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Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.

Mahen R, Koch B, Wachsmuth M, Politi AZ, Perez-Gonzalez A, Mergenthaler J, Cai Y, Ellenberg J - Mol. Biol. Cell (2014)

Bottom Line: Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells.Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B.We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.

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Construction and validation of genome-edited cell lines expressing AURKB-GFP. (A) Schematic of fluorescent gene–tagging systems used to create AURKB-GFP. (i) ZFNs or TALENs cause DNA double-strand breaks at the C-terminus of the AURKB locus, before repair with a donor construct containing EGFP (arrow). (ii) Plasmids containing AURKB-GFP as either the full mouse gene (BAC) or as cDNA are randomly integrated into the genome. (B) Flowchart of assays used to construct genome-edited cell lines. Junction PCR was not used to screen plasmid-based systems since the genomic locations are unknown. (C) Fluorescence-activated cell sorting of GFP-positive cells. Gates were drawn based on comparison to nonfluorescent wild-type parental cells. (D) Fluorescence confocal microscopy maximum-intensity z-projections of genome-edited AURKB-GFP cells (clone HZ2). (E) Junction PCR screening of ZFN genome-edited AURKB-GFP clonal cells. Stars denote clones with all alleles successfully targeted. (F) Western blot screening of nocodazole-arrested ZFN AURKB-GFP cells with anti-AURKB antibody. (G) Southern blot screening of ZFN AURKB-GFP cells using a probe in AURKB intron 2 after Kpnl and Nsil digestion. (H) Mitotic timing from live-cell imaging; cells were automatically classified into cell cycle stages using mCherry-H2B as previously described (Held et al., 2010). n >355 cells from two experiments.
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Figure 1: Construction and validation of genome-edited cell lines expressing AURKB-GFP. (A) Schematic of fluorescent gene–tagging systems used to create AURKB-GFP. (i) ZFNs or TALENs cause DNA double-strand breaks at the C-terminus of the AURKB locus, before repair with a donor construct containing EGFP (arrow). (ii) Plasmids containing AURKB-GFP as either the full mouse gene (BAC) or as cDNA are randomly integrated into the genome. (B) Flowchart of assays used to construct genome-edited cell lines. Junction PCR was not used to screen plasmid-based systems since the genomic locations are unknown. (C) Fluorescence-activated cell sorting of GFP-positive cells. Gates were drawn based on comparison to nonfluorescent wild-type parental cells. (D) Fluorescence confocal microscopy maximum-intensity z-projections of genome-edited AURKB-GFP cells (clone HZ2). (E) Junction PCR screening of ZFN genome-edited AURKB-GFP clonal cells. Stars denote clones with all alleles successfully targeted. (F) Western blot screening of nocodazole-arrested ZFN AURKB-GFP cells with anti-AURKB antibody. (G) Southern blot screening of ZFN AURKB-GFP cells using a probe in AURKB intron 2 after Kpnl and Nsil digestion. (H) Mitotic timing from live-cell imaging; cells were automatically classified into cell cycle stages using mCherry-H2B as previously described (Held et al., 2010). n >355 cells from two experiments.

Mentions: To address this issue, we created HeLa cell lines stably expressing C-terminal monomeric enhanced GFP (mEGFP) fusions of the key mitotic kinase Aurora kinase B (AURKB-GFP) using four different transgene methods (Figure 1A). We used genome editing by ZFNs (Bibikova et al., 2003) or TALENs (Miller et al., 2011) to target recombination of EGFP from a donor plasmid containing regions of homology to endogenous loci (Figure 1Ai). In addition, we performed random integration of BACs or cDNA plasmids (Figure 1Aii; Poser et al., 2008). For each transgene method (Figure 1A), we created several monoclonal or pooled HeLa lines stably expressing AURKB-GFP, which we validated with a series of assays (Figure 1B; see Materials and Methods for detailed protocols). Briefly, after introduction of the tag, expressing cells were isolated by fluorescence-activated cell sorting (FACS; Figure 1C) and then screened by fluorescence microscopy for specific localization to mitotic organelles as expected for AURKB (Figure 1D; Terada et al., 1998). We distinguished homozygous from heterozygous clones (AURKB has three alleles in HeLa cells; Landry et al., 2013) for the genome-editing methods (Figure 1B; In vitro) by genomic PCR (Figure 1E and Supplemental Figure S1A), Western blot (Figure 1F and Supplemental Figure S1B), and Southern blot (Figure 1G and Supplemental Figure S1, C and D).


Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.

Mahen R, Koch B, Wachsmuth M, Politi AZ, Perez-Gonzalez A, Mergenthaler J, Cai Y, Ellenberg J - Mol. Biol. Cell (2014)

Construction and validation of genome-edited cell lines expressing AURKB-GFP. (A) Schematic of fluorescent gene–tagging systems used to create AURKB-GFP. (i) ZFNs or TALENs cause DNA double-strand breaks at the C-terminus of the AURKB locus, before repair with a donor construct containing EGFP (arrow). (ii) Plasmids containing AURKB-GFP as either the full mouse gene (BAC) or as cDNA are randomly integrated into the genome. (B) Flowchart of assays used to construct genome-edited cell lines. Junction PCR was not used to screen plasmid-based systems since the genomic locations are unknown. (C) Fluorescence-activated cell sorting of GFP-positive cells. Gates were drawn based on comparison to nonfluorescent wild-type parental cells. (D) Fluorescence confocal microscopy maximum-intensity z-projections of genome-edited AURKB-GFP cells (clone HZ2). (E) Junction PCR screening of ZFN genome-edited AURKB-GFP clonal cells. Stars denote clones with all alleles successfully targeted. (F) Western blot screening of nocodazole-arrested ZFN AURKB-GFP cells with anti-AURKB antibody. (G) Southern blot screening of ZFN AURKB-GFP cells using a probe in AURKB intron 2 after Kpnl and Nsil digestion. (H) Mitotic timing from live-cell imaging; cells were automatically classified into cell cycle stages using mCherry-H2B as previously described (Held et al., 2010). n >355 cells from two experiments.
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Related In: Results  -  Collection

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Figure 1: Construction and validation of genome-edited cell lines expressing AURKB-GFP. (A) Schematic of fluorescent gene–tagging systems used to create AURKB-GFP. (i) ZFNs or TALENs cause DNA double-strand breaks at the C-terminus of the AURKB locus, before repair with a donor construct containing EGFP (arrow). (ii) Plasmids containing AURKB-GFP as either the full mouse gene (BAC) or as cDNA are randomly integrated into the genome. (B) Flowchart of assays used to construct genome-edited cell lines. Junction PCR was not used to screen plasmid-based systems since the genomic locations are unknown. (C) Fluorescence-activated cell sorting of GFP-positive cells. Gates were drawn based on comparison to nonfluorescent wild-type parental cells. (D) Fluorescence confocal microscopy maximum-intensity z-projections of genome-edited AURKB-GFP cells (clone HZ2). (E) Junction PCR screening of ZFN genome-edited AURKB-GFP clonal cells. Stars denote clones with all alleles successfully targeted. (F) Western blot screening of nocodazole-arrested ZFN AURKB-GFP cells with anti-AURKB antibody. (G) Southern blot screening of ZFN AURKB-GFP cells using a probe in AURKB intron 2 after Kpnl and Nsil digestion. (H) Mitotic timing from live-cell imaging; cells were automatically classified into cell cycle stages using mCherry-H2B as previously described (Held et al., 2010). n >355 cells from two experiments.
Mentions: To address this issue, we created HeLa cell lines stably expressing C-terminal monomeric enhanced GFP (mEGFP) fusions of the key mitotic kinase Aurora kinase B (AURKB-GFP) using four different transgene methods (Figure 1A). We used genome editing by ZFNs (Bibikova et al., 2003) or TALENs (Miller et al., 2011) to target recombination of EGFP from a donor plasmid containing regions of homology to endogenous loci (Figure 1Ai). In addition, we performed random integration of BACs or cDNA plasmids (Figure 1Aii; Poser et al., 2008). For each transgene method (Figure 1A), we created several monoclonal or pooled HeLa lines stably expressing AURKB-GFP, which we validated with a series of assays (Figure 1B; see Materials and Methods for detailed protocols). Briefly, after introduction of the tag, expressing cells were isolated by fluorescence-activated cell sorting (FACS; Figure 1C) and then screened by fluorescence microscopy for specific localization to mitotic organelles as expected for AURKB (Figure 1D; Terada et al., 1998). We distinguished homozygous from heterozygous clones (AURKB has three alleles in HeLa cells; Landry et al., 2013) for the genome-editing methods (Figure 1B; In vitro) by genomic PCR (Figure 1E and Supplemental Figure S1A), Western blot (Figure 1F and Supplemental Figure S1B), and Southern blot (Figure 1G and Supplemental Figure S1, C and D).

Bottom Line: Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells.Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B.We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.

Show MeSH
Related in: MedlinePlus