The overall goal of this procedure is to separate mouse embryonic, facial ectoderm, and mechy. This is accomplished by first isolating CD one E 10.5 embryos. The second step of the procedure is to treat the whole embryos with dys.
Phase two. After the enzymatic treatment, the facial prominences are isolated and the final step is to carefully separate the facial derm from mesenchyme. The isolated facial derm and mesenchyme are then ready for further experimentation.
The main advantage of this technique over existing methods like dis space treatment of dissected facial prominence, is that treatment of the whole embryo with DYS space keeps both the facial accident and intact. This method can help answer key questions in the mammalian developmental biology field, such as how differential gene expression, epigenetic modification, and transcription factor binding can regulate the molecular crosstalk between facial derm and mesenchyme. During embryogenesis Plan on four to six hours between now and the isolation of the tissues, clean the dissection area and tools for 70%ethanol.
And if the samples are for RNA isolation, use RNAs away. It is critical that the forceps are sharp. Additionally, for RNA isolation, the solution should be treated with ethyl percarbonate.
Now euthanize a pregnant CD one female using approved protocols to collect E 10.5 embryos. Spray the abdomen with 70%ethanol, open the abdomen. Use one set of forceps to pull the uterus and another to tear the meso nutrium away from the uterus.
Remove the uterus from the body and repeat the process on the opposite uterine horn. Briefly rinse the uterus with ice cold PBS in a 10 centimeter Petri dish to wash out the blood. Transfer the uterus into a new dish with ice cold PBS.
Separate it into one embryo fragments by cutting it between the implantation sites. Then transfer one embryo into ice cold PBS under a stereo microscope. Starting at one of the cut sites.
Use the fine forceps to tear off the muscular wall of the uterus to expose the embryo with the yolk sack. Then peel off the yolk sack and amnion. Transfer the dissected embryo to a six centimeter Petri dish with PBS on ice, and repeat the procedure until all embryos have been collected.
Before treating the embryos for dys paste two, wash them with ice cold PBS in a six centimeter Petri dish. Replace the PBS with 10 milliliters of ice cold diluted dys Pastes two in PBS. Cover the dish and incubate it at 37 degrees Celsius for about 25 minutes.
Adjust the time according to the enzymatic activity of the dys phase two, which can vary to confirm the digestion look under a stereo microscope and check that the derm has become loose. This is typified by a clear gap between this tissue layer and the underlying mechy. If it is not loose, incubate the tissue for another five minutes.
When the digestion is complete, put the samples on ice. Keep them chilled from now onward, except during dissections after the treatment with dys. Phase two, the facial derm is easy to peel off.
Use a disposable glass pipette to transfer a treated embryo into ice cold PBS in a six centimeter dish under a stereo microscope. Use forceps to hold the embryo and find forceps to carefully cut the boundaries of the facial prominences attached to the head. Pay special attention to contamination from other ectoderm, discard, or potentially contaminated tissue to dissect the facial prominences out intact.
Start the dissection from one side First, dissect under the MNP. Then proceed with the MXP and the FNP. Repeat this process from the other side to complete the isolation at this point if needed, separate and isolate the three facial prominences with all the tissues readied under three x magnification.
Separate the facial derm and mechy as quickly as possible. First, use a long glass pasta pipette to gently pipette the facial prominences up and down five times. The ectoderm is now easily removed.
Use one forceps to hold the facial prominences and a second forceps to peel them off the ectoderm very slowly. This is the most difficult step of this procedure. Makes certain that the forceps are very sharp.
And when gently peeling the odum from the magic climb, do it very slowly. Also, always adjust your position as necessary. Using a new long glass pasta pipette.
Transfer the facial derm into a 1.5 milliliter tube with fresh PBS on ice. Transfer the mesenchyme to another 1.5 milliliter tube with fresh PBS on ice. Now wash the samples with PBS next centrifuge.
The samples of four degrees Celsius of 500 G and for three minutes aspirate the PBS using long glass pasta pipettes. The samples can now be processed for a chip assay or RNA extraction or protein extraction. After the dys phases two treatment, the ectoderm of the embryo tends to be loose.
The facial prominences should remain intact after the dissection. The isolated facial ectoderm should be clear and free of mechy tissue to determine the efficacy of the protocol. Cross-contamination was tested for on the molecular level.
RT PCR was performed on each tissue for the following, for brain express sick. Three, ODEM specific CD CDH one and Mechy specific. So 10, the results confirmed that the tissues expressed the expected genes and were free of cross-contamination.
While attempting these procedure, it's important to remember to keep the samples on eyes at all times except for the DASE two treatment and dissections After its development. This technique paved the way for researchers in the field of craniofacial development to explore differential gene expression, molecular signal, crosstalk, and downstream target identification in embryonic facial derm and mechy with relevance to facial development and or facial clefting. Following this procedure are the methods like chipsy arming as ASIC and protein detection can be performed in order to answer additional questions like how gene expression differences in the differential transcription factor binding distinguishes the odum and amazing climb.
