In situ detection of frameshift mutations in mouse cells.
A reporter gene system that allows in situ detection of cells that have suffered a specific frameshift mutation was developed. To construct the reporter gene, the open reading frame of a human placental alkaline phosphatase (PLAP) gene was disrupted by insertion of either 5 or 7 G:C basepairs, which formed mutant alleles carrying 9 and 11 consecutive G residues, respectively. The mutant PLAP genes did not produce alkaline phosphatase activity in cultured mouse cells in transient transfection assays. Several cell lines that contained integrated copies of the mutant PLAP genes were made. Histochemical staining of fixed cells showed that these cell lines contained a small number of cells that expressed PLAP activity and bound antibodies directed against PLAP. Cells carrying the allele with 11 consecutive G residues (G11 allele) acquired PLAP activity at a rate between 2 x 10(-3) and 2 x 10(-4) events per cell per generation, depending on the cell line. Cells carrying the allele with 9 consecutive G residues (G09 allele) acquired PLAP activity at a rate between 2 x 10(-5) and 2 x 10(-6) events per cell per generation, depending on the cell line. Cultures of PLAP+ cells were derived from cell lines carrying PLAP mutant genes. All the cells in these cultures had PLAP activity and bound anti-PLAP antibody. PLAP mRNA levels were the same in cultures where all cells were PLAP+ and in cultures where less than 1% of the cells expressed PLAP activity. DNA sequence analysis of PLAP+ cells showed that the G11 allele reverted by losing one basepair, and the G09 allele reverted by gaining one basepair.
Cao
J
J
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, OH 45267-0524, USA.
DePrimo
S E
SE
Hersh
M N
MN
Stringer
J R
JR
D000469:Alkaline Phosphatase / Q000235:genetics; D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D005260:Female; D016368:Frameshift Mutation; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D016366:Open Reading Frames; D010641:Phenotype; D010920:Placenta / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D004247:DNA; D000469:Alkaline Phosphatase