Extensions of MADM (Mosaic Analysis with Double Markers) in Mice
Figure 2
Random integration-based approach to expand MADM to other mouse chromosomes.
A and B) Schematic representations of MADM precursor (pMADM) constructs. A) pMADMα contains the CA promoter, FRT-flanked MADM GT and TG cassettes and a single polyadenylation signal (pA). The cassette containing the floxed neomycin phosphotransferase gene (loxP-pPGK-Neo-pA-loxP) is placed in the introns of both cassettes. pMADMα can be converted into either GT or TG via partial Flp-mediated recombination in ES cells. B) pMADMβ construct contains the CA promoter driving the βgeo gene (a lacZ and neomycin-phosphotransferase fusion) flanked by FRT5 and FRT. pMADMβ can be converted into any transgene, including a GT or TG cassette via Flp- and FRT5/FRT-mediated cassette exchange in ES cells. These MADM cassettes contained a hygromicin resistance gene (H) that was removed by φC31 integrase-mediated recombination (see Figure S1) before performing the experiments shown in D. C) Distribution of pMADM transgene intergenic integration sites in the mouse genome. Each centromere is represented by a blue circle, and mapped insertion sites are indicated by triangles (Mb, mega base pair). The pMADMα insertion site used to establish MADM-10 (Figure 2D) is represented by the blue triangle located close to the centromere of Chr. 10. All the other triangles represent the insertion sites of pMADMβ transgenes based on the 5′ genomic sequence amplified by Splinkerette PCR. Insertion sites that were mapped close to centromeres, and were independently confirmed by both 5′ and 3′ genomic PCR, are represented by red triangles. The insertion located ∼39 Mb from the centromere of Chr. 1 (indicated by an asterisk) was used to establish MADM-1. D) Representative epifluorescence images of tissue sections with genotypes indicated on top and tissue identity on the bottom. The sections were unstained or stained only with DAPI to label nuclei (blue). The creation of fluorescent cells was Cre-dependent. Scale bars, upper row of images: 100 µm, lower row: 50 µm.