Figure 1. Extension of MADM to all 19 mouse autosomes.

(A) Summary of the MADM principle. For MADM, two chimeric split marker genes containing partial coding sequences of EGFP and tdT are inserted into identical genomic loci of homologous chromosomes. Following Cre-recombinase-mediated interchromosomal (trans) recombination during mitosis, the split marker genes are reconstituted and functional green and red fluorescent proteins expressed. As a result, green GFP⁺, red tdT⁺, and yellow GFP⁺/tdT⁺ cells appear sparsely, due to an inherently low stochastic interchromosomal recombination rate, within the genetically defined cell population expressing Cre recombinase. Introduction of a mutant allele distal to the MADM cassette results in a genetic mosaic with homozygous mutant cells labeled in one color (e.g., green GFP⁺) and homozygous wild-type sibling cells in the other (e.g., red tdT⁺). Heterozygous cells appear in yellow (GFP⁺/tdT⁺).

(B) Expansion of MADM to all mouse autosomes. Transgenic mice with MADM cassettes inserted close to the centromere have been generated for all 19 mouseautosomes. The directionality (forward, centromere-telomere; reverse, telomere-centromere) of marker gene transcription is indicated.

(C) MADM labeling scheme for cassettes inserted in forward direction. MADM experiments involving forward cassettes require that the mutant allele of acandidate gene must be linked to the T-G MADM cassette in order for mutant cells to be labeled in green upon a G2-X MADM event.

(D) MADM labeling scheme for cassettes inserted in reverse direction. MADM experiments involving reverse cassettes require that the mutant allele of a candidate gene must be linked to the G-T MADM cassette in order for mutant cells to be labeled in green upon a G2-X MADM event.

(E) Generation of recombinant MADM chromosomes. To genetically link a mutant allele of a candidate gene of interest to the corresponding chromosome containing the T-G MADM cassette (i.e., forward orientation), it is necessary to first cross mice bearing the T-G MADM cassette with mice bearing the mutant allele. Resulting F1 transheterozygous offspring are then backcrossed to mice homozygous for the T-G MADM cassette. In F2, recombinant offspring emerge from meiotic recombination events in the germline. These F2 recombinants now contain both the MADM cassette (in homozygous configuration) and the mutant allele linked on the same chromosome. For experimental MADM mice, F2 recombinants are crossed with mice bearing the G-T MADM cassette and a Cre driver of interest.

(F) Calculation of predicted meiotic recombination frequency. The probability for meiotic recombination resulting in the linkage of the MADM cassette with the mutant allele can be estimated by the genetic distance of the MADM cassette to the location of the mutant allele divided by two.

See also Figures S1–S4 and Table S1.