature, transfer each eluate into a new eph tube.
Repeat the elucian process and combine the eluate for each sample in the same eph tube. To reverse the cross links, add 20 microliters of five moer sodium chloride to each 500 microliter eluate. For the previously reserved input control add 40 microliters of five moer sodium chloride per milliliter.
Heat the EITs in a 65 degree Celsius water bath for four hours. To overnight run 10 microliters of each sample in the input control on a 2%aeros gel next to size marker spanning 0.1 to one kilobase pair to check the DNA fragment size. The DNA will appear as a smear and not a sharp band.
Recover the DNA from each 500 microliters sample using a kaya quick gel extraction kit. Begin by adding 600 microliters of qg buffer to optimize DNA absorption with the silica membrane in a kaya quick spin column and then add 200 microliters of isopropanol prior to loading the column. Check the mixture to determine whether any SDS precipitate is present.
If SDS precipitation has occurred, the samples should be warmed in a 42 degree Celsius water bath until the precipitate disappears. Then apply the samples to the columns and elute the DNA per manufacturer's instructions. SDS can interfere with the activity of PCRT polymerase and should therefore be removed completely before performing PCR.
Incubate each DNA sample on ice to precipitate any residual SDS and centrifuge at 14, 000 G for one minute at four degrees Celsius. Transfer the SANE to new einor tubes. The DNA EITs can be detected either by conventional PCR or by quantitative real-time PCR or QPCR with primer specific For each sequence to be analyzed, use five to 10 microliters from the total DNA IIT for one PCR or QPCR reaction, PCR and QPCR conditions will vary.
However, the limited amount of starting material requires additional PCR cycles for conventional PCR detection, start with 40 cycles and adjust as needed. Using this protocol, chips have been performed from both E 8.5 and E 9.5 embryos. Chip purified DNAs were analyzed by conventional PCR and by quantitative realtime PCR.
The results demonstrate that the myogenic and regulatory factor is present on the myogenic and promoter in E 8.5 and E 9.5 embryos. In contrast, there was no indication of myogenic and binding to the myogenic and promoter in the oak sac where myogenic is not expressed. The interferon gamma promoter, which contains sequences matching the myogenic binding site was used as a negative sequence control as expected Myogenic was not bound to the interferon gamma promoter in any of the tissue samples tested.
The results indicate that Myogenic is bound at the Myogenic promoter in the somites of E 8.5 and E 9.5 embryos as myo Genin is specifically expressed in the somites At these stages, After watching this video, you should have a good understanding of how to isolate and prepare EA 0.5 mouse embryos for chromatin precipitation assess that will identify regulatory factors found to tissue specific genes in developing tissues.
