Figure 1. Recessive pedigree scheme for genome-wide mutations. A male mouse is treated with ENU, which induces many DNA lesions in the spermatogonial stem cells of the testis. After mitosis and meiosis, the testis will become populated with clones of mutagenized sperm. Thus, the testis represents a mosaic of different ENU-induced mutations (schematically indicated as dots; top left). New mutations will be transmitted to the first-generation offspring and become fixed in the germ line. These mice become founders of a pedigree to test for new recessive mutations (solid gray box; m). Males are mated to a wild-type female (open circle) to generate offspring that may carry the new mutation (solid gray) or may be wild type (open shapes). Without genetic markers, the genotype of the animal is not known (m?), since the mutation is recessive. Therefore, multiple daughters are mated back to the first-generation founder father to generate offspring that may carry a new mutation. A pedigree is normally flagged as a mutant if more than one third-generation offspring exhibits the same phenotype.

Figure 2. Recessive pedigree scheme using a balancer chromosome. The balancer chromosome used in this pedigree is a Cre–loxP engineered inversion on mouse Chr 4. The end points of this inversion are about 30 cM apart and effectively suppress the recovery of viable recombinant products over the interval. One end of the inversion is tagged with the tyrosinase minigene (Tyr), which confers a dark pigment to an otherwise white coat. The other end is tagged with the K14-agouti minigene, which confers a yellowish pigment to a pigmented mouse coat. Mice homozygous for the balancer are a dark chocolate color (Bal/Bal). When crossed with a Tyr –/– (white) mouse, the offspring that are heterozygous for the balancer are a grayish color (e.g. m/Bal). Male C57BL/6Brd Tyr –/– mice are mutagenized with ENU to generate many different mutations in their germ line (two representative germ-line mutations are shown as blue and red mosaics). When the ENU-treated males are mated to females that are homozygous for the balancer (shaded in black), the resulting first-generation offspring will all be heterozygous for the balancer chromosome and will possibly inherit an ENU-induced mutation from the male. If the new mutation lies on Chr 4, it is balanced with the coat color marked inversion, which will suppress recombination and allow for selection based on coat color in subsequent generations. ENU-induced mutations that are not on Chr 4 will also be transmitted but will segregate randomly from the coat color. First-generation males and females are then mated to mice homozygous for the balancer, resulting in two types of offspring: chocolate-colored animals that do not carry a new mutation on Chr 4 (shaded black) or grayish animals that are heterozygous for the balancer and potentially a new ENU-induced mutation on Chr 4 (shaded black and red or blue). Heterozygous offspring from this second generation are brother–sister mated to screen for new recessive mutations carried in their germ line. Mice carrying potential recessive mutations on Chr 4 (m/m) can be recognized by their white coat color and can be screened for clinically relevant phenotypes. If no white animals are produced in the third generation, it indicates the presence of an autosomal recessive lethal mutation. These lethal mutations can be easily maintained by brother–sister mating the heterozygous gray mice (m/Bal). Any chocolate-colored animal (Bal/Bal) does not carry an ENU-induced mutation on Chr 4; however, a new genome-wide mutation may be observed in this population and may be further characterized using the mating scheme diagrammed in Figure 1. The incorporation of the coat color tag allows for the selection of mutations localized on a single chromosome, which are mapped upon their isolation by linkage to the coat color marker, while it also allows for recovery of mutations that segregate geneome-wide.