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

Larson JS, Yin M, Fischer JM, Stringer SL, Stringer JR - BMC Mol. Biol. (2006)

Bottom Line: Other clones exhibited LOH near the centromere, but were disomic for chromosome 6.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, OH 45267-0524, USA. larsonjs@gmail.com

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.

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FACS dot plots (106 events) showing fluorescent profiles of three R26CY MEF cell lines (A-C) and two ES cell lines (D, E). Arrows in panel A indicate "dim" and "bright" events in MEF plots. Circles in panels D and E indicate areas where monochromatic variants would be expected to be located.
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Figure 1: FACS dot plots (106 events) showing fluorescent profiles of three R26CY MEF cell lines (A-C) and two ES cell lines (D, E). Arrows in panel A indicate "dim" and "bright" events in MEF plots. Circles in panels D and E indicate areas where monochromatic variants would be expected to be located.

Mentions: Nearly all of the cells in each population exhibited both CFP and YFP fluorescence (bright cells) (Figures 1A–C). However, CFP signal intensities tended to be lower than YFP intensities, which was expected because CFP is intrinsically less bright [19]. A few percent of the cells exhibited little if any fluorescence of either color (dim cells). The nature of these cells was not investigated. Intact embryos appeared to express both fluorescent proteins uniformly and ubiquitously (Figure 2). Nevertheless, embryos could have contained a small number of cells that fail to express either fluorescent protein.


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

Larson JS, Yin M, Fischer JM, Stringer SL, Stringer JR - BMC Mol. Biol. (2006)

FACS dot plots (106 events) showing fluorescent profiles of three R26CY MEF cell lines (A-C) and two ES cell lines (D, E). Arrows in panel A indicate "dim" and "bright" events in MEF plots. Circles in panels D and E indicate areas where monochromatic variants would be expected to be located.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1621078&req=5

Figure 1: FACS dot plots (106 events) showing fluorescent profiles of three R26CY MEF cell lines (A-C) and two ES cell lines (D, E). Arrows in panel A indicate "dim" and "bright" events in MEF plots. Circles in panels D and E indicate areas where monochromatic variants would be expected to be located.
Mentions: Nearly all of the cells in each population exhibited both CFP and YFP fluorescence (bright cells) (Figures 1A–C). However, CFP signal intensities tended to be lower than YFP intensities, which was expected because CFP is intrinsically less bright [19]. A few percent of the cells exhibited little if any fluorescence of either color (dim cells). The nature of these cells was not investigated. Intact embryos appeared to express both fluorescent proteins uniformly and ubiquitously (Figure 2). Nevertheless, embryos could have contained a small number of cells that fail to express either fluorescent protein.

Bottom Line: Other clones exhibited LOH near the centromere, but were disomic for chromosome 6.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, OH 45267-0524, USA. larsonjs@gmail.com

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.

Show MeSH
Related in: MedlinePlus