The overall goal of this procedure is to study X chromosome inactivation in differentiated female mouse embryonic stem cells by means of simultaneously combined DNA and RNA fluorescent in C two hybridization or D-N-A-R-N-A fish. This is accomplished by first inducing differentiation in female mouse embryonic stem cells. As during this process, which simulates normal post implantation embryonic development, X chromosome inactivation is initiated, resulting in enhanced transcription and accumulation of cyst RNA, coding the future inactive X chromosome.
The second step is to fix the differentiated embryonic stem cells using paraldehyde. Next, prepare the probes for detecting both DNA from the X chromosome and cyst RNA by labeling DNA fragments with biotin or digo genin. Using Nick translation.
The final step is to perform the D-N-A-R-N-A FISH procedure. The differentiated embryonic stem cells are perme, denatured, and hybridized to the labeled fish probes, which are subsequently detected using fluorescently conjugated antibodies that detect the biotin or diggen in haps. Ultimately, immunofluorescent microscopy is SU to show the location of the two chromosomes in female cells and to visualize the expression of cyst RNA from the X chromosome that undergoes inactivation.
The main advantage of this technique compared to other methods like FISH methods where RNA and DNA are subsequently detected is that using our protocol, we are able to detect both types of nucleic acid simultaneously in the same specimen within less than two days. Although combined DNA and RNA fish can be challenging to to perform as conditions which are optimal to detect DNA or RNA differ significantly. Using the herein presented protocol, we are able to detect exist RNA and DNA derived from both X chromosomes in virtually all cells analyzed.
This has allowed us to study X chromosome in activation as it is happening during the differentiation of female mouse embryonic stem cells or abnormal female mouse development. Although this method can provide insights into the X in activation process, which is an important and fascinating process occurring during early mouse embryonic development, it can likely also be applied to other biological systems where it is important to detect RNA transcripts which are derived from the native chromatin as non-coding RNAs are more and more being appreciated for the importance in biological systems and often single cell approaches are favored over alternative approaches where whole populations of cells are being assessed. We foresee that this protocol will be helpful for a broad scientific community.
Female mouse embryonic stem cells or E ESL are grown under standard ESL conditions on gelatinized culture dishes coated with mouse embryonic fibroblasts ORMs to begin the procedure for induction of differentiation. Remove ES medium from the ESL culture and wash twice with cell culture PBS Add 1.5 milliliters of TRIPSIN EDTA prewarm to 37 degrees Celsius and incubate the cells at 37 degrees Celsius for seven minutes. After 3.5 minutes, shake the dish gently to break up the colonies.
Inactivate the trypsin EDTA by adding 3.5 milliliters of differentiation media to the cells, pipette cells up and down to obtain a single cell solution and collect them in a 15 milliliter tube. Spin for five minutes at 200 Gs.After collecting cells in 10 milliliters of differentiation medium, add the cell suspension to a non gelatinized 10 centimeter culture dish and incubate for one hour. In a cell culture incubator, MES will be able to adhere to the non gelatinized culture dish, whereas ES cells will remain in suspension.
In the meantime, place sterile glass cover slips into six well plates add 0.2%gelatin and incubate for a minimum of five minutes. At room temperature after one hour, collect the snat of the prepl ESL culture, which will mainly consist of non adhering e ESLs. After diluting the E ESLs plate into six well plates containing cover slips.
Begin this procedure by removing differentiation medium from differentiation cultures and washing twice with cell culture PBS. Be sure to add PBS carefully to prevent the cells from being washed away. Fix cells by adding 4%para formaldehyde PBS and incubating for 10 minutes at room temperature.
After 10 minutes, remove the para formaldehyde and wash the cover slips three times with 70%ethanol. To prepare the DNA probes for labeling, first dilute one microgram of each respective DNA in water in a total volume of 16 microliters. Then add four microliters of ox genin or biotin labeling solution mixed by vortexing and incubate at 16 degrees Celsius for 90 minutes.
This will incorporate the biotin or dig oxygen labeled nucleotides by NIC translation. In the meantime, prepare G 50 columns. To remove non-integrated nucleotides, take a two milliliter syringe, remove the stamper and add sterilized cotton into the syringe.
Add G 50 balls and solution to fill the syringe. Then place a syringe in a 15 milliliter tube centrifuge at 642 GS for one minute. After centrifugation, approximately 80%of the syringe should be filled with G 50 After 90 minutes, add 40 microliters of water to each NIC translation reaction mixture and transfer the reaction to the G 50 column.
Add an empty tube under the column and centrifuge at 642 Gs for two minutes. Collect the flow through in a reaction tube. Measure the volume of the flow through with a pipette and then add water to reach a total volume of 200 microliters.
To precipitate the flow through, add salmon sperm, DNA yeast TRNA mouse caught 1D NA and two molar sodium acetate mixed by vortexing. Add 533 microliters of ice cold 100%ethanol and shake the tube centrifuge at 16, 200 Gs for 30 minutes at four degrees Celsius. At the completion of centrifugation, remove the supra natant and wash the pellet twice with 70%ethanol centrifuge at 16, 200 Gs for five minutes at four degrees Celsius and air dry the pellet.
When the pellet is dry. Add 50 microliters, a 50 plus hybridization solution and incubate at 37 degrees Celsius for 30 minutes. To facilitate dissolving prior to permeation rehydrate fixed cells uncover slips by removing the 70%ethanol and adding cell culture PBS to the cells.
Incubate for five minutes, repeat a total of three times so perme mobilize the cells. Remove cell culture PBS and add 0.2%pepin to the six Well plates. Incubate the plates in a water bath at 37 degrees Celsius for four minutes.
After that, remove the pepin and inactivate with RNA free water. Postfix the cells by incubating them with 4%paraform aldehyde PBS for five minutes at room temperature after fixation, wash twice with cell culture or PBS for five minutes. Dehydrate the cells by incubating with 70%ethanol for three minutes, 90%ethanol for three minutes and a hundred percent ethanol for three minutes.
After that transfer the cover slips out of the six well plates and add dry the cover slips for several minutes. Age the cells by incubating the cover slips at 65 degrees Celsius for one hour on a hot plate. In the meantime, pre hybridized fish probes with mouse coat 1D NA by adding together for every cover slip to be analyzed.
25 microliters of 50 plus hybridization solution. One microliter of biotin labeled cyst Probe one microliter of digo oxygen and labeled BAC probe detecting the X chromosome and 0.5 microliters of mouse coat 1D NA mixed by vortexing and in nature at 99 degrees Celsius for five minutes. Incubate immediately at 37 degrees Celsius for 45 minutes to allow the cot 1D NA to hybridize to repeat sequences present in the probe.
Once the cells on the cover slips have aged spot 50 microliters of de maturation buffer on a glass slide and put the cover slips on top of the cells facing toward the de maturation buffer. Incubate at 65 degrees Celsius for two minutes. Postfix the cells by transferring the cover slips back to the six well plates prefilled with ice cold, 70%ethanol and incubate for five minutes.
Dehydrate the cells by subsequent incubation in 70%ethanol for three minutes, 90%ethanol for three minutes and 100%ethanol for three minutes. Remove the cover slips from the six well plates and let them air dry for several minutes. Spot pre hybridized Probe on a glass slide and incubator a cover slip on top.
Place the slides in a humidified chamber filled with 50 milliliters of 50%IDE two XSSC and incubate overnight at 37 degrees Celsius. After the overnight incubation transfer cover slips from glass slides to six wall plates that have been filled with two XSSC prewarm to 42 degrees Celsius. Incubate at 42 degrees Celsius for five minutes.
Remove two XSSC and add 50%form MI two XSSC. Incubate for 10 minutes at 42 degrees Celsius. Wash with 50%form IDE two X-S-S-C-A total of three times for 10 minutes at 42 degrees Celsius per wash.
After the third wash, remove the 50%form my two XSSC and wash the slides with tris saline tween or TST twice for five minutes at room temperature for blocking spot 50 microliters of tris saline BSA or T-S-P-S-A. Her cover slip on new glass slides and place the cover slips upside down on the glass slide incubate for 30 minutes at room temperature in a TST humidified dark chamber. After 30 minutes, transfer the cover slips back into the six well plates and wash twice for five minutes with TST for the first antibody Incubation spot, 50 microliters of sheep antigen and antibody per cover slip on new glass slides and place cover slips upside down on the glass slide incubate for 30 minutes at room temperature in A TST humidified dark chamber.
Subsequently, the cover slips are incubated with a series of fluorescently labeled antibodies to enhance the original signal as detailed in the accompanying manuscript. After incubation with a series of fluorescently labeled antibodies, dehydrate the cells by incubation for three minutes each in 70%ethanol, 90%ethanol and 100%ethanol. Remove the cover slips from the six well plates and let them air dry for several minutes.
Finally, spot a drop of mounting medium with dappy on each glass slide. Add a cover slip upside down on top and seal the slides with nail varnish. Using this protocol for combined D-N-A-R-N-A FISH X chromosome inactivation can be visualized in differentiating female ES cells.
This figure shows a representative example of A-D-N-A-R-N-A fish experiment where both cyst RNA and a region of the X chromosome are simultaneously detected. Cyst, RNA is a non-coding RNA that is essential to the process of X chromosome inactivation. In differentiating female ESL here cyst, RNA is detected using a biotin labeled CD NA probe, which is visualized using antibodies conjugated to rumine and indicated in red.
The X chromosome is detected using a xigen in labeled BAC probe, which is visualized using antibodies conjugated to FITC indicated in green nuclei are counter stained with DPI indicated in blue. In almost every cell, two x chromosomes are detected. Both large cyst accumulations on the inactive X chromosomes or cyst clouds and basal cyst expression from the active X chromosome or cyst Pinpoints are detected.
The cyst pinpoint are not detected in every nucleus because upon differentiation, this basal cyst expression is ceased on the future active X chromosome. Note that some of the pinpoint signals derived from the X chromosome are located within cyst clouds, thereby showing that the cyst cloud is indeed localizing along and coding the X chromosome Once mastered. This technique can be done in less than two days and with a reliable outcome if it is performed properly.
After watching this video, you should have a good understanding of how to induce differentiation of female mouse embryonic stem cells, how to fix these cells, and how to prepare them to allow you to perform combined D-N-A-R-N-A fish. Remember that it is important for the success of your experiments to keep temperatures and incubation times constant as our indicated in this protocol. Also, it is important to work in a clean environment.
If you're using a different cell type or studying a different biological system, some optimizing might be required to provide you with an optimal result. Good luck with your experiments.
