Rapid transcriptional assay for the expression of two distinct reporter genes by microinjection.
We have developed a technique in which gene expression from multiple reporter plasmids introduced by needle microinjection can be monitored simultaneously in individual cells by double-label indirect immunofluorescence. With constitutively active viral promoters, expression from lacZ or chloramphenicol acetyl transferase (CAT) reporter genes can be detected within as little as 30 min after injection. Expression from such strong promoters reaches a maximum level after about 2 hr. In place of the constitutive promoter, promoters containing different enhancer elements respond as expected to different stimuli, allowing for the comparison of two defined transcriptional control elements in living cells. Reporter expression can be analyzed temporally and can be compared to expression of endogenous genes. This technique is complementary to transfection and allows for the targeted analysis of expression in specific cells, for example, in a mixed cell population, and for the analysis of expression in cells that are available only in small numbers.
Alberts
A S
AS
Biomedical Sciences Graduate Program, University of California, School of Medicine, La Jolla 92093-0636.
Frost
J A
JA
Thorburn
A M
AM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D017930:Genes, Reporter; D016762:Genes, fos; D005821:Genetic Techniques; D006801:Humans; D008845:Microinjections; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D016755:Proto-Oncogene Proteins c-jun; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase