Journal: Genome Research

Article Title: Efficient and flexible tagging of endogenous genes by homology-independent intron targeting

doi: 10.1101/gr.246413.118

Figure Lengend Snippet: Homology-independent generic intron tagging enables efficient and easy generation of endogenous fusions. ( A ) Illustration of the tagging approach: Double-strand breaks are generated in the intron and donor resulting in the addition of a synthetic exon and fusion of the tag to the coding sequence. ( B ) Using a small donor composed of the mNG2 11 epitope flanked by splice acceptor and donor sites results in efficient tagging of CANX , CBX1 , VIM , and ACTB at the indicated introns (all sgRNA “target 1”), as observed by flow cytometry ( upper panels, colored by density) and by confocal microscopy ( lower panels). Percentages in the dot plots represent the green population as a subset of the total. ( C ) All transfection mix components are required for tagging of CANX intron 14, sgRNA target 1 (14.1), and of CBX1 intron 3, targets 1 and 2 (3.1 and 3.2). The table indicates which component was removed, and bar plots represent the relative percentage of fluorescence-positive cells compared to the full mix. ( D ) Tagging using a full-length mClover3 fluorophore as a donor. ( E ) Tagging of CANX and CBX1 in HeLa cells, H9 human embryonic stem cells (hESC), and HAP1 cells. All images are maximum projections of Z -stacks, and scale bars correspond to 10 µm.

Article Snippet: The following antibodies were used: α-CANX (Novus Biologicals, NBP2-53352, 1:1000), α-GAPDH (Cell Signaling 2118, 1:2000), IRDye 680LT Goat anti-Rabbit (LI-COR 926-68021, 1:10,000), and IRDye 800CW Goat anti-Mouse (LI-COR 926-32210, 1:10,000).

Techniques: Generated, Sequencing, Flow Cytometry, Confocal Microscopy, Transfection, Fluorescence

Journal: Genome Research

Article Title: Efficient and flexible tagging of endogenous genes by homology-independent intron targeting

doi: 10.1101/gr.246413.118

Figure Lengend Snippet: Successful tagging is mostly determined by the choice of intron. ( A ) Tagging with mNG2 11 across introns in ACTB and CANX . Bar plots represent the percent of fluorescence-positive cells for each sgRNA position. ( B ) Expression mean and standard error for positive cells in each location. Sample sizes are proportional to the bar plots in A . ( C ) Gel image showing the amplification of donor-to-genomic DNA junctions, as illustrated in the right -hand diagrams. Expected band sizes for insertion of a single copy of donor are circled in green. In the diagrams, black arrows represent primer sites for amplification and red arrows represent primer sites for sequencing in D . The last lane corresponds to a PCR reaction with primers for CANX intron 14, target 1, but without a template. ( D ) Sanger sequencing of donor-to-genomic DNA junctions shows dephasing at the donor and genomic DNA junction, which indicates indels at the integration site.

Article Snippet: The following antibodies were used: α-CANX (Novus Biologicals, NBP2-53352, 1:1000), α-GAPDH (Cell Signaling 2118, 1:2000), IRDye 680LT Goat anti-Rabbit (LI-COR 926-68021, 1:10,000), and IRDye 800CW Goat anti-Mouse (LI-COR 926-32210, 1:10,000).

Techniques: Fluorescence, Expressing, Amplification, Sequencing

Journal: Genome Research

Article Title: Efficient and flexible tagging of endogenous genes by homology-independent intron targeting

doi: 10.1101/gr.246413.118

Figure Lengend Snippet: A modified donor allows for easy selection of tagged cells. ( A ) Schematic of donor constructs without and with a blasticidin resistance ( BSD ) gene. ( B ) Enrichment of fluorescence-positive HEK293 mNG2 1–10 cells tagged with mNG2 11 -BSD(−/+) at CANX intron 14 and CBX1 intron 3 after blasticidin treatment. Data represent mean ± SEM ( n = 3). ( C ) Dot plots of total HEK293 cell populations tagged with mNG2 11 or with mNG2 11 -BSD(−/+) and selected for 12 d. Plots are colored by density. ( D ) Confocal microscopy of total cell populations as in C . Images are maximum projections of Z -stacks, and scale bars correspond to 10 µm. ( E ) Western blot of clonal HAP1 lysates tagged with mClover3 only or mClover3-BSD(−/+) at CANX intron 14, target 1. The values below the anti-CANX blot indicate total levels of the major CANX band (tagged and untagged) relative to levels in wild-type (w.t.) cells.

Article Snippet: The following antibodies were used: α-CANX (Novus Biologicals, NBP2-53352, 1:1000), α-GAPDH (Cell Signaling 2118, 1:2000), IRDye 680LT Goat anti-Rabbit (LI-COR 926-68021, 1:10,000), and IRDye 800CW Goat anti-Mouse (LI-COR 926-32210, 1:10,000).

Techniques: Modification, Selection, Construct, Fluorescence, Confocal Microscopy, Western Blot

Journal: Nucleus

Article Title: Mutations causing Greenberg dysplasia but not Pelger anomaly uncouple enzymatic from structural functions of a nuclear membrane protein

doi: 10.4161/nucl.1.4.12435

Figure Lengend Snippet: Localization of the lamin B receptor in human control fibroblasts. (A) Lamin B receptor staining (gp-anti-LBR_N-term) of the nuclear rim and in addition in the cytoplasm in control fibroblasts. The non-nuclear LBR showed a partial co-localization with the ER membrane protein calnexin (green), indicating a localization of LBR in this compartment. Scale bar 10 µm. (B) Nuclear (N) and cytoplasmatic (C) extracts from human control skin fibroblasts showed a strong LBR expression in both the nuclear and the cytoplasmatic fraction by three different LBR antibodies. The nuclear protein lamin B2 and the cytoplasmatic alpha tubulin confirmed separation of the nuclear and cytoplasmatic fractions.

Article Snippet: The following antibodies were used for immunoblot analysis on PVDF membranes: as described above the polyclonal gp-anti-LBR_N-term, monoclonal rb-anti-LBR_N-term, monoclonal mouse-anti-LBR_C-term, further a rabbit polyclonal calnexin antibody (catalogue number GTX13504, Acris Antibodies, Herford, Germany), a mouse monoclonal alpha-tubulin antibody (ab7291, abcam, Cambridge, USA), and a mouse monoclonal anti-lamin B2 antibody (clone X223, catalogue number 65147C, Progen, Heidelberg, Germany).

Techniques: Staining, Expressing