MASTR: A Technique for Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination Using Conditional Floxed Alleles in Mice

Zhimin Lao,G Praveen Raju,C Brian Bai,Alexandra L Joyner

doi:10.1016/j.celrep.2012.07.004

Abstract

Mosaic mutant analysis, the study of cellular defects in scattered mutant cells in a wild-type environment, is a powerful approach for identifying critical functions of genes and has been applied extensively to invertebrate model organisms. A highly versatile technique has been developed in mouse: MASTR (mosaic mutant analysis with spatial and temporal control of recombination), which utilizes the increasing number of floxed alleles and simultaneously combines conditional gene mutagenesis and cell marking for fate analysis. A targeted allele (R26(MASTR)) was engineered; the allele expresses a GFPcre fusion protein following FLP-mediated recombination, which serves the dual function of deleting floxed alleles and marking mutant cells with GFP. Within 24 hr of tamoxifen administration to R26(MASTR) mice carrying an inducible FlpoER transgene and a floxed allele, nearly all GFP-expressing cells have a mutant allele. The fate of single cells lacking FGF8 or SHH signaling in the developing hindbrain was analyzed using MASTR, and it was revealed that there is only a short time window when neural progenitors require FGFR1 for viability and that granule cell precursors differentiate rapidly when SMO is lost. MASTR is a powerful tool that provides cell-type-specific (spatial) and temporal marking of mosaic mutant cells and is broadly applicable to developmental, cancer, and adult stem cell studies.

Highlights

In order to study the full repertoire of in vivo functions of a gene and to effectively model certain human diseases, it is necessary to generate tissue-specific conditional mutants at defined times during development, disease progression or homeostasis
Mosaic mutant analysis is a powerful innovation applied to studies in model organisms that allows the behaviors of individual mutant cells to be analyzed in a wild type (WT) cellular environment, and distinguishes the cell autonomous functions of genes
A frequent problem faced in interpreting the phenotypes produced through conditional mutagenesis approaches, especially with CreER alleles, is that not all cells in a given tissue undergo recombination, and the mosaic mutant cells are often indistinguishable from WT cells

Summary

Introduction

In order to study the full repertoire of in vivo functions of a gene and to effectively model certain human diseases, it is necessary to generate tissue-specific conditional mutants at defined times during development, disease progression or homeostasis. Conditional mutagenesis in mouse can circumvent the lethality caused by germline null mutations, and allow the primary function of a gene to be determined in a specific organ or cell type without potential confounding secondary effects in other tissues. In Drosophila, the elegant MARCM approach (Mosaic Analysis with a Repressible Cell Marker) combined with mutant alleles has revolutionized studies of cellular gene function by allowing mutant cells to be induced and marked in specific tissues at a defined time point using the FLP sitespecific recombinase (Lee et al, 2000). A similar approach has been engineered in mice (MADM; Mosaic Analysis with Double Markers), and involves rare inter-chromosomal recombination events induced by CRE recombinase that produce a small number of cells marked with GFP (Zong et al, 2005). A new technique for mosaic mutant analysis would be of high impact to the mouse genetics field

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