Figure 1.
CRISPR/Cas9 allows efficient and complete endogenous knock-in of DD-tagged TCOF1.
(A) The scheme for CRISPR/Cas9-mediated homologous recombination to insert antibiotic resistance and a DD tag to the N-terminus of TCOF1 is shown. The genomic sequence used in the donor plasmid homology arms is shown in blue. (B) PCR genotyping to detect knock-in was performed with the primers indicated on the diagram, and reveals wild-type (960 bp), blasticidin resistance (BlastR) knock-in (1790 bp), and puromycin resistance (PuroR) knock-in (1990 bp) bands. ¼ of the clone 4 PCR was also run to illustrate the lack of wild-type allele detection in the other clone PCRs.
Figure 2.
DD-TCOF1 expression is regulated by the concentration of Shield-1.
(A–C) The Shield-1 concentration- and time-dependent expression of TCOF1 was examined as described in Materials and Methods. Although the EtOH vehicle was kept constant as described, a control without EtOH vehicle was also included for the parental wild-type 293Ts in panel A. As wild-type TCOF1 runs at about 220 kDa, the addition of the 12 kDa DD tag results in small upward shift for knock-in cells. Two-fold dilutions of high TCOF1-expressing sample were included on each gel to allow for standard curves to quantitate relative TCOF1 and β-tubulin. Relative TCOF1 values normalized to β-tubulin are shown below each panel. The data represents one of three experiments with similar results.
Figure 3.
DD-TCOF1 retains the nucleolar localization of wild-type TCOF1.
Knock-in cells in Shield-1 or ethanol vehicle alone and parental wild-type 293T cells were stained and imaged as described in Materials and Methods. A single representative z-slice confocal image is shown. The visible grayscale cell boundaries represent F-actin (stained with phalloidin).
Figure 4.
Destabilization of TCOF1 results in dispersal of nucleolin.
(A) The relative fluorescence intensities across the indicated lines are shown in the graph, and the arrows indicate the position of nucleoli as defined by a hole in the DAPI stain. Representative nuclei from knock-in cells in Shield-1 or ethanol vehicle alone are shown. (B) The aggregation of nucleolin was measured by the coefficient of variation (CV, stdev/mean) of nucleolin voxel intensities within DAPI-defined nuclei in 3D reconstructions, as described in Materials and Methods. As examples, this CV metric was 0.95 for the +Shield nucleus and 0.71 for the –Shield nucleus in panel A. The population means are indicated by the red lines. There is significantly less nucleolin aggregation in knock-in cell nuclei in the absence of Shield-1, p<0.0001, 2-tailed Student's t-test, n = 272 for +Shield and n = 230 for –Shield.
Figure 5.
Destabilization of TCOF1 impairs cell growth.
The growth of knock-in cells in the presence or absence of Shield-1 was compared as described in Materials and Methods. EtOH represents the vehicle control. Error bars represent SD of 3 replicate samples. The knock-in cell count in the absence of Shield-1 was significantly less than in the presence of Shield-1 except on the first day post-seeding: ns, not significant; *, p<0.05; **, p<0.01, 2-tailed Student's t-test at each time point. The growth of knock-in cells in the presence or absence of Shield-1 was compared two other times with similar results.