Abstract
Mosaic Analysis with Double Markers (MADM) is a mouse genetic system that allows simultaneous gene knockout and fluorescent labeling of sparse, clonally-related cells within an otherwise normal mouse, thereby circumventing embryonic lethality problems and providing single-cell resolution for phenotypic analysis in vivo. The clonal efficiency of MADM is intrinsically low because it relies on Cre/loxP-mediated mitotic recombination between two homologous chromosomes rather than within the same chromosome, as in the case of conditional knockout (CKO). Although sparse labeling enhances in vivo resolution, the original MADM labels too few or even no cells when a low-expressing Cre transgene is used or a small population of cells is studied. Recently, we described the usage of a new system, MADM-ML, which contains three mutually exclusive, self-recognizing loxP variant sites as opposed to a single loxP site present in the original MADM system (referred to as MADM-SL in this paper). Here we carefully compared the recombination efficiency between MADM-SL and MADM-ML using the same Cre transgene, and found that the new system labels significantly more cells than the original system does. When we established mouse medulloblastoma models with both the original and the new MADM systems, we found that, while the MADM-SL model suffered from varied tumor progression and incomplete penetrance, the MADM-ML model had consistent tumor progression and full penetrance of tumor formation. Therefore MADM-ML, with its higher recombination efficiency, will broaden the applicability of MADM for studying many biological questions including normal development and disease modeling at cellular resolution in vivo.
Highlights
Many human diseases, including cancer and some neurodegenerative diseases, are a consequence of genetic mosaicism, i.e., the pathogenic cells contain modified genomes compared to the normal cells in the body
We report the detailed characterization of Mosaic Analysis with Double Markers (MADM)-ML in comparison to MADM-SL based on the cell labeling efficiency in cerebellar granule neuron precursors (GNPs)
While the MADM-SL based medulloblastoma model provided some interesting insights toward tumor progression, we found that tumor penetrance was quite low, most likely due to the low number of initial mutant cells
Summary
Many human diseases, including cancer and some neurodegenerative diseases, are a consequence of genetic mosaicism, i.e., the pathogenic cells contain modified genomes compared to the normal cells in the body. To gain insights into aberrations occurring at the single cell level during pathogenic processes, we created a mouse genetic mosaic system termed MADM that allows simultaneous gene knockout and fluorescent labeling of sparsely distributed cells within an otherwise normal animal [1]. The capability of generating a small number of fluorescently labeled mutant cells allows one to study cell-autonomous gene functions and developmental lineage relationships with single-cell resolution in vivo. MADM generates a pair of GFP and RFP labeled daughter cells from a colorless progenitor cell via Cre/loxPmediated mitotic recombination between two homologous chromosomes followed by G2-X type chromatid segregation (Figure 1). It should be noted that, if an alternative type of segregation, called G2-Z occurs after the mitotic recombination, one yellow and one colorless cell will be produced, both of which are still heterozygous for the gene of interest (Figure 1) and are relatively less useful for phenotypic analysis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
