Abstract
Mosaic Analysis with Double Markers (MADM) is a method for generating genetically mosaic mice, in which sibling mutant and wild-type cells are labeled with different fluorescent markers. It is a powerful tool that enables analysis of gene function at the single cell level in vivo. It requires transgenic cassettes to be located between the centromere and the mutation in the gene of interest on the same chromosome. Here we compare procedures for introduction of MADM cassettes into new loci in the mouse genome, and describe new approaches for expanding the utility of MADM. We show that: 1) Targeted homologous recombination outperforms random transgenesis in generation of reliably expressed MADM cassettes, 2) MADM cassettes in new genomic loci need to be validated for biallelic and ubiquitous expression, 3) Recombination between MADM cassettes on different chromosomes can be used to study reciprocal chromosomal deletions/duplications, and 4) MADM can be modified to permit transgene expression by combining it with a binary expression system. The advances described in this study expand current, and enable new and more versatile applications of MADM.
Highlights
Mosaic animals contain cells with different genotypes
Design of new Mosaic Analysis with Double Markers (MADM) cassettes The original version of MADM relied on the DsRed2 fluorescent protein as one of the two markers [10]
The new TG cassette is compatible with any GFPN-terminus-intron-XATG-less cassette, where XATG-less is any gene without the start codon (Figure 1B, bottom right)
Summary
Mosaic animals (genetic mosaics) contain cells with different genotypes. Phenotypic analysis of genetic mosaics has become an indispensible tool in modern genetics. Interchromosomal recombination techniques depend on recombination between chromatids after DNA replication in the G2 phase of the cell cycle to generate sibling cells of different genotypes. The common and key feature of these approaches is that they create cells with different genotypes in vivo and at the same time label those cells with unique markers that strictly correlate with the genotype. To enable such concomitant in vivo genetic manipulation and labeling in mammals, we have established Mosaic Analysis with Double Markers (MADM) in mice (Figure 1A) [10]. To expand the utility and versatility of MADM, we present here modifications and new applications of the technique, and compare different procedures for establishment of MADM-ready chromosomes
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