Genetic mosaic dissection of candidate genes in mice using mosaic analysis with double markers

Tissue-wide Effects Overrule Cell-intrinsic Gene Function in Cortical Projection Neuron Migration

“…Since these Control-MADM mice did not carry any loss-of-function (LOF) allele all cells were considered as ‘Control’ with some sparse cells expressing the fluorescent GFP and tdT markers enabling single cell tracing (Figure 1A). In GeneX -MADM with Emx1 -Cre, a genetic mutation was coupled to one MADM cassette by meiotic recombination [see Materials and Methods (Amberg and Hippenmeyer, 2021; Contreras et al, 2021) for details] in a way that sparse homozygous mutant cells were always labeled in green (GFP + ) and wild-type cells in red (tdT + ), respectively (Figure 1B). In KO/cKO- GeneX -MADM with Emx1 -Cre, the mutant allele of the gene of interest was coupled to both MADM cassettes resulting in full (KO) or conditional (cKO) tissue-specific knockout ( Emx1 + projection neuron lineage) with sparse fluorescent labeling in respective experimental MADM mice (Figure 1C).…”

Tissue-wide Genetic and Cellular Landscape Shapes the Execution of Sequential PRC2 Functions in Neural Stem Cell Lineage Progression

“…Thus, assuming a purely cell-autonomous function of PRC2 in proliferating RGPs, we predicted a dramatic loss of Eed mutant cells in comparison to wild-type cells when analyzed in a sparse genetic mosaic as provided by the MADM system (Figure 1C). To test the above, we generated mosaic Eed -MADM ( MADM-7 GT/TG,Eed ;Emx1 Cre/+ ) mice with sparse green GFP + homozygous Eed -/- mutant cells, yellow (GFP + /tdT + ) heterozygous Eed +/- , and red ( tdT + ) Eed +/+ wild-type cells in an otherwise unlabeled heterozygous background (Figures 1C, 1H, 1I and S1D-S1E) (Amberg and Hippenmeyer, 2021). Contrary to our expectation, we found no reduction of green Eed -/- mutant when compared to red Eed +/+ control neurons and thus a g/r ratio of ∼1 at P21 (Figure 1O).…”

“…Calculation of recombination probability and recombination of Eed-flox allele onto chromosomes carrying the MADM cassettes was performed according to standard techniques. These are described in detail elsewhere (Amberg and Hippenmeyer, 2021) and are summarized in Figure S1. All MADM-based analyses were carried out in a mixed C57BL/6, CD1 genetic background, in male and female mice without sorting experimental cohorts according to sex.…”

“…(C) MADM scheme for Eed conditional knockout (cKO). The Eed conditional allele was introduced distal to both, the GT-MADM and the TG-MADM cassettes, via meiotic recombination (Amberg and Hippenmeyer, 2021; Contreras et al, 2021). Following Cre-mediated interchromosomal recombination and mitosis, sparsely labelled GFP + (green), tdT + (red) and GFP + tdT + (yellow) homozygous Eed mutant cells will be generated in an unlabeled homozygous Eed mutant environment.…”

“…(E) MADM scheme for sparse mosaic Eed knockout. The Eed conditional allele was introduced distal to the TG-MADM cassette via meiotic recombination (Amberg and Hippenmeyer, 2021; Contreras et al, 2021). Upon G2-X event one GFP + homozygous Eed mutant cell and one tdT + homozygous wild-type cell will be generated in an unlabeled heterozygous environment.…”