The pGL3 Luciferase Reporter Vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. These factors may be cis-acting, such as promoters and enhancers, or trans-acting, such as various DNA-binding factors. The backbone of the pGL2 Luciferase Reporter Vectors was redesigned for the pGL3 Vectors for increased expression, and contains a modified coding region for firefly (Photinus pyralis ) luciferase that has been optimized for monitoring transcriptional activity in transfected eukaryotic cells. The assay of this genetic reporter is rapid, sensitive and quantitative. In addition, the Luciferase Reporter Vectors contain numerous features aiding in the structural characterization of the putative regulatory sequences under investigation.
I. Description 1
II. pGL3 Vector Maps and Sequence Reference Points .2
III. Product Components .6
IV. Cloning Methods.6
A. Cloning Strategies .6
B. Preparation of pGL3 Vectors and Insert DNA for Cloning .6
C. Transformation Protocols for pGL3 Vectors .7
D. Isolation of Plasmid DNA.7
V. Transfection of Mammalian Cells8
VI. Assay of Luciferase Activity9
VII. Generation of Nested Deletions 10
VIII. Generation of Single-Stranded DNA and Site-Specific Mutations .10
A. Production of Single-Stranded DNA 10
B. Generation of Site-Specific Mutations .11
IX. Sequencing of Luciferase Reporter Vectors 11
X. Appendix .12
A. Common Structural Elements of the pGL3 Luciferase Reporter Vectors .12
B. Advantages of the pGL3 Vectors .13
C. Description of Reporter Vector Changes .13
D. Distinguishing Features of the pGL3 Luciferase
Reporter Vectors15
E. Mapping Genetic Elements Located Within DNA Fragments 15
F. Composition of Buffers and Solutions .16
G. References 17
H. pGL3-Basic Vector Restriction Sites and Sequence19
I. pGL3-Enhancer Vector Restriction Sites and Sequence .24
J. pGL3-Promoter Vector Restriction Sites and Sequence29
K. pGL3-Control Vector Restriction Sites and Sequence.34
Experienced User’s Protocol 40
