Development of an enhanced GFP-based dual-color reporter to facilitate genetic screens for the recovery of mutations in mice

Abstract

Mutagenesis screens to isolate a variety of alleles leading to null and non-null phenotypes represent an important approach for the characterization of gene function. Genetic schemes that use visible markers permit the efficient recovery of chemically induced mutations. We have developed a universal reporter system to visibly mark chromosomes for genetic screens in the mouse. The dualcolor reporter is based on a single vector that drives the ubiquitous coexpression of the enhanced GFP (EGFP) spectral variants yellow and cyan. We show that widespread expression of the dual-color reporter is readily detected in embryonic stem cells, mice, and throughout developmental stages. CRE-loxP-and FLPe-FRT-mediated deletion of each color cassette demonstrates the modular design of the marker system. Random integration followed by plasmid rescue and sequence-based mapping was used to introduce the marker to a defined genomic location. Thus, single-step placement will simplify the construction of a genomewide bank of marked chromosomes. The dual-color nature of the marker permits complete identification of genetic classes of progeny as embryos or mice in classic regionally directed screens. The design also allows for more efficient and novel schemes, such as marked suppressor screens, in the mouse. The result is a versatile reporter that can be used independently or in combination with the growing sets of deletion and inversion resources to enhance the design and application of a wide variety of genetic schemes for the functional dissection of the mammalian genome.

Developmental fate and lineage commitment of singled mouse blastomeres

“…Long-term separated blastomeres preferentially contribute to the trophectoderm of the blastocyst To determine whether the observed gene expression patterns observed correlate with the developmental behavior of LS blastomeres, we aggregated separated blastomeres, from a transgenic mouse that abundantly expresses green fluorescent protein (GFP) in each cell (Frank et al, 2003), with zona-free intact eight-cell stage embryos (i8C) (Fig. 4A).…”

Mice with the Enhanced Green Fluorescent Protein Gene Knocked in to Chromosome 11 Exhibit Normal Transmission Ratios

“…It has been suggested that the GFP-based fluorescent coat color markers should offer significant benefits when applied to various screening strategies to recover induced mutations in the mouse (Frank et al, 2003;Klysik, 2002). Consistently, the EGFP knocked-in strain described in this paper, in combination with previously generated K14-agouti tagged Trp53-Egfr inversion, should facilitate tracing of the tagged chromosome in genetic crosses to uncover recessive phenotypes on mouse chromosome 11.…”

“…Large segmental inversions, or balancers (Frank et al, 2003;Justice et al, 1999;Kile et al, 2003;Klysik et al, 2003Klysik et al, , 2004Rinchik, 2000), constitute effective tools that allow one to address ∼2% of the genome in a single mutagenesis experiment. Balancers were first observed in Drosophila melanogaster stocks causing self-perpetuating maintenance of lethal mutations located on homologous chromosomes (Muller, 1918).…”

“…Double recombinants would be viable, but multiple crossovers occur rather rarely on distances not exceeding 30 cM (Rinchik, 2000). of the inversion segment may be helpful in certain designs of phenotypic screens (Frank et al, 2003;Kile et al, 2003), this feature becomes dispensable in other designs (Klysik, 2002). …”

Moving forward with chemical mutagenesis in the mouse

“…An alternative strategy uses radiation mutagenesis to induce deletion complexes surrounding an integrated drug selection cassette (You et al 1997; Thomas et al 1998). Furthermore, the K‐14‐agouti transgene or enhanced green fluorescent protein (EGFP) based transgenic strategies can serve as visible markers to couple with the chromosomal aberration resources (Zheng et al 1999; Frank et al 2003). Thus, marked, regionally directed mutagenesis screens – once restricted to deletion complexes surrounding classic coat colour and dysmorphology loci – can now be applied across the genome.…”