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Abstract
Functional and mechanistic studies of Wnt signaling have been severely hindered by the inaccessibility of bioactive proteins. To overcome this long-standing barrier, we engineered and characterized a panel of Chinese hamster ovary (CHO) cell lines with inducible transgenes encoding tagged and un-tagged human WNT1, WNT3A, WNT5A, WNT7A, WNT11, WNT16 or the soluble Wnt antagonist Fzd8CRD, all integrated into an identical genomic locus. Using a quantitative real-time bioluminescence assay, we show that cells expressing WNT1, 3A and 7A stimulate Wnt/beta-catenin reporter activity, while the other WNT expressing cell lines interfere with this activation. Additionally, in contrast to WNT3A, WNT1 only exhibits activity when cell-associated, and thus only signals to neighboring cells. The reporter assay also revealed a rapid decline of Wnt activity at 37°C, indicating that Wnt activity is highly labile. These engineered cell lines will reduce the cost of making and purifying Wnt proteins and serve as a continuous, reliable and regulatable source of Wnts to research laboratories around the world.
Highlights
Wnt signaling pathways are among the most important and most complex described in developmental biology
Engineering Wnt-producing inducible CHO lines (iCHO) cell lines We previously described a Dox-controlled transgene expression system in mammalian cells in which a transgene was efficiently targeted to a genomic locus [18]
We introduced a Blasticidin drug resistance gene (BSD) into the donor exchange vector and optimized the recombinase mediated cassette exchange (RMCE) selection scheme with two rounds of drug selection: first, Ganciclovir treatment selects for the absence of the HyTK cassette; second, Blasticidin treatment selects for the presence of the transgene cassette (Fig. S1A)
Summary
Wnt signaling pathways are among the most important and most complex described in developmental biology. There are nineteen Wnt ligands that signal via receptors of the Frizzled family (ten members), Lrp co-receptors (two members) and receptor tyrosine kinases Ryk (one member) and Ror (two members) [1]. In the canonical Wnt signaling pathway, Wnt binding to Frizzled, a seven-pass transmembrane receptor, and Lrp5/6, a single-pass co-receptor, triggers a cascade of events resulting in accumulation and nucleartranslocation of the transcriptional activator beta-catenin. Alternative mechanisms of Wnt signaling, often referred to as ‘noncanonical,’ do not stimulate beta-catenin-mediated gene transcription, but rather trigger changes in cell morphology, motility and polarity [6]. These alternative mechanisms can involve the Ryk and/or Ror receptors and are often associated with repression of canonical Wnt signaling. It should be noted that certain non-canonical Wnts can activate betacatenin in certain contexts [9,10,11,12]
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