Fertilized one-celled eggs for microinjection are produced by mating a donor female and a stud male. Choosing the right strain of mouse for egg production is pivotal, since the ease and efficacy of generating transgenic mice is highly strain-dependent. Brinster et al. (10) compared C57BL/6J inbred eggs and C57BL/6J x CBA/J hybrid eggs in terms of parameters, such as egg yield and survival after injection. Overall, the experiments on hybrid eggs were eightfold more efficient than inbred eggs. Inbred zygotes should only be used when the genetic background of the host animal needs to be carefully controlled. We use F1 animals generated from matings between CBA/J and C57BL/6J.
Immature (12-14 g, 4-5 wk old) F1 hybrid females are superovulated and mated with F1 hybrid males. Ten immature superovulated females will yield at least 250 viable eggs (F2 zygotes). Fifty breeding pairs (CBA/J male and C57BL/6J female) can provide 30 F1 females/wk. Alternatively, immature F1 hybrid females can be purchased from a reputable commercial and institutional supplier. Twenty F1 hybrid studs caged individually are required to mate with the immature females and are replaced every 8 mo to a year.
Estrous females (0.5 d pc) are used as surrogate mothers to nurture surviving eggs to birth. Females can be of any strain with good maternal characteristics, but need to be sexually mature and >19 g in weight. We use C57BL/6J x CBA/J F1 females. Estrous females are made pseudopregnant by mating with vasectomized males.
Sexually mature males are sterilized by vasectomy and used to engender pseudopregnancy in sexually mature females. Any strain of mouse can be used, but Parkes and Swiss males are particularly suitable because they perform well. Twenty to 30 vasectomized males, mated on alternate days with females analyzed for their stage in the estrous cycle, should be able to provide at least five pseudopregnant females a day. Sexually mature male mice (about 2 mo old) are used. The vasectomy procedure is described in Subheading 4. of this chapter.
If only a very low number of fetuses develop in the pseudopregnant recipient, the resultant fetuses may be too large for natural delivery and a Caesarean section must be performed. The pups are fostered to mothers with natural pups of a similar age.
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