Liu H S, Feliciano E S, Stambrook P J
Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, OH 45267-0521.
Proc Natl Acad Sci U S A. 1989 Dec;86(24):9951-5. doi: 10.1073/pnas.86.24.9951.
Bacterial beta-galactosidase, encoded by the lacZ gene, serves as a sensitive cytochemical marker in eukaryotic cells and tissues. In transient expression experiments, human and simian cells stain blue 48 hr after transfection with a plasmid containing a lacZ gene, whose expression is directed by a simian virus 40 promoter containing a synthetic lactose operator sequence. Transfection efficiency was about 0.6%. Incorporation of an operator sequence within the promoter permits regulation of beta-galactosidase gene expression by the lacI gene product, the lac repressor. When cells were cotransfected with the lacZ plasmid and a second plasmid containing the lacI gene, beta-galactosidase activity was extinguished. Its activity could be reestablished to original levels upon application of isopropyl beta-D-thiogalactoside to transfected cells. A cell line that stably carries both the lacI and lacZ genes was efficiently induced to synthesize beta-galactosidase after isopropyl beta-D-thiogalactoside administration. In transient expression experiments and in stably transfected lines, repression and induction of beta-galactosidase activity were predominantly at the transcriptional level.
由lacZ基因编码的细菌β-半乳糖苷酶，在真核细胞和组织中作为一种灵敏的细胞化学标记物。在瞬时表达实验中，用含有lacZ基因的质粒转染人细胞和猴细胞48小时后，细胞会染成蓝色，该lacZ基因的表达由含有合成乳糖操纵序列的猴病毒40启动子指导。转染效率约为0.6%。在启动子中引入操纵序列可通过lacI基因产物（即lac阻遏物）来调节β-半乳糖苷酶基因的表达。当细胞与lacZ质粒和含有lacI基因的第二个质粒共转染时，β-半乳糖苷酶活性会被消除。在转染细胞中应用异丙基β-D-硫代半乳糖苷后，其活性可恢复到原始水平。在给予异丙基β-D-硫代半乳糖苷后，稳定携带lacI和lacZ基因的细胞系被有效诱导合成β-半乳糖苷酶。在瞬时表达实验和稳定转染的细胞系中，β-半乳糖苷酶活性的抑制和诱导主要发生在转录水平。
