Why Use ENU as a Mutagen 
    ENU is an alkylating agent (See Figure) that is a powerful mutagen in mouse spermatogonial stem cells, producing single locus mutation frequencies of 6 X 10-3 to 1.5 x 10-3, equivalent to obtaining a mutation in a single gene of choice in one out of every 175 to 655 gametes screened. The analysis of 61 sequenced germline mutations from 24 genes reveals that ENU predominantly modifies A.T base pairs, with 44% A.T to T.A transversions, 38% A.T to G.C transitions, 8% G.C. to C.G transversions, 5% A.T to C.G transitions, and 2% G.C to T.A transitions  (See Figure). When translated into a protein product, these changes result in 64% missense mutations, 10% nonsense mutations, and 26% splicing errors. Because it is a point mutagen, ENU can induce many different types of alleles. Loss of function mutations, viable hypomorphs of lethal complementation groups, antimorphs, and gain-of function mutations have been isolated in mouse mutagenesis screens. Missense changes are a common finding in many human disease mutations, therefore the ENU mutations will complement and extend the information provided by targeted gene disruptions.

Balancer Chromosomes
    Generation of chromosome rearrangements using Cre/loxP: Two lambda mouse genomic libraries have been constructed that contain the selectable markers required for two step targeting events. One contains the selectable marker neomycin (Neo), the 5’ end of Hypoxanthine phosphoribosyltransferase (Hprt), a loxP site and the Tyrosinase minigene (Ty). The second library contains the selectable marker puromycin (Puro), the 3’ end of Hprt, a loxP site, and the K14-Agouti transgene. If the loxP sites are inserted in the same orientation, recombination after Cre transfection will produce a deletion, and HAT resistant, Puro sensitive, Neo sensitive ES cells. If the loxP sites are inserted in opposite orientation, recombination after Cre transfection will result in an inversion, with HAT resistant, Puro resistant, Neo resistant ES cells. (See Figure)

Using a deletion scheme to isolate mutations
    To limit the extent of the screen, a Cre/loxP engineered deletion that encompasses the Smith Magenis homology region is being used in a two-generation mutagenesis scheme.
    After regaining fertility, ENU-treated males are mated to homozygous Re/Re females. The Re mutation marks the non-mutagenized chromosome, with the caveat that recombination can occur between a new linked mutation and Re.
    G1 animals, heterozygous for ENU mutagenized chromosomes and Re are mated to mice hemizygous for a yellow-tagged deletion.
    The resulting classes of offspring can be readily identified: 1) the mutant class is yellow and straight-haired, and, if missing, indicates the likelihood of a lethal mutation, 2) a carrier class that is wild type, and can be used to recover any lethal mutations, 3) two curly-haired classes of mice (black and yellow) that are uninformative and can be immediately discarded.  (See Figure)

Using balancer chromosomes to isolate mutations
    Chromosomes carrying Cre/loxP engineered inversions (balancer chromosomes) are being used in genetic screens to isolate the mutations.
    The first balancer to be engineered breaks in Trp53 and Wnt3, a distance of about 24 cM. We are using this balancer in a screening protocol to isolate mutations.
    Wild type males (C57BL/6J, black) are injected with a 3 X 100 mg/kg dose of ENU. The balancer chromosome contains an inversion that suppresses recombination over a reasonable interval, 20 – 30 cM, is marked with the dominant K14-agouti transgene conferring yellow coat color, and is homozygous lethal due to disruption of one or more lethal genes at its endpoints. After regaining fertility, ENU-treated males are mated to females carrying the balancer chromosome (yellow).
    G1 animals that are yellow are mated with animals heterozygous for the balancer chromosome and Rex, which confers a dominant wavy coat (yellow, wavy lines).
    Three classes of offspring can be identified in the second generation, and the fourth class, which is homozygous for the balancer chromosome, dies (upside down).
    The useful G2 animals are the yellow, straight-haired animals, which are brother-sister mated.
    The G3 offspring are easily classified as 1) the wild type mutant class, which if missing, indicate the likelihood of a linked lethal mutation, and 2) a carrier class used to rescue any lethal mutations, which carries the balanced point mutation, ideal for stock maintenance.  (See Figure)

Phenotype Screen 
    If a lethal or visible mutation is not present at weaning, Test animals are held to the age of 3 months to observe for later acting traits. At the age of 3 months, the animals are X-rayed to observe skeletal morphology and bone density, then bled to perform a Complete Blood Count (CBC) with differential, which can identify abnormalities in red blood cell and white blood cell numbers or morphology, as well as platelet abnormalities. Following the bleed, animals are necropsied and observed for internal organ defects. These screens are designed to isolate additional models of human diseases.

Mutations Isolated
    Preliminary experiments show that we are isolating three classes of mutations: 1) Dominant mutations observed as visible traits in the G1 offspring of ENU mutagenized males, 2) Recessive lethal and visible mutations segregating with Chromosome 11, observed in the G3 test class of animals, and 3) Recessive visible mutations segregating genome-wide. Each of these types of mutations is provided in the mutation database.