Targeted mutagenesis in mice, a powerful tool for the analysis of gene function and human disease, makes extensive use of 129 mouse substrains. Although all are named 129, we document that outcrossing of these substrains, both deliberate and accidental, has lead to extensive genetic variability among substrains and embryonic stem cells derived from them. This clearer understanding of 129 substrain variability allows consideration of its negative impact on targeting technology, including: homologous recombination frequencies, preparation of inbred animals, and availability of appropriate controls. Based on these considerations we suggest a number of recommendations for future experimental design.

The generation of transgenic mice by pronuclear microinjection and their subsequent breeding has been more efficient with F1 or F2 zygotes than with zygotes of inbred strains because inbred mice generally have a relatively poor reproductive performance (1). However, an inbred genetic background is preferable for genetic analyses such as the transfer of one allele of a mouse gene into a strain carrying a different allele (2). Likewise, for insertional mutagenesis experiments, inbred strains eliminate ambiguity caused by different genetic backgrounds and segregating markers in the progeny. As reported here, the inbred strain FVB/N is a good breeder with large litters, and the fertilized eggs of this strain have large prominent pronuclei, which facilitate microinjection of DNA.The ancestor of FVB/N is an outbred colony of Swiss mice N:GP (NIH general purpose mouse) established at the National Institutes of Health in 1935. From the N:GP colony, a second colony N:NIH (NIH mouse) was established in the early 1940s. In 1966, a project was begun to develop two populations of N:NIH mice. Mice were inoculated with pertussis vaccines, followed by a challenge with histamine diphosphate. Two strains were selected for sensitivity and resistance and were designated as histamine sensitivity factor sensitive (HSFS/N) and histamine sensitivity factor resistant (HSFR/N), respectively. In the early 1970s, a group of mice at the eighth inbred generation from HSFS/N line were determined to carry the Fv-lb allele for sensitivity to the B strain of Friend leukemia virus, in contrast to N:NIH mice, which were sensitive to the N strain of this virus (Fv-J ") (F.L., unpublished results). These mice were then inbred and offspring were selected for Fv-Jb homozygosity. To avoid confusion with the HSFS/N strain that is Fv-1 ", the Fv-J b strain was designated as FVB for Friend virus B-type susceptibility. This strain has been maintained since the late 1970s as an inbred strain without selection for either pertussis vaccine sensitivity or virus type. In this report, we provide a detailed characterization of the genetic background of the FVB/N strain and the advantages of using the strain to generate and study transgenic mice.MATERIALS AND METHODS Mice. FVB/N mice (F38) were obtained from the National Institutes of Health Animal Genetic Resource.Pronudear Measurement. Embryos were obtained by in vitro fertilization of superovulated oocytes as described (3).Embryos developing pronuclei between 6 and 7 hr postinsemination were cultured an additional 5-6 hr and photographs were taken with Nomarski optics. Pronuclear volumes were calculated from their diameters measured along the equatorial planes perpendicular to the location of the polar bodies and excluding the zonae pellucidae. Only embryos exhibiting both pronuclei were used for analysis.Generation of Transgenic Mice. Pronuclear microinjections were performed by standard techniques (1). Mice were maintained on a cycle of light from 6:00 a.m. to 8:00 p.m.Superovulation was induced by administra...

Sperm from C57BL/6J, DBA/2J, BALB/cJ, 129S3/SvImJ, and FVB/NJ inbred mice were cryopreserved in 3% skim milk/18% raffinose cryoprotectant solution. The post-thaw sperm from all strains were evaluated for their viability and fertility by comparing them against B6D2F1 sperm used as a control. The protocol used for freezing mouse sperm was effective in different strains, because the motility was decreased by 50% after cryopreservation similar to other mammalian sperm. However, the progressive motility and the fertility of each inbred strain were affected differently. The C57BL/6J, BALB/cJ, and 129S3/SvImJ strains were the most affected; their fertility (two-cell cleavage) decreased from 70%, 34%, and 84% when using freshly collected sperm to 6%, 12%, and 6% when using frozen/thawed sperm, respectively. Live newborns derived from frozen/thawed sperm were obtained from all strains in the study. These results corroborate the genetic variation among strains with regard to fertility and susceptibility to cryopreservation.

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Mouse half ovaries were cryopreserved and orthotopically transplanted into ovariectomized recipients genetically identical to ovary donors except for the coat color gene. Fertility was reestablished in 57% of the female recipients, which became pregnant in an average of 40 days after transplantation of frozen-thawed half ovaries. These experiments demonstrate that ovary cryopreservation can be a very useful option for banking mouse germplasm, or managing subfertile animal colonies, when embryo or sperm freezing cannot be used or is not cost effective.