Four groups of mice (see Note 2) are required:
1. Immature (12-14 g, 4-5 wk old) F1 hybrid females: These mice act as egg (zygote) donors for microinjection. The eggs are harvested following a superovulation protocol and fertilization (see Chapter 17). We use F1 animals generated from matings between C57BL/6J and CBA/J mice; other hybrids have been successfully used.
A typical microinjection session will employ 10 F1 hybrid females, giving approx 250 injectable eggs. To provide 30 F1 females/wk, it will be necessary to maintain 50 breeding cages, each containing a male (CBA/J) and a female (C57BL/6J). Since many more females than males are generally required, the number of female progeny can be maximized by placing two females with each male and culling male progeny at birth. The frequency of litter production can be maximized by taking advantage of the postpartum estrus in female mice; thus, males should not be removed from the cage following parturition. In general, the breeding colony should be carefully monitored to provide both accurate dates for weaning and superovulation, and fecundity data. Alternatively, it may be more convenient to dispense with a breeding colony and purchase a suitable supply of F1 hybrid females from an outside source.
2 Mature (2-12 mo) F1 hybrid males—"studs": Sexually mature F1 hybrid males (C57BL/6J x CBA/J) are required for mating with the immature female Fls to provide fertilized F2 zygotes. A subcolony of at least 20
studs should be individually housed (for example, 33 x 15 x 13 cm cages) and mated on alternate days. Although these animals should act as good studs for up to 1 yr, a record of the plugging rate (see Chapter 13) will indicate duds that need to be replaced from time to time. Studs can be obtained either from the breeding colony or purchased. A stock of males may be caged together provided that they have been simultaneously weaned and not exposed to females. However, potential studs should be individually caged at least 1 wk prior to mating, since sperm production may be suppressed in subordinate males within a group.
3. Mature (>6 wk, >19 g), pseudopregnant females: These mice (0.5 d pc) act as recipients for the microinjected F2 eggs. The state of pseudopreg-nancy, in which the eggs will develop in an environment free from "competing" fertilized eggs, is achieved by mating mature estrous females with sterile (vasectomized, see Section 2.1., step 4) male mice. The females may be conveniently taken from the F1 colony or may be of another strain with equally good maternal characteristics. A subcolony of at least 30 mature females will be required to provide five pseudopregnant recipients/d, since in a randomly cycling colony, approx 25% of the mice will exhibit estrus on a particular day (see below). Ovulating females are chosen for mating with the vasectomized males following an examination of vaginal characteristics. A scheme to facilitate the identification of ovulating female mice is described in Chapter 13. As an alternative to vaginal examination and if there are sufficient vasectomized males, it is possible to establish a larger number of matings (20-30) to obtain sufficient pseudopregnant animals from a randomly cycling group of females. If sufficient recipients are not obtained on a particular day, injected eggs may be maintained in culture while a second series of matings is set up. Conversely, extra pseudopregnant mice should be saved and reused for matings 10-14 d later.
4. Sterile stud males (2-24 mo): Sterile males, usually vasectomized (see Chapter 15), are required for the production of pseudopregnant recipient mothers. Between 20 and 30 individually caged mice of any strain (Parkes or Swiss are excellent performers) are required. They should be mated on alternate days only. Animals that consistently fail to produce plugs should be replaced.
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