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Large-scale preparation of fluorescence multiplex host cell reactivation (FM-HCR) reporters

Nature Protocols volume 16, pages 4265–4298 (2021)Cite this article

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Abstract

Repair of DNA damage is a critical survival mechanism that affects susceptibility to various human diseases and represents a key target for cancer therapy. A major barrier to applying this knowledge in research and clinical translation has been the lack of efficient, quantitative functional assays for measuring DNA repair capacity in living primary cells. To overcome this barrier, we recently developed a technology termed ‘fluorescence multiplex host cell reactivation’ (FM-HCR). We describe a method for using standard molecular biology techniques to generate large quantities of FM-HCR reporter plasmids containing site-specific DNA lesions and using these reporters to assess DNA repair capacity in at least six major DNA repair pathways in live cells. We improve upon previous methodologies by (i) providing a universal workflow for generating reporter plasmids, (ii) improving yield and purity to enable large-scale studies that demand milligram quantities and (iii) reducing preparation time >ten-fold.

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Fig. 1: Schematic representation of FM-HCR workflow.
Fig. 2: Schematic representation of using transcriptional mutagenesis as a basis for FM-HCR reporter plasmids.
Fig. 3: Schematic representation of the FM-HCR reporter preparation protocol and gel electrophoretic analysis of intermediates and products.
Fig. 4: Gating scheme for identifying single cells and representative scatter plots of samples requiring compensation.
Fig. 5: Scheme for setting compensation in a four-color experiment.
Fig. 6: Gating scheme for excluding false-positive fluorescent events in FM-HCR experiments.
Fig. 7: Representative four-color FM-HCR experiment with three FM-HCR reporters.

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Data availability

Source data are provided with this paper. All other data supporting the approach described in this protocol are available from the corresponding authors upon reasonable request. Starting plasmids will be deposited in Adgene. Small amounts of prepared FM-HCR reporter plasmids can be shared for pilot and feasibility studies upon reasonable request.

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Acknowledgements

This work was supported by National Institutes of Health grants 1U01ES029520, P30ES000002 and 5P01CA092584.

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Author notes

  1. These authors contributed equally: C. G. Piett, T. J. Pecen.

Authors and Affiliations

  1. Department of Environmental Health, JBL Center for Radiation Sciences, Harvard T. H. Chan School of Public Health, Boston, MA, USA

    C. G. Piett, T. J. Pecen, D. J. Laverty & Z. D. Nagel

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  1. C. G. Piett
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Contributions

C.G.P., T.J.P. and D.J.L. prepared samples, designed and conducted experiments and developed the method. They were supervised by Z.D.N. All authors contributed to the writing and editing of the manuscript and approved the final version.

Corresponding author

Correspondence to Z. D. Nagel.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Bennet van Houten and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Nagel, Z. D. et al. Proc. Natl Acad. Sci. USA 111, E1823–E1832 (2014): https://doi.org/10.1073/pnas.1401182111

Chaim, I. A. et al. Proc. Natl Acad. Sci. USA 114, E10379–E10388 (2017): https://doi.org/10.1073/pnas.1712032114

Nagel, Z. D. et al. Cancer Res. 77, 198–206 (2017): https://doi.org/10.1158/0008-5472.can-16-1151

Extended data

Extended Data Fig. 1 Gel electrophoretic analysis and flow cytometric validation of GFP_Hx, mPlum_A-8oxoG and mOrange_8oxoG-C reporter plasmids.

a, Analytical digest and flow cytometric validation of GFP_Hx plasmid. Lane 1: NEB 1-kb MW ladder; lane 2: pMax_GFP_C289T ccDNA; lane 3: HIA overnight extension reaction for GFP_Hx; lane 4: pMax_GFP_C289T ccDNA; lane 5: pMax_GFP_C289T after a 45-min ApaLI digestion at 37 °C, which cleaves two restriction sites, resulting in two linear DNA fragments; lane 6: GFP_Hx ccDNA after T5 Exo and PEG purification steps; and lane 7: GFP_Hx after a 45-min ApaLI digestion at 37 °C, in which the Hx lesion blocks ApaLI cleavage of one restriction site, leaving a single linearized fragment. At right: flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in WT HAP cells compared to MPG−/− HAP cells. b, Analytical digestion and flow cytometric validation of mPlum_A-8oxoG plasmid. Lane 1: HIA overnight extension reaction for mPlum_A-8oxoG; lane 2: pMax_mPlum ccDNA; lane 3: pMax_mPlum after a 45-min Fpg endonuclease digestion at 37 °C; lane 4: mPlum_A-8oxoG after T5 Exo and PEG purification steps; lane 5: mPlum_A-8oxoG after a 45-min Fpg endonuclease digestion at 37 °C, resulting in plasmid nicking and upward mobility shift. At right: flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in WT HAP cells compared to MUTYH−/− HAP cells. c, Analytical digestion and flow cytometric validation of mOrange_8oxoG-C plasmid. Lane 1: NEB 1-kb MW ladder; lane 2: pMax_mOrange_A215C ssDNA; lane 3: pMax_mOrange_A215C ocDNA; lane 4: pMax_mOrange_A215C ccDNA; lane 5: pMax_mOrange_A215C after a 45-min Fpg endonuclease digestion at 37 °C; lane 6: mOrange_8oxoG-C ccDNA; and lane 7: mOrange_8oxoG-C after a 45-min Fpg endonuclease digestion at 37 °C, which introduces a nick at the 8oxoG lesion, resulting in upward mobility shift. At right: flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in WT MEF cells compared to OGG1−/− MEF cells. Error bars represent s.e.m. from three to four biological replicates; differences of statistical significance (*, P < 0.05; ***, P < 0.005; ****, P < 0.0001) were determined by unpaired two-tailed t test.

Source data

Extended Data Fig. 2 Gel electrophoretic analysis and flow cytometric validation of mPlum_O6-MeG and BFP_U reporter plasmids.

a, Analytical digestion and flow cytometric validation of mPlum_O6-MeG plasmid. Lane 1: NEB 1-kb MW ladder; lane 2: pMax_mPlum_C207G/T208C ccDNA; lane 3: pMax_mPlum_C207G/T208C after a 45-min PspOMI digestion at 37 °C, resulting in linear pMax_mPlum_C207G/T208C starting plasmid; lane 4: mPlum_O6-MeG plasmid after T5 Exo and PEG purification steps; lane 5: mPlum_O6-MeG after a 45-min PspOMI digestion at 37 °C, in which the O6 group on the guanine blocks linearization by PspOMI, leaving predominantly ccDNA product (note: upon extended digestion or when excess enzyme is present, some linearized DNA will result). At right: flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in MGMT-deficient TK6 cells compared to TK6 cells complimented with stable MGMT expression. b, Analytical digestion and flow cytometric validation of BFP_U plasmid. Lane 1: NEB 1-kb MW ladder; lane 2: pMax_BFP_A191G ccDNA; lane 3: pMax_BFP_A191G ocDNA; lane 4: pMax_BFP_A191G after a 5-min UDG digestion at 37 °C, followed by a 30-min APE1 digestion at 37 °C; lane 5: BFP_U after a 5-min UDG digestion at 37 °C, followed by a 30-min APE1 digestion at 37 °C, resulting in UDG excising the incorporated uracil, followed by APE1 nicking the abasic site, resulting in an upward gel mobility shift; lane 6: BFP_U plasmid after T5 Exo and PEG purification steps. At right: Flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in WT MEF cells compared to UNG−/− MEF cells. Error bars represent s.e.m. from three to four biological replicates; differences of statistical significance (*, P < 0.05; **, P < 0.005; ***, P < 0.005) were determined by unpaired two-tailed t test.

Source data

Extended Data Fig. 3 Gel electrophoretic analysis and flow cytometric validation of mOrange_GG plasmid.

a, Gel electrophoretic analysis of mOrange_GG plasmid. Lane 1: NEB 1-kb MW ladder; lane 2: PMax_mOrange_G299C ccDNA; lane 3: HIA overnight extension reaction for mOrange_GG; lane 4: mOrange_GG after a 3-h digestion with T5 Exo; lane 5: mOrange_GG after PEG precipitation step; and lane 6: final mOrange_GG ccDNA after T5 Exo and PEG purification steps. b, Flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in TK6 cells compared to MMR-deficient MT1 lymphoblastoid cells. Error bars represent s.e.m. from three to four biological replicates; differences of statistical significance (***, P < 0.005) were determined by unpaired two-tailed t test.

Source data

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Piett, C.G., Pecen, T.J., Laverty, D.J. et al. Large-scale preparation of fluorescence multiplex host cell reactivation (FM-HCR) reporters. Nat Protoc 16, 4265–4298 (2021). https://doi.org/10.1038/s41596-021-00577-3

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