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Transgenic Core Facility
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The use of hormones in females prior to mating, to stimulate the release of greater numbers of eggs, has been used successfully in this facility only on the CD-1 strain. It does increase two-to threefold the number of embryos obtained, but this is somewhat countered by an increase in the percentage of eggs that are not fertilized. Nonetheless, it is an economical way to maximize yield. It is most effective when young (4-6 week) females and only the most productive males are used. Aliquots of pregnant mares’ serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) are made up at the appropriate concentrations and stored at 80˚ C. It is important to allow the hormone to thaw gently at room temperature - accelerating the thawing seems to reduce the efficacy of the hormones. 1) Using a 1 cc syringe with a 30 G x 1/2” needle, inject each CD-1 female with 0.1cc of PMSG intraperitoneally (the female’s estrus cycle can be ignored when using the hormones). Make a note of the injection time. 2) After 46-48 hours, inject the same mice with 0.1 cc of HCG intraperitoneally. Aliquots of HCG, need to be diluted 1:10 with sterile saline before injection. 3) Once they are injected with HCG, place females with intact CD-1 males, as with natural matings. 4) Check females for “plugs” the following morning. Back to Top Back to Protocol Page
2. Isolation of Blastocysts from Donor 1) Remove the uterine horns of female mice 72 hours after they have “plugged” and place them in a 35 x 10 mm petri dish. 2) Place one uterus in a deep depression slide and wet with flushing and holding media (FHM), then cut the cervical end of the uterus just below the junction of the horns as well as between the oviduct and ovary on each horn. 3) Insert a 1 cc syringe with a 30 G 1/2 “ needle into the cervical end of the uterus and thread the needle through one of the horns. Flush approximately 1 cc of FHM through the horn. You will see the oviduct expand as the fluid flushes through. Repeat on the other horn. 4) Remove the uterus from the slide and allow 2-3 minutes for the blastocysts to settle at the bottom of the dish. Carefully search the entire depression for blastocysts and transfer each with a mouth pipetter into culture dish of FHM. Rinse the blastocysts and transfer to a drop culture of KSO with amino acid media. 5) Place drop culture in an incubator at 5% CO2 and 37˚ C. Back to Top Back to Protocol Page
3. ES Cell Injection into Blastocysts 1) Cover
the bottom of a deep depression slide with blastocyst injection media (BIM)
provided by the researcher and made using their serum.
Cover the media with a thin layer of mineral oil. 2) Place the slide on the stage of the microinjection scope. Fill injection needle and holding pipet with BIM. Lower holding pipet onto the slide their tips touch the bottom of the injection chamber. 3) Using a mouth pipet, transfer expanded blastocysts into injection slide. 4) Transfer
ES cells into slide. Keep reserve of
cells on ice. 5) Load ES cells into needle. Choose 15-20 small, round, healthy cells of uniform size. 6) Use holding pipet to position and hold a blastocyst by adjusting the suction of the pipet, using a 60 ml syringe. The egg should be positioned so that the larger of the cell masses is located away from the needle. Pull back slightly on the syringe controlling the holding pipet. Be careful not to harm the blastocyst. 7) Focus on the needle. Bring the blastocyst to be injected into the same plane by using the micromanipulator controls. Push the tip of the needle gently into the blastocyst. When the needle is in the blastocyst, push on the 10 cc syringe controlling the needle until 10-18 ES cells have been injected. Once you have successfully injected the blastocyst, slowly remove the needle. Back to Top Back to Protocol Page
4. Implantation of 3.5 day Embryos into Surrogate Mother This procedure takes place entirely in the cubicle room located in the animal facility. You will need: Sterilized surgical pack Mouth pipet Several transfer pipets 30 G 1/2” needle 1) Take previously injected blastocysts (0-24 hours post-injection) and place them in a culture dish filled with flushing and holding media (FHM). 2) Take blastocysts, sterilized surgical pack, and mouth pipet to surgery area. 3) Use Nembutol or Ketamine/Xylazine to anesthetize a pseudo-pregnant female CD-1 mouse (plugged 48 hours prior to implant. Inject 0.3-0.5 cc intraperitoneally, according to weight. This provides 20-30 minutes of surgical plane anesthesia. 4) Using a pair of small surgical scissors and a pair of forceps, gently expose the ovary and uterine horn through the dorsal body wall between the last rib and the pelvis. Use a small clamp attached to a fat pad to keep the ovary in position. 5) Load
the injected blastocysts into a mouth pipet. First
draw up a small amount of FHM, then two
small air bubbles, then 1-6 blastocysts, and finally one more small air
bubble. 6) Use a dissection scope to focus on the uterine horn of the mouse. Use a 27 G 1/2” needle to make a hole in the uterine horn near the ovary and away from any major blood vessels. Insert mouth pipet into hole and gently blow the blastocysts into the uterine horn. Remove pipet and look to make sure that all the air bubbles have been pushed into the uterine horn. 7) Remove clamp and allow the ovary to return to the body cavity. Close the skin incision using surgical staples. Do not implant more than 12 blastocysts into each female. 8) Visually check for pregnancy in 10 days. Back to Top Back to Protocol Page
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Transgenic Core Facility 2160 South First Avenue, Maywood Illinois 60153 |
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©1995-2008 Loyola University Health
System. All rights reserved.
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