Hi, my name is Har Moreno from the Reproductive Genetics and Stem Cell Laboratory of Mississippi State University. Hi, my name is Clara St.Also for the reproductive genetics and stem cell laboratory of Mississippi State University. Premortal germinal cells or PGC are the progenitors of armonia and voia in the ovarian testis respectively.
In mouse embryos. PGC migrate from the hin gut and across the genital ridge at 8.5 to 9.5 days. Postum PGC arrive at the genital ridge at 10.5 to 11.5.
TAs Postum and begin forming the gonadal reach in D female. The pg cs migrate to the co lateral end of the kidney to begin forming the female gonadal reach. In contrast in the male PGCs migrate to either side of the bladder.
To begin forming. The male alridge Ger cells are an excellent research model to study onic developmental pathways in the presence of oxidative stress as a cause of infertility and cancer. So Let's get started.
Today we will show you how to collect and isolate PV Cs from developing gonadal ridges at 10.5 to 11.5 days Postum mouse embryos gonadal ridges are dissociated by mechanical destruction to implant planted on mouth embryo fibroblast figure layer. After 10 days of culture under optimized conditions, embryonic germinal colonies will show embryonic phenotypes. Also after passages they can be identified by their expression of chloro potent markers.
The following materials are needed for this step on sterile dissection. Package that contain sterile fields, forceps, scissors, razor blades, antimicrobial soaps, saline solutions, Petri dish filled with PBS Pep on ice and gauze a pregnancy five seven BL six female mouth at 10.5 to 11 days, Postum is euthanized by cervical dislocation. Clean the abdomen with antimicrobial soap, then shave it after.
Wash the abdomen using saline solutions. Dry it with a sterile gauze. Cover the mouse with a new sterile field.
Make a ventral incision using forceps and dissect some scissors. Identify and remove the entire uterus from the abdominal cavity. Mouth embryos will be visible inside the uterus.
Very carefully. Put through the ligaments that are still attached to the uterine horn. Complete the dissection by making a transverse cut along the vagina.
Transcribe the uterus to a Petri dish seal with GPVS and keep it on ice. Separate the extra embryonic tissues from the mouth embryos. Using a sterile scalpel and forcep.
Transfer the mouth embryos into a fresh Petri dish filled with DPBS. Measurable length of the mouth embryo. We found sizes differed according to the embryo age.
For example, 8.5 days post coum measured in average six millimeters, 10.5 days post coum measured in average 11 millimeters and 12.5 days post coum measured in average 16 millimeters. Remove their tails through DNA extraction. The following materials are needed for the dissection stereo microscope biosafety cabinet PDS sterile scalpel.
Sterile fine forceps, sterile fine teasing needle with handle petri disc filter paper sheets. Place a filter paper in a Petri dish, then place your mouth embryo on top of the field paper to dry the embryo and immobilize it for dissection. Using a sterile scalpel, make a transverse cut of the mouth embryo above the origin of the umbilical cord under a stereo microscope.
Using sterile fine forceps in a sterile fine teasing needle. Remove the intestines and liver so that the urinary system is visible. The bladder is located on a medial plane.
The embryo can be identified as male because the gonadal bridges are located on either side of the bladder. Using sterile fine forceps in a sterile fine needle, remove the right male gona ridge by cutting away the tissue that support it. Use the same procedure to remove the left male genital ridge.
Transfer both male genital ridges to a small Petri dish containing fresh DPDS microscopically. You can visualize the male gonadal ridge attached to the miso ne brace. The male gona ridges should be striped large and oval in shape under a stereo microscope.
Remove the intestines using sterile fine forceps and a sterile fine teasing needle. The kidneys are located laterally in the abdominal cavity and the bladder is located more medial and codal in comparison to the kidney. The female gonadal ridge is different from the male gonadal ridge because of its morphology and anatomical location.
The female gonadal ridges are firmly attached to the codal lateral end of the kidney. Using sterile fine forceps and a sterile fine needle. Remove the left female gona ridge by cutting away the tissues that attach it to the kidney.
Repeat the same procedure to remove the right female gonadal ridge. Transfer both female genital ridges to a small Petri dish containing fresh PDS microscopically. You can visualize the male gonadal ridge attached to the Neso Nera.
These female go natal ridge should be spotted having elongated shape and be smaller when compared to the male go natal ridges using sterile, fine and sharp needles. Separate the female gona ridges from the meso and other somatic tissue using sterile, fine and sharp needles. Separate the male gonadal ridges from the meso neros and other somatic tissues.
It is important to take out the meso Neros from ne Natal ridge because it is a somatic tissue that will overgrow in the PGC derivation process. Collect the gona ridge in a 0.5 microliter drop of fresh DPVS with collagenase distaste solutions. Under stereo microscopes, gonad tissues are cut into small pieces using sterile fine and sharp needles.
Petri dishes are transferred to an incubator at 37 degrees Celsius for 15 minutes. After 15 minutes, the tissue can be dissociated by pipetting using pulled glass capillary pipettes with different diameters between 40 to a hundred microns. Break up the pieces by pipetting up and down.
This will form small slumps. These small clumps are washed in 0.5 microliters of DPDX after washing. These are transferred to Eend cubes at 2000 RPM for five minutes.
At room temperature after cent fu, the supernatant is removed and the pellet is rended in 0.5 milliliters of supplemented knockout media free warmed at 37 degrees Celsius 24 hours. Before this process, you should prepare a mily inactivated mouth embryo fibroblasts meth CCF one feeder layer immediately before plating the PGC. The mass media must be removed from the prepared mass feeder layer.
Then add point milliliters of the supplemented knockout media previously warmed at 37 degrees Celsius onto the meth feeder layer. Under stereo microscopes using a symmetrical distribution, small pumps of PT GC are plated over the meth feeder layers. If these pieces are close, they tend to aggregate and make dense colonies that attach poorly or begin differentiating culture.
Dishes must be labeled with embryonic germ cell line name, passage number, and date. Carefully transfer the culture dishes in an incubator at 37 degrees Celsius and 5%COC after 10 days post isolation, you can identify embryonic germinal cell colonies based on their characteristic morphology. Also, after the first passage, they can be identified by their expression of flu potent markers such as alpha lime phosphatase, OP four SSEA one, and so two.
Today we just joined you How to collect insolate and culture. Embryonic germinal cells from PGC as 10.5 11.5 days posts. Coum mouse Characterization of embryonic germinal cells as precursor of GA meat is essential for recess related to gametogenesis in fertility and cancer.
Thank you for watching our video. Good luck in your experiment.
