Detecting lacZ gene expression in living cells with new lipophilic, fluorogenic beta-galactosidase substrates.
Current methods for detecting lacZ expression in transformed cells are limited because they require such harsh conditions that viability of the cells after detection is drastically reduced. To overcome this problem, we developed a series of new substrates for detection of lacZ expression in living cells under standard culture or physiological conditions. After incubation with these fluorogenic substrates, cultured lacZ-positive mammalian cells appear morphologically normal, continue to divide, and retain the fluorescent product. Because the product is so well retained, fluorescence intensity can be quantitatively related to the level of gene expression. We have demonstrated this correlation using transformed yeast cells bearing various plasmids, each containing the lacZ gene and a unique promoter sequence with known capabilities for promoting gene expression in yeast.
Zhang
Y Z
YZ
Molecular Probes, Inc., Eugene, Oregon 97402.
Naleway
J J
JJ
Larison
K D
KD
Huang
Z J
ZJ
Haugland
R P
RP
D000818:Animals; D016923:Cell Death; D002478:Cells, Cultured; D005452:Fluoresceins / Q000138:chemical synthesis / Q000378:metabolism*; D005697:Galactosides / Q000138:chemical synthesis / Q000378:metabolism*; D015870:Gene Expression; D007763:Lac Operon; D008322:Mammals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods*; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000378:metabolism*
D005452:Fluoresceins; D005697:Galactosides; D011993:Recombinant Fusion Proteins; C038848:fluorescein-digalactoside; D001616:beta-Galactosidase