The overall goal of this procedure is to perform nascent RNA FISH on sections of post-implantation embryos as well as on short-term cultures of ectoplacental cones. With this technique, single cell analysis of transcriptional activity can be evaluated in our bionic tissues and explained to study the dynamic changes in gene expression that occur during in vitro development. This method can help answer key question in the field of development, epigenetics, and cell biology.
Here, we use it to study X chromosome inactivation and imprinting in exo-bionic tissue, the trophoblast giant cell. To begin, isolate E7 conceptus'in PBS in a 60 millimeter dish. First, dissect the embryo.
Begin with forceps. Pierce the sample and open and tear the two sides of the decidua apart. Then, shell out the embryo.
Use the fine forceps tips in a scissor-like fashion to carefully separate the ectoplacental cone, or EPC, from the embryo proper. The goal is a perfectly clean sample without maternal tissue, chorion, or yolk sac. Next, prepare flame-sterilized 12 millimeter round cover slips and place them in four well plates.
Load each well with one cover slip and 500 microliters of EPC medium. Now, deposit the EPC at the center of the cover slip and press it gently into position using the forceps. Central placement of the EPCs is critical.
Culture the loaded plates for three to five days at 37 degrees Celsius under 5%carbon dioxide. From the X plants, there will be a monolayer outgrowth of flattened TGCs. After incubation, wash the cells on the cover slips with PBS for five minutes.
Then, fix the cells for 10 mintues in 3%paraformaldehyde at room temperature. Then, treat the cells with ice-cold permeabilization solution. Incubate them on ice for five minutes.
Once permeabilized, rinse the cells three times with 70%ethanol. To store the cells at this point, keep them in 70%ethanol at minus 20 degrees Celsius in four well plates sealed with parafilm. To begin, precipitate three to five microliters of the fluorescently-labeled probe with five micrograms of salmon sperm DNA in a one to 10 volume of three molar sodium acetate at pH 5.2 and three volumes of 100%ethanol.
Next, spin the mixture at 16, 000 times G in a chilled centrifuge for 25 minutes. Then, wash the pellet with 70%ethanol. Following the wash, repeat the spin for five more minutes.
Now, discard the supernatant and dry the pellet using a SpeedVac for two minutes. Once dry, re-suspend the pellet of probe in 100%formamide. Then, put the probe solution at 37 degrees Celsius with shaking for 30 minutes.
Next, denature the probe at 75 degrees Celsius for seven minutes. Once denatured, quench the probe on ice and add an equal volume of 2X hybridization solution. Now, dehydrate the cover slips by sequentially washing them with 80%ethanol, then 95%ethanol, then twice in 100%ethanol.
Each bath should be five minutes long. Once dehydrated, dry the cover slips completely. Once dry, apply the probe in hybridization solution onto the cells and sandwich them with a slide.
Now, place the preparations in a humidified chamber and incubate them overnight at 37 degrees Celsius in the dark. The following day, wash the cells. Use one milliliter of denaturing solution to loosen and remove the cover slip.
Take care not to scrape the cells. Then, place the cover slips cell side up into a four well plate containing warm denaturing solution and transfer the plate to 42 degrees Celsius for seven minutes. Change the denaturing solution twice for a total of three seven-minute washes at 42 degrees Celsius.
Keeping the preparations at 42 degrees Celsius, wash the cells three times in 2X SSC for five minutes per wash. Now, counterstain nuclei by washing the cells in 2X SSC with DAPI for three minutes at room temperature. To remove the counterstain, use two rinses with room temperature 2X SSC for 30 seconds per rinse.
Finally, mount the wet cover slips to a slide using 5 microliters of mounting medium. Avoid generating any bubbles as this can lead to local bleaching of fluorescent signal, and wipe off any excess mounting media. Then, seal the slides with nail polish.
TGCs were identified from E7 sections using DAPI staining. They are extremely large compared to other cells at this stage, and easily visible in the conceptus. RNA FISH was performed to study X chromosome inactivation in this extra embryonic lineage.
The TGCs were stained with a probe to Xist and a probe for the Atrx gene. The Xist probe was labeled with SG to produce a green signal and the Atrx probe was labeled with a zar to produce a red signal. The inactive X chromosome is coated with Xist while the active X chromosome shows only a pinpoint of low transcription at the Xist locus.
By comparison, the nascent Atrx transcripts are only associated with the active X.Using a similar procedure, secondary TGCs were derived from the EPC and identified using DAPI staining. Because these cells can be heterogeneous in size, only the largest ones were analyzed to be certain of their lineage. The cells were labeled with three probes:Xist with the green SG signal, and probes to two X-linked genes:G6pd and Huwe1, visualized with red and magenta.
G6pd appeared to be monoallelically expressed from the active X.Huwe1, however, appeared to be expressed from both alleles, indicating it escapes from X inactivation. After watching this video, one should have a good understanding of how to obtain trophoblast giant cells from ectoplacental cone and decidua of gestation, and to study transcriptional activity in embryonic tissues at the single cell level by RNA FISH. While carrying out this procedure, it is important to remember that all steps should be learned in RNA-free conditions.
Following this procedure, other methods like immunofluorescence and DNA FISH can be performed to study protein in presence and distribution as well as gene locus position. It is also feasible to perform RNA FISH and immunofluorescence image sample, and then peform DNA FISH to colocalize the signals.
