Visualizing loss of heterozygosity in living mouse cells and tissues.
Loss of heterozygosity (LOH) in somatic cells can contribute to the genesis of cancer, but little is known about the frequency with which LOH occurs in normal cells of the body. To detect LOH in situ, we studied mouse shYFP embryonic stem (ES) cells and cells of the intestinal epithelia derived from these ES cells. shYFP ES cells are heterozygous at the ROSA26 locus. One copy of the locus carries a gene encoding a yellow fluorescent protein (YFP), while the other copy harbors an shRNA gene that produces a short hairpin RNA (shRNA) molecule that causes degradation of YFP mRNA. Nearly all cells in shYFP populations were faintly fluorescent, but brightly fluorescent cells arose at a rate of approximately 10(-5)bright cells/generation. Bright cells lacked the gene encoding the shRNA and contained two copies of the YFP gene. Comparison of these results to previous data on LOH in ES cells that lacked interfering shRNA showed that LOH in shYFP cells was not influenced by the presence of the shRNA. Bright cells were also seen in intestinal villi of chimeric mice made by injecting blastocysts with shYFP cells. These data demonstrate that this approach can detect LOH and suggest that it will allow detection of LOH in a broad array of tissues and cell types in transgenic mice made from shYFP cells.
Fischer
Jared M
JM
University of Cincinnati, Department of Molecular Genetics, Biochemistry and Microbiology, Cincinnati, OH 45267-0524, USA. fischerjm@gmail.com
Stringer
James R
JR
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D007421:Intestine, Small / Q000166:cytology / Q000378:metabolism; D019656:Loss of Heterozygosity; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D008164:Luminescent Proteins; D012333:RNA, Messenger