Archive for category: Protocols

Mouse Surgery Protocol ( Click to Download )

A. Mice, like most species have a circadian rhythm. Investigators should be aware that this may affect biological data and should standardize the time of day that samples/measurements are taken to avoid this affect. The standard light/dark cycle at the University of Massachusetts is ???. The adult mouse weighs approximately 40 grams and this small size and resulting large surface area/body weight ratio makes them susceptible to changes in environmental conditions. The core body temperature is easily affected by small changes in temperature which may modify the physiologic responses of the animal. The acute hearing of mice makes them highly sensitive to ultrasounds and high pitched noises inducing a stress response that has been empirically related to annibalism of pups by their dams. The well developed sense of smell is used to detect pheromones used in social interactions. The poor vision of mice makes them unable to detect color and red light is often used to observe animals during the dark cycle.

B. Basic Biological Data

Mouse Surgery Protocol ( Click to Download )

A. Oocytes for pronuclear injection are collected from females that have been hormonally stimulated to cause superovulation. The eggs are flushed from the oviducts following euthanasia of the donor. After pronuclear injection of the DNA, the embryos are surgically injected into the oviducts of recipient females.

B. Transgenic mice will be screened by removal of approximately 1 cm of the tail tip from which DNA is then extracted. This procedure inflicts minimal discomfort, and therefore, requires no anesthetic. Usually bleeding is not a problem, however, the vessels can be electrically cauterized if needed.

Mouse Surgery Protocol ( Click to Download )

A. Estrus cycle and hormone profiles

1. Vaginal smears (method and stages from Boot, Kwa and Ropcke, 1981 in Mammary Tumors in the Mouse, Hilgers & Sluyser, Eds.; see ref #2679) – A cotton swab is used to collect cells from the vagina daily. The cells are swabbed onto a slide, air-dried, then stained according to the usual H&E protocol. It should be noted that vaginal stimulation can induce pseudopregnancy, but mice are less susceptible to this than rats.
   1. Diestrus: Smear contains predominantly leukocytes, some epithelial cells with stained nuclei.
   2. Proestrus
      1. Stage 1: Leukocytes dominate, but partly degenerated; fragmentation and cytolysis are observed. The vaginal contents are scanty, as a result the smears are thinly spread.
      2. Stage 2: Leukocytes still constitute ~90% of cells. Among the epithelial cells, some appear to have no stainable nuclei indicative of keratinization.
      3. Stage 3: Proportion of leukocytes decline rapidly such that leukocytes should be less than 60% and the epithelial cells comprising about 40%. About ¼th of the epithelial cells should have no stained nuclei. The vaginal content is increasing leading to thicker smears.
      4. Stage 4: Total disappearance of the leukocytes, while ~50% of the epithelial cells are keratinized. The keratinized cells may still be quite small. The smears continue to thicken.
   3. Estrus
      1. Stage 1: Thick smears in which the keratinized cells predominate. Stained nuclei should be present in <10% of cells. This stage corresponds to ovulation.
      2. Stage 2: The smears contain only keratinized cells. They have a large, flattened form characteristic of full estrus.
   4. Metestrus
      1. Stage 1: The same as above, but leukocytes appear (<10% of cells). They may have a degenerated appearance.
      2. Stage 2: The leukocytes now number up to 40% of cells.
      3. Stage 3: Leukocytes increase to ~90% of cells.
      4. Stage 4: The smears are now very thick with vaginal contents becoming “pasty” or “milky”. Leukocytes dominate and stain clearly showing their characteristic clumping in large masses around the epithelial cells. The epithelial cells are still of the large keratinized type with a few smallers cells with stained nuclei be present a well.
      5. Stage 5: Thick smears composed of largely leukocytes, characteristically surrounding the epithelial cells. About half of the epithelial cells have stained nuclei as the keratinized layer of the epithelium dissolves rapidly.
      6. Stage 6: A transition to diestrus; this stage is still set apart because of the thick smear and the typical arrangement of the leukocytes around epithelial cells that now all have stained nuclei.
2. Hormonal profiles during estrous cycle in mice
3. Hormonal profiles during pregnancy in mice – See McCormack and Greenwald, 1974 (Ref #2675). Compare with humans reported by Tulchinsky et al., 1972. (Ref #2676)
4. Hormonal levels during pseudopregnancy – See Christov et al., 1993 (Ref#1869).

B. Growth of the mouse mammary gland – Methods for estimating gland development and responses to treatments is reviewed by Ceriani and Hilgers, 1981. (In Mammary Tumors in the Mouse, Hilgers & Sluyser, Ed.; Ref # jj2680)

1. DNA assay
2. Morphometric methods
3. Effects of hypophyseal transplants

C. Superovulation – See Fowler & Edwards, 1957 (jj2765)

1. Prepare hormone solutions and store as 1 ml aliquots at -70^oC.
   1. PMSG (Sigma #G4877)
      1. Dissolve in 5 ml DPBS, then add to 35 ml DPBS to yield a final concentration of 5 IU/200 µl.
   2. hCG (Sigma #CG-5)
      1. Dissolve in 10 ml DPBS, then add to 190 ml DPBS to yield a final concentration of 5 IU/200 µl.
2. Inject 200 µl i.p. of PMSG.
3. Inject 200 µl i.p. of hCG 48 h later.
4. Cycle changes based on data from rats. For hormone profiles of natural rat see p 620 in “The Physiology of Reproduction, Volume II, Second Edition“ by Knobil and Neil, editors. In the section of this text entitled “Endocrinology of Reproduction” they review the superovulation procedures and effects. PMSG has both FSH and LH activities whereas hCG is primarily LH-like. Therefore, superovulation will cause multiple fllicles to develop, ovulate, then form corpus lutea. Therefore, there will be an exaggeration of the hormonal profile during estrus and subsequent phases. The luteal period will have extraordinary levels of progesterone due to multiple C.L.’s. For experimental data refer to data from C. Snelham (CS-I-p51). In this experiment samples were taken at the following times: 18 h post-hCG for estrus; 36 h post-hCG for early metestrus; 60 h post-hCG for late metestrus; and 100 h for disestrus.
   1. Estrus: Mice will ovulate at 12 post-injection. (12-36 h post-hCG) High FSH levels, but levels of Progesterone, Estradiol, Prolactin and LH decline to baseline within 5 h post-ovulation).
   2. Diestrus I: 48 h post-hCG (37-60 h post-hCG) Rising levels of Estradiol and Progesterone; basal for rest. Luteal phase is apparent(?)
   3. Diestrus II: 72 h post-hCG (61-84 h post-hCG) Progesterone levels drop within 5 h following luteolysis. Estradiol levels continue to increase.
   4. Proestrus: 96 h post-hCG (85-108 h post-hCG). Estradiol continues to increase. Prolactin also increases during mid-proestrus, but appears to precede the Progesterone release that coincides with the LH surge.

D. Estrogen + Progesterone Priming (Vonderhaar procedure)

E. Hormonal treatments for altering mammary development

1. Ductal development (Nandi, 1960; see Ref#jj0632)
   1. 1 µg estradiol for 30 days was sufficient to induce ductal elongation in hypophysectomized and ovariectomized.
   2. 2.5 µg estradiol + 1 mg progesterone for 30 days was sufficient to induce ductal branching.
   3. These hormones are soluble in ethanol at approximately 1 mg/ml. However, mice can tolerate no more than approximately 50 µl of ethanol. Therefore, most investigators appear to make aqueous suspensions in PBS. A volume of 100-200 µl was used for sub-cutaneous injections into the dorsal region over the front shoulders.
2. Long-term treatments to mimic prophylactic effects of pregnancy
   1. Preparations for daily injections
      1. Dissolve estrogen in a stock solution of 2 mg/ml of 100% ethanol.
      2. Dissolve progesterone in a stock solution of 1 mg/ml in sesame oil. This is accomplished by incubating the mixture overnight at 60^oC in a glass scintillation vial with a magnetic stirrer and covered with foil. Be sure to add the oil, start stirring, then add the progesterone SLOWLY.
      3. These are combined to yield final doses of 1 µg E and 1 mg P in 100 µl volume.
      4. Notes from Lakshmi Sivaraman: Regarding the EP injections, my lab mates working with the PRKO and PRAKO mice still inject the animals with 1µgE/1mgP (made up in sesame oil) and look for gene expression changes as early as 4h-6h. At least I am sure they do RPAs. So long or short term the steroids are made up in sesame oil, not PBS.
   2. Preparation for beeswax implants for use in rats (Sivaraman et al., 1998; see Ref # jj2448)
      1. Dissolve estrogen in a stock solution of 2 mg/ml of 100% ethanol.
      2. Dissolve progesterone in a stock solution of 1 mg/ml in sesame oil. This is accomplished by incubating the mixture overnight at 60^oC in a glass scintillation vial with a magnetic stirrer and covered with foil. Be sure to add the oil, start stirring, then add the progesterone SLOWLY.
      3. Appropriate volumes are mixed with beeswax to yield pellets containing 20 µg E + 20 mg P. One pellet is implanted s.c. into rats to mimic mammary gland development observed in pregnancy.

F. Glucocorticoid treatment to inhibit involution

1. Previous doses reported
   1. Lund et al., 1996 (#jj2194) – 0.5 mg/g BW/day; made as a suspension in PBS to provide a longer-lasting treatment (personal communication); delivered by daily injection s.c.
   2. Li et al., 1997 (#jj2238) – 7.5 mg/mouse/day; delivered by daily injection s.c.
   3. Feng et al., 1995 (#jj2282) – used hydrocortisone or progesterone to inhibit involution; delivered as pellets/
2. Solubility characteristics of hydrocortisone 21-acetate (Sigma #H4126)
   1. DMSO: 400 mg/ml; used 18.75 µl to deliver 7.5 mg/day