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A Laboratory Guide to the Mammalian Embryo by David K. Gardner, Michelle Lane, Andrew J. Watson

By David K. Gardner, Michelle Lane, Andrew J. Watson

This publication pulls jointly the complete variety of mobile tradition, biochemical, microscopic, and genetic thoughts to review the early mammalian embryo. beforehand, there hasn't ever been this sort of complete compendium, although there were extra concentrated books of protocol, reminiscent of Manipulating the Mouse Embryo, from chilly Spring Harbor. This e-book is meant to attract all constituencies, from uncomplicated experimental technology to scientific and animal technological know-how purposes.

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Remove all adhering tissue and the bursa and transfer the cleaned ovaries to a fresh dish of L15 medium. 2. Bisect the ovaries and transfer pieces to individual dishes of L15. Keep at 37°C. 3. Using 2 × 25-G needles, gently tear the tissue into smaller pieces. Follicles for culture can then be identified from these pieces and dissected free of surrounding tissue. Acupuncture needles (30 G) mounted into a holder are useful tools for dissection, as these have a fine point and no barb. 4. 1% BSA solution to prevent sticking) to a fresh L15 dish before selection.

Females are then placed with males and allowed to remain overnight. Mating can be detected by the presence of a vaginal plug the next morning. Fertilized embryos can then be collected (see below). Response to superovulation procedures vary significantly between strains of mice. We typically give a dose of 5 IU per mouse for mice 3–4 weeks old. Hormone solutions can be prepared by reconstituting lyophilized powder in sterile saline at a concentration of 50 IU/ml. 1 ml per mouse. Hormones can be stored for 2–3 weeks at –20°C or at –80°C for 2–3 months.

The female reproductive tract contains significant levels of glycosaminoglycans (31), the levels of which can vary during the menstrual/estrous cycle (32). One glycosaminoglycan of interest is hyaluronan. Hyaluronan is a high molecular-mass polysaccharide and can be obtained endotoxin- and prion-free from a yeast fermentation procedure. In the mouse hyaluronan levels in the uterus increase at the time of implantation (33). It has been shown in the pig (34) and cow (35) that hyaluronan can act in synergy with albumin in an embryo culture system.

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