Expression and loss of alleles in cultured mouse embryonic fibroblasts and stem cells carrying allelic fluorescent protein genes

Jon S. Larson, Moying Yin, Jared Fischer, Saundra L. Stringer, James R. Stringer

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Background: Loss of heterozygosity (LOH) contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cell's phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors. Results: As a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS) showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10-4 and appeared to be produced at a rate of approximately 10-5 variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6. Conclusion: Allelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors influencing expression from allelic genes. Similar approaches will allow these phenomena to be studied in tissues.

Original languageEnglish (US)
Article number36
JournalBMC Molecular Biology
Volume7
DOIs
StatePublished - Oct 16 2006
Externally publishedYes

Fingerprint

Loss of Heterozygosity
Fibroblasts
Stem cells
Alleles
Proteins
Centromere
Chromosomes, Human, Pair 6
Clone Cells
Phenotype
Chromosomes
Sorting
Flow Cytometry
Clone cells
Fluorescence
Mouse Embryonic Stem Cells
Tissue
Embryonic Stem Cells
Mitosis
Genetic Recombination
Population

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Expression and loss of alleles in cultured mouse embryonic fibroblasts and stem cells carrying allelic fluorescent protein genes. / Larson, Jon S.; Yin, Moying; Fischer, Jared; Stringer, Saundra L.; Stringer, James R.

In: BMC Molecular Biology, Vol. 7, 36, 16.10.2006.

Research output: Contribution to journalArticle

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