Detection of mutation in transgenic CHO cells using green fluorescent protein as a reporter.
A novel approach was developed for rapidly estimating the frequency of specific mutations in genetically engineered Chinese hamster ovary (CHO) cells. We designed double-transgenic CHO cell lines that contain a transgene consisting of the sequence coding for green fluorescent protein under the control of a tetracycline (Tet) responsive promoter and a second transgene coding for the constitutively expressed Tet repressor. Cultures of these CHO cells were treated with gamma-radiation, N-methyl-N-nitrosourea or methyl methanesulfonate, and the fluorescence of individual cells from both control and treated cultures was measured by flow cytometry. The treatments increased the number of highly fluorescent cells, those with presumed mutations in the Tet-repressor gene. Mutant cells from gamma-radiation-exposed cultures were isolated by fluorescence-activated cell sorting, cultured, and individual clones expanded. A PCR-based analysis indicated that the highly fluorescent expanded cells had lost the transgene coding for the Tet repressor, suggesting that the system mainly detects large genetic alterations. A similar approach may be useful for making high-throughput in vivo models for mutation detection.
Dobrovolsky
Vasily N
VN
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, 3900 NCTR Road, HFT-120, Jefferson, AR 72079, USA. vdobrovolsky@nctr.fda.gov
McGarrity
Lynda J
LJ
Morris
Suzanne M
SM
Heflich
Robert H
RH
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004274:DNA, Recombinant / Q000235:genetics; D005434:Flow Cytometry; D005720:Gamma Rays / Q000009:adverse effects; D017930:Genes, Reporter; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008770:Methylnitrosourea / Q000633:toxicity; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics
D004274:DNA, Recombinant; D008164:Luminescent Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D049452:Green Fluorescent Proteins; D008770:Methylnitrosourea; D008741:Methyl Methanesulfonate