Lentiviral vectors to probe and manipulate the Wnt signaling pathway.
BACKGROUND
The Wnt signaling pathway plays key roles in development, adult tissue homeostasis and stem cell maintenance. Further understanding of the function of Wnt signaling in specific cell types could benefit from lentiviral vectors expressing reporters for the Wnt pathway or vectors interfering with signaling.


RESULTS
We have developed a set of fluorescent and luminescent lentiviral vectors that report Wnt signaling activity and discriminate between negative and uninfected cells. These vectors possess a 7xTcf-eGFP or 7xTcf-FFluc (Firefly Luciferase) reporter cassette followed by either an SV40-mCherry or SV40-Puro(R) (puromycin N-acetyltransferase) selection cassette. We have also constructed a vector that allows drug-based selection of cells with activated Wnt signaling by placing Puro(R) under the control of the 7xTcf promoter. Lastly, we have expressed dominant-negative Tcf4 (dnTcf4) or constitutively active beta-catenin (beta-catenin(4A)) from the hEF1alpha promoter in a SV40-Puro(R) or SV40-mCherry backbone to create vectors that inhibit or activate the Wnt signaling pathway. These vectors will be made available to the scientific community through Addgene.


CONCLUSIONS
These novel lentiviruses are efficient tools to probe and manipulate Wnt signaling. The use of a selection cassette in Wnt-reporter viruses enables discriminating between uninfected and non-responsive cells, an important requirement for experiments where selection of clones is not possible. The use of a chemiluminescent readout enables quantification of signaling. Finally, selectable vectors can be used to either inhibit or activate the Wnt signaling pathway. Altogether, these vectors can probe and modulate the Wnt signaling pathway in experimental settings where persistence of the transgene or gene transfer cannot be accomplished by non-viral techniques.
Fuerer
Christophe
C
Howard Hughes Medical Institute, Department of Developmental Biology, Stanford University School of Medicine, Stanford, California, United States of America.
Nusse
Roel
R
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D015398:Signal Transduction; D014162:Transfection; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D060508:Wnt3 Protein
D011500:Protein Synthesis Inhibitors; D051153:Wnt Proteins; D060508:Wnt3 Protein; D049452:Green Fluorescent Proteins; D011691:Puromycin; D008156:Luciferases