- Mar 2021
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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Homology directed repair (HDR) assayEach variant was introduced into a HA-FLAG-tagged full-length PALB2 complementary DNA (cDNA) expression in the pOZC plasmid by site-directed mutagenesis using pfu turbo. Variants were verified by Sanger sequencing. Cotransfection of PALB2 expression constructs and the I-SceI expression plasmid into B400/DR-GFP reporter cells was performed at a 5:1 molar ratio using Xtremegene 9 transfection reagent (Roche). At least two independent clones containing each variant were analyzed in duplicate. PALB2 expression and transfection efficiency was verified by western blotting. Green fluorescence protein (GFP) expressing cells were quantified by fluorescence-activated cell sorting. Fold increases in GFP-positive cells, which are equivalent to HDR fold change, were normalized and rescaled relative to a 1:5 ratio derived from the p.Y551X pathogenic variant control and the wild-type PALB2 control.
AssayGeneralClass: BAO:0003061 reporter protein
AssayMaterialUsed: CLO:0036938 tumor-derived cell line
AssayDescription: Stable expression of wild type and variant PALB2 cDNA constructs in Trp53 and Palb2-null mouse cell line containing DR-GFP reporter; I-SceI endonuclease introduces a double-stranded break in the reporter construct and efficient repair results in GFP expression, which is detected by flow cytometry
AssayReadOutDescription: Homology directed repair (HDR) activity fold change, measured as GFP-positive cells and normalized relative to wild type PALB2 (set to 5.0) and the p.Y551X truncating variant (set to 1.0).
AssayRange: scaled score
AssayNormalRange: >4.4
AssayAbnormalRange: ≤1.7 for "deleterious" variants and ≤2.4 for "hypomorphic"variants
AssayIndeterminateRange: >2.4-<4.4
ValidationControlPathogenic: 7
ValidationControlBenign: 4
Replication: At least 2 independent clones per variant, each analyzed in duplicate
StatisticalAnalysisDescription: Not reported
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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CRISPR-LMNA HDR assayU2OS were seeded in 6-well plates at 200 000 cells per well. Knockdown of PALB2 was performed 6–8 h later with 50 nM siRNA using Lipofectamine RNAiMAX (Invitrogen). Twenty-four hours post-transfection, 1.5 × 106 cells were pelleted for each condition and resuspended in 100 μL complete nucleofector solution (SE Cell Line 4D-Nucleofector™ X Kit, Lonza) to which 1μg of pCR2.1-mRuby2LMNAdonor, 1 μg of pX330-LMNAgRNA2, 1 μg of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-PALB2 construct, and 150 ρmol siRNA was added. Once transferred to a 100 ul Lonza certified cuvette, cells were transfected using the 4D-Nucleofector X-unit, program CM-104, resuspended in culture media and split into 2 60-mm dishes. One dish was harvested 24 h later for protein expression analysis as described above while cells from the other were trypsinised after 48 h for plating onto glass coverslips. Coverslips were fixed with 4% paraformaldehyde and cells analyzed for expression of mRuby2-LMNA (indicative of successful HR) by fluorescence microscopy (63×) a total of 72 h post-nucleofection. Data are represented as mean relative percentages ± SD of mRuby2-positive cells over the YFP-positive population from 3 independent experiments (total n >300 YFP-positive cells per condition).
AssayGeneralClass: BAO:0003061 reporter protein
AssayMaterialUsed: CLO:0009454 U-2 OS cell
AssayDescription: U2OS cells were treated with PALB2 siRNA and synchronized to G1/S phase by double thymidine block. Cells were then co-transfected with peYFP-PALB2 expressing PALB2 variants (or empty vector), pCR2.1-mRuby2LMNAdonor, and pX330-LMNAgRNA, which generates mRuby2-Lamin A/C fusion if HDR is successful.
AssayReadOutDescription: Mean relative percentages of mRuby2-positive cells over the YFP-positive population relative to the wild type condition.
AssayRange: %
AssayNormalRange: Not reported
AssayAbnormalRange: <40%
AssayIndeterminateRange: 41%-77%
ValidationControlPathogenic: 1
ValidationControlBenign: 3
Replication: Three independent experiments, each with n > 300 YFP-positive cells per condition
StatisticalAnalysisDescription: One-way ANOVA followed by Dunnett's post hoc analysis
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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A cell-based functional assay for PALB2 variants
AssayGeneralClass: BAO:0003061 reporter protein
AssayMaterialUsed: CLO:0037317 mouse embryonic stem cell line
AssayDescription: Stable expression of wild type and variant PALB2 cDNA constructs in Trp53 and Palb2-null mouse cell line containing DR-GFP reporter; I-SceI endonuclease introduces a double-stranded break in the reporter construct and efficient repair results in GFP expression, which is detected by flow cytometry
AssayReadOutDescription: Relative homologous recombination (HR) efficiency represented as mean percentages of GFP-positive cells among the mCherry-positive cells relative to wild type, which was set to 100%
AssayRange: %
AssayNormalRange: HR levels comparable to that of cells expressing wild type PALB2; no numeric threshold given
AssayAbnormalRange: HR levels ≤40% of wild type
AssayIndeterminateRange: Not reported
ValidationControlPathogenic: 12
ValidationControlBenign: 9
Replication: 2 independent experiments
StatisticalAnalysisDescription: Not reported
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