Biological Characteristics and Data (Mice)

Mouse Surgery Protocol ( Click to Download )

Mouse Surgery Protocols
(edited 1/11/96)

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

Adult body weight:male 20-40gm
Adult body weight:female 20-40gm
Body surface area 10.5(wt. in grams)
Life span 1.5-3 years
Food consumption 15 gm/100 gm/day
Water consumption 15 ml/100 gm/day
Breeding onset:male 50 days
Breeding onset:female 50-60 days
Gestation Period 19-21 days
Body Temperature 36-37 C
  36.5-38.0 C
Heart rate 500-600 beats per minute
  325-780 beats per minute

DMBA Treatment

Mouse Surgery Protocol ( Click to Download )

Mouse Surgery Protocols
(edited 1/11/96)

A. Dimethylbenzanthracene (DMBA, Calbiochem) will be dissolved in cottonseed oil to yield a 0.25% (w/v/) solution. A dose of 0.2 ml will be administered by gastric intubation once each week for 3 consecutive weeks starting when the mice are 8 weeks old. DMBA is made up fresh and just before use. The carcinogen in solution is light sensitive so the flask containing the solution is wrapped in aluminum foil. We ensure safe handling by using carcinogen dedicated balance, flask and spatula. Wear gloves and administer carcinogen to mice in a hood and use disposable diapers or coverings on any surface area where carcinogen is placed or used. The room should be low light or reflected light. The flask, spatula and feeding needle are rinsed in acetone and the solution put in a dedicated bottle exposed to light. Eventually the filled bottle is collected by environmental safety and the material appropriately disposed. The half life of DMBA in oil solution is approximately 30 minutes when exposed to normal ambient laboratory lighting conditions. The bottle containing the powdered carcinogen is kept in a locked cabinet. The cages containing the mice receiving carcinogen are handled specially for four weeks following carcinogen feeding. All feces, cage shavings and uneaten food are collected and incinerated at >1800 degrees. The carcinogen shows up in feces and urine for up to 10 days after treatment, so we build in a safety factor of an additional 20 days.

Embryo Transfer and Screening Transgenic Mice

Mouse Surgery Protocol ( Click to Download )

Mouse Surgery Protocols
(edited 1/11/96)

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.

Hormone BrdU Aministsration

hormone_BrDU ( Click to Download )

•Bed the animals on SaniChips, rather than corn pellets, to prevent any interference from phytoestrogens for 5-7 days before the hormone treatment.

•Two days before sacrifice, inject mice with 100 μl of E+P under the skin between the shoulder blades. [The final dose given is 1 μg E2 and 1 mg P]

•Two hours before sacrifice, inject the mice with BrdU intraperitoneally (10 μl per gram of body weight….ie: 20 gram mouse would get 200 μl).

•Remove the mammary glands, spread on parafilm paper squares, and place in tissue cassettes. Fix for 2 hours at 4oC in cold 4% paraformaldehyde.

•After 2 hours, rinse the tissue with PBS and store in 70% ethanol at 4oC. The tissue could also be stained with Carmine Alum overnight at this point (see Carmine Whole mount protocol).



•ß-Estradiol 3-benzoate, Sigma E-8515

[1,3,5[10]-Estratriene-3,17 ß-diol 3-benzoate]

•Progesterone, Sigma P-0130


•Sesame oil, Sigma S-3547

E2 stock: [10 mg/ml] stock in sesame oil

10 mg E2 powder in 1 ml sesame oil.

Stir with mild heat (≤ 60oC) until dissolved.

E+P for injections: [10 μg/ml] E2 Benzoate + [10 mg/ml] Progesterone in sesame oil

100 mg progesterone powder

10 μl of [10 mg/ml] E2 stock

10 ml sesame oil

Stir until dissolved.

BrdU: BromodeoxyUridine: Amersham RPN201 or Sigma B-5002


Make up at [3 mg/ml] in PBS or saline. Store aliquots at -20oC.

Inject 10 μl per gram of bodyweight.

4% PFA:

1. Working in a fume hood, heat 450 ml of ddH2O to 60oC.

2. With stirring, add 20 g of paraformaldehyde powder to the heated water. Cover and maintain ≤ 60oC on a hot plate.

3. Add 5 drops of 2N NaOH (1 drop per 100 ml). The solution should clear within a couple of minutes (There will be some fine particles that will not go away). Do not heat solution above 70°C.

4. Remove from heat and add 50 ml of 10X PBS. Adjust pH to 7.2; you may have to add some HCl. Filter and place on ice.

Hormone Treatment of Mice

Mouse Surgery Protocol ( Click to Download )

Mouse Surgery Protocols
(edited 1/11/96)

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 -70oC.
    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 60oC 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 60oC 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

Irradiation of Mice

Mouse Surgery Protocol ( Click to Download )

Mouse Surgery Protocols
(edited 1/11/96)

A. Gamma-irradiation will be administered at the dose rate of 400 Rads/minute using 137Cs source located in Room 112 of Paige laboratory. Total whole body doses will not exceed 500 Rad. This will be accomplished by placing single mice in a cardboard box (10 cm x 5 cm x 4 cm) that provides ample ventilation. The box is placed into the irradiator for <75 seconds to achieve the desired dose. Mice will be sacrificed within 24 hours and tissues used for biochemical and histological analyses.

B. The doses are sublethal and result in no acute or chronic effects on the mice (Storer, J.B. 1975. Acute Responses to Ionizing Radiation. In: Biology of the Laboratory Mouse, 2nd Edition, pp429-433.) Any discomfort should be minimal as the animals will be maintained <48 hours following exposure to radiation.

Mouse Estrous Cycle

Stage Ovary Uterus Vagina Smear
Diestrus Small follicles only are present with large corpora lutea from the previous ovulation. These secrete for only a very short time unless pregnancy or pseudopregnancy intervene. Small and anaemic, low motility, lumen small and slit-like. Cells of the uterine mucosa columnar; polymorphonuclear leucocytes in stroma; endometrial glands collapsed, atrophic. Epithelium thin, mitotic figures infrequent. Leucocytes abundant in stroma, migrate through the epithelium into vaginal lumen. Stringy mucous in which are entangled many leucocytes and a few nucleated epithelial cells.
Proestrus Some follicles grow rapidly. Become more vascular, water content increases, organ distends. Contractility more pronounced. Epithelial cells become higher (continuing into estrus). Leucocytes disappear from mucosa. Endometrial glands hypertrophy. Epithelum thickens, numerous mitoses in inner layers. Old layers of epithelium line the lumen. Leucocytes no longer migrate through the epithelium. Superficial epithelial cells slough off into lumen. Largely small, round, nucleated epithelial cells, singly or in sheets. None to few leucoytes.
Estrus Ovulation in the rat is spontaneous and occurs about 10 hours after the beginning of estrus. “Heat” (receptivity) lasts about 13 hours. Usually 10-20 eggs ovulated each time. gains maximum vascularisation. Epithelial cells reach maximum development. No leucocytes. Outer layer of epithelial cells become cornified and sloughed into the lumen. In early estrus these cells retain their nuclei, but in later stages no nuclei visible and the cells are irregular, flat, cornified plates. The skin around the vaginal orifice becomes swollen. Contains hundreds of large cornified cells (squames) with degenerate nuclei. Towards the end of estrus the smear becomes “cheesy” – masses of adherent cornified cells.
Metestrus Many corpora lutea, which secrete only for a very short time, and small follicles. Epithelium continues vacuolar degeneration and replacement. Leucocytes in stroma. Decrease in size and vascularity. Deeper layers of the estrous epithelium now line the lumen, the older, superficial layers having become cornified and sloughed off. Reduction of mitotic activity in epithelium. Leucocytes in stroma and migrating through the epithelium into the lumen. Many leucocytes and a few cornified cells

Protocol For Genotyping ERKO Mice


UY83 (P1); UV182 (P2); WR115 (P3); WR111 (P4)


PCR Mixes (for 10 samples):

Solution for Primer Mix A 1X 11X
PCR Supermix 22.5ml 247.5ml
P1/P2 0.5ml /0.5ml 5.5ml/5.5ml
Solution for Primer Mix B 1X 11X
PCR Supermix 22.5ml 247.5ml
P3/P4 0.5ml /0.5ml 5.5ml/5.5ml
  • Mix well by vortexing.
  • Aliquot 23.5ml of mix A and mix B into designated tubes (20 total= 10A + 10B).
  • Add 2μl of DNA to the appropriate A and B aliquots. (Note: There should be an A and B for each DNA sample)
  • Mix well by pipetting the sample up and down 3 times leaving no bubbles.
  • Add 40ml of mineral oil to the top of the sample in the tube.

PCR Conditions:

  • 94oC for 1 min @ File 45 (1) or 43 (2), link to…
  • (94oC for 30 sec, 55oC for 30 sec, 72oC for 1 min) X30 @ File 46 (1) or 44 (2), link to…
  • 4oC soak @ File 99

Gel Electrophoresis:

  • Use 1% agarose gel + 5ml of Ethidium bromide.
  • 10ml PCR product + 2ml of loading buffer (6X)
  • 7ml of DNA ladder.
  • Run at 100 Volts.

Identifying the Genotype:

HT (-/+) KO (/) WT(/)
Primer mix A: 387bp 387bp
Primer mix B: 255bp 255bp 815bp

Cell line Xenograft Injections (PJ Keller)

Xenograft Injection Prep ( Click to Download )

Preparing cells for injection:

  1. Determine the total volume of injections (plan on 40 µl per gland plus enough for 1-2 extra glands per cell line/condition).
  2. Thaw out enough matrigel on ice for 50% of the total volume of injections.
  3. Trypsinize cells to a single cells suspension and count.
  4. Put 1 x 106 cells per gland plus enough for 1-2 extra glands per cell line/condition in a conical tube and pellet.
    1. Optional:  wash 1X with PBS or media
  5. Make a solution of 50% Matrigel and 50% media, keep on ice (if cell pellet is going to take up significant volume, you could account for this by making 60% matrigel and 40% media)
  6. Aspirate all media from pellets and resuspend in matrigel/media mix; use enough volume for 30-40 µl per injection.  Try to take into account the approximate volume of the pellet to the best of your ability to estimate when resuspending.
  7. Keep cells in Matrigel:media mix on ice until injection.