Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice.
A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow direct-repeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 +/- 0.1 per 106 cell divisions. Interestingly, the rate is approximately 10-fold greater in fibroblasts derived from embryonic tissue. We observe an approximately 15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo.
Hendricks
Carrie A
CA
Biological Engineering Division, Massachusetts Institute of Technology, Cambridge 02139, USA.
Almeida
Karen H
KH
Stitt
Molly S
MS
Jonnalagadda
Vidya S
VS
Rugo
Rebecca E
RE
Kerrison
G Foster
GF
Engelward
Bevin P
BP
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D004249:DNA Damage; D018076:DNA, Complementary; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008938:Mitosis / Q000235:genetics*; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid
D001426:Bacterial Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria