Development of the first Gateway firefly luciferase vector and use of reverse transcriptase in FLOE (Fluorescently Labeled Oligonucleotide Extension) reactions.
To study promoters we usually use primer extension to map the transcription start site and a panel of PCR generated deletion mutants. This strategy is complex and time-consuming. Therefore, we decided to improve it by using Gateway and FLOE (Fluorescently Labeled Oligonucleotide Extension). In this report we developed the first luciferase reporter "destination vector" (GW luc basic) for the Gateway technology and tested its efficacy, accuracy and background level by transfecting two distant cell lines (THP1 monocytic and SH-SY5Y neural cells). This vector is a real advantage for the cloning of many PCR fragments and sustains reporter activity also in THP1 cells, which are known to be problematic for transfection/expression. FLOE is a straightforward method to map transcription start sites but a bias in the capillary electrophoretic migration pattern of ROX weight markers has been reported: ROX markers migrated as if they were some bp longer. We hypothesized that this could depend on the use of different enzymes for the two principal reactions (DNA polymerase for the dideoxy chain terminated reaction on DNA and reverse transcriptase for the primer extension on RNA). Therefore, we used the same reverse transcriptase enzyme on both reactions, demonstrating that the reported bias is not due to the use of different enzymes but is an intrinsic feature of the ROX markers. The proposed procedure is important not only because of the timeliness but also for the global impact on the study of the first layer of the gene regulation.
Del Vecchio
Igor
I
Zuccotti
Annalisa
A
Canneva
Fabio
F
Lenzken
Silvia Carolina
SC
Racchi
Marco
M
D000818:Animals; D002460:Cell Line; D048888:Fireflies; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D049409:Luciferases, Firefly / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D014162:Transfection
D009841:Oligonucleotides; D049409:Luciferases, Firefly; D012194:RNA-Directed DNA Polymerase