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. 2013;8(3):e58395.
doi: 10.1371/journal.pone.0058395. Epub 2013 Mar 13.

Use of a Molecular Genetic Platform Technology to Produce Human Wnt Proteins Reveals Distinct Local and Distal Signaling Abilities

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Free PMC article

Use of a Molecular Genetic Platform Technology to Produce Human Wnt Proteins Reveals Distinct Local and Distal Signaling Abilities

Jennifer L Green et al. PLoS One. .
Free PMC article

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.

Conflict of interest statement

Competing Interests: The authors have the following conflicts to declare: JLG, YCL and GMW are named as inventors on U.S. Patent Application No. 13/538,755 Methods for Improved Production of Bioactive WNT Proteins. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Generation of Wnt-expressing iCHO clones.
A) Schematic of RMCE strategy. Parental CHO cells containing TetR-KRAB, rtTA and a genomic acceptor cassette, located downstream of the dihydrofolate reducatse (DHFR) locus, were co-transfected with a plasmid containing the incoming exchange cassette and a plasmid encoding Cre recombinase. Upon expression of Cre, the L3 and 2L recognition sequences in the genome are recombined with the L3 and 2L recognition sequences in the incoming exchange cassette. This results in excision of HyTK, thus rescuing Ganciclovir sensitivity, and insertion of the Wnt-expression cassette, which confers Blasticidin (BSD) resistance. B) Anti-FLAG western blot of cell lysates shows expression of FLAG-hWNT3A and FLAG-hWNT5A in three different iCHO clones. * Indicates non-specific band. Protein loading was visualized by Ponceau staining (bottom). C) Cells were treated with 0, 0.1 or 1.0 µg/mL Dox. Anti-FLAG western blot of cell lysates shows expression of FLAG-hWNT3A and FLAG-hWNT5A. Near maximal expression was achieved with 0.1 µg/mL Dox. D) iCHO cells were grown in the presence of 0.25 µg/mL Dox for three days at which point CM and cell lysates (L) were collected. Wnts and mFZ8CRD were immunoprecipitated from CM using anti-FLAG sepharose. While two species of FLAG-hWNT3A were visible in the cell lysate, only one form was visible in the CM, suggesting that the smaller species is not secreted.
Figure 2
Figure 2. Activity of iCHO-produced Wnt Proteins.
The graphs on the left depict induction of SUPERTOPFLASH (STF) activity in real-time bioluminescence monitoring assays. Values from the 24 hour timepoint were extracted for statistical analysis and are presented in the charts on the right.A) CM was collected from iCHO cells grown in the presence of Dox and 100 µL was added to reporter cells (mouse L + STF) in a 96 well plate. The volume of CM was kept constant such that ‘FLAG-hWNT3A:Par’ contained 50 µL FLAG-hWNT3A CM and 50 µL CM from parental CHO cells. Thus, ‘FLAG-hWNT3A:FLAG-hWNT5A’ CM and ‘FLAG-hWNT3A:Par’ each have an equal amount of FLAG-hWNT3A CM. Arrow points to FLAG-hWNT1 in CM. B, D–G) 293A-STF cells and iCHO cells were seeded together in a 96 well plate in the presence of Dox. Accumulation of luciferase activity is delayed compared to CM, presumably because it takes some time for the iCHO cells to produce Wnt following Dox stimulation. The total number of iCHO cells per well was kept constant such that ‘FLAG-hWNT3A’ contained 100% FLAG-hWNT3A cells and ‘FLAG-hWNT3A:Par’ contained 50% FLAG-hWNT3A cells and 50% Parental CHO cells. C) Anti-WNT3A and anti-WNT5A western blots comparing the levels of tagged and untagged WNTs in cell lysates and conditioned media. Quantification is shown below, FLAG staining was normalized to non-specific bands.
Figure 3
Figure 3. Wnt production, secretion and stability.
A) Anti-WNT3A western blots comparing the levels of mWNT3A produced by L cells or iCHO cells in cell lysates and conditioned media. B) Coomassie stain of purified hWNT3A and mWNT3A separated by SDS-PAGE. C) 293A-STF cells were treated with 200 ng/mL purified hWNT3A or mWnt3A protein that was either fresh from 4°C, or pre-incubated at 37°C for 6 hr or 24 hr.
Figure 4
Figure 4. Different Wnt proteins display distinct activities.
The graphs on the left depict activity in real-time bioluminescence monitoring assays. Values from the 24 hour timepoint were extracted for statistical analysis and are presented in the charts on the right.A–C) 293A-STF cells and CHO cells were seeded together in a 96 well plate in the presence of Dox, the total number of CHO cells per well was kept constant.
Figure 5
Figure 5. WNT1 is retained on the cell surface.
A) Right - Western blot detecting the FLAG epitope comparing the amounts of hWNT1, hWNT3A and hWNT5A in cell lysates and conditioned media. Arrow points to hWNT1. Left - Quantification of relative WNT protein levels in CM. B) WNT3A CM was diluted to approach the WNT concentration of WNT1 CM (FLAG-WNT3A 50%), and then diluted further (FLAG-WNT3A 25%, FLAG-WNT3A 10%). In each case, WNT3A CM elicited a STF reporter response, while undiluted WNT1 CM did not. C) Flow cytometry analysis detecting the FLAG epitope comparing the amount of different WNTs on the surface of live iCHO cells following Dox induction.
Figure 6
Figure 6. WNT1 acts locally.
A) iCHO cells expressing untagged hWNT1 or hWNT3A were co-cultured with 293A-STF cells at varying cell densities. The ratio of hWNT3A-induced STF activity to hWNT1-induced STF activity is shown for three timepoints. The rate of reporter induction by WNT3A is higher than WNT1 at low cell densities, but not at high cell densities. B) The BSTG cell line was generated to distinguish iCHO cells (uncolored) from 293 reporter cells (blue). iCHO cells were seeded at 1000 cells per 10 cm plate and allowed to form colonies for 4 days prior to the addition of BSTG cells. Dox was added to plates after 24 hours to induce Wnt expression. Images were taken 48 hours later. iCHO colonies were located based on cell morphology and confirmed by corresponding gaps in the blue channel. The BSTG cell line is not clonal and cells exhibit varying levels of blue fluorescence. Unprocessed GFP images taken with identical exposure settings are shown, additional unprocessed images are shown in Fig. S6. C) Flow cytometry analysis of the cells used in co-culture imaging experiments. The plots show GFP expression of 50,000 live BSTG cells. The bar graphs depict the number of GFP+ cells in the ‘P4’ gate (left) and the geometric mean GFP intensity of those cells (right).

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