8. RNase/DNase-free pipette tips.
9. DAPI: 100 ng/mL 4
0 ,6-Diamidino-2-phenylindole diluted
in PBS.
10. Sealing film.
2.8 Immunofluorescence (IF)
1. Nuclease-free water.
2. 4% PFA: 4% (w/v) Paraformaldehyde diluted in PBS.
3. 0.5% TritonX-100: 0.5% (v/v) TritonX-100 diluted in PBS.
4. 0.05% TritonX-100: 0.05% (v/v) TritonX-100 diluted in PBS.
5. Blocking buffer: 1% (w/v) BSA, 10% (v/v) FBS in PBS.
6. Human anti-centromere antibodies (see Note 12).
7. Anti-human secondary antibodies labeled with Alexa647 that
are optically distinct from MTSb-targeting MBs (see Note 12).
8. RNase/DNase-free pipette tips.
9. DAPI: 100 ng/mL 4
0 ,6-Diamidino-2-phenylindole diluted
in PBS.
2.9 Microscope
and Imaging Software
1. Inverted wide-field fluorescence microscope equipped for
high-magnification digital imaging.
2. A CCD camera.
3. Proper filter sets for DAPI, EGFP, TRITC, Cy5, TAMRA, and
ATTO647N imaging.
4. A filter set enabling simultaneous dual-color imaging (see
Note 13).
5. Image acquisition software.
6. Image analysis software such as Fiji [31] (see Note 14).
7. MATLAB (Version R2014b 64-bit, MathWorks) (see
Note 14).
3 Methods
3.1 Cloning
For efficient co-transfection and co-expression of all system components (sgRNA-MTS, a transfection indicator (EGFP or BFP),
and dCas9) in HEK293 cells, a single plasmid containing all three
components should be generated. The steps and details are as
follows:
1. Generate pdCas9-C1 by inserting the PCR product of dCas9
from pSLQ1658-dCas9-EGFP into AgeI/BamHI-digested
pEGFP-C1 vector (see Subheading 2.3, Primer pair 1).
2. For telomere labeling, generate a mammalian expression vector
termed SL2-sgTelo-MTSa/EGFP/pdCas9-C1, in which
sgRNA, EGFP, and dCas9 are expressed under the control of
362
Xiaotian Wu et al.
9. DAPI: 100 ng/mL 4
0 ,6-Diamidino-2-phenylindole diluted
in PBS.
10. Sealing film.
2.8 Immunofluorescence (IF)
1. Nuclease-free water.
2. 4% PFA: 4% (w/v) Paraformaldehyde diluted in PBS.
3. 0.5% TritonX-100: 0.5% (v/v) TritonX-100 diluted in PBS.
4. 0.05% TritonX-100: 0.05% (v/v) TritonX-100 diluted in PBS.
5. Blocking buffer: 1% (w/v) BSA, 10% (v/v) FBS in PBS.
6. Human anti-centromere antibodies (see Note 12).
7. Anti-human secondary antibodies labeled with Alexa647 that
are optically distinct from MTSb-targeting MBs (see Note 12).
8. RNase/DNase-free pipette tips.
9. DAPI: 100 ng/mL 4
0 ,6-Diamidino-2-phenylindole diluted
in PBS.
2.9 Microscope
and Imaging Software
1. Inverted wide-field fluorescence microscope equipped for
high-magnification digital imaging.
2. A CCD camera.
3. Proper filter sets for DAPI, EGFP, TRITC, Cy5, TAMRA, and
ATTO647N imaging.
4. A filter set enabling simultaneous dual-color imaging (see
Note 13).
5. Image acquisition software.
6. Image analysis software such as Fiji [31] (see Note 14).
7. MATLAB (Version R2014b 64-bit, MathWorks) (see
Note 14).
3 Methods
3.1 Cloning
For efficient co-transfection and co-expression of all system components (sgRNA-MTS, a transfection indicator (EGFP or BFP),
and dCas9) in HEK293 cells, a single plasmid containing all three
components should be generated. The steps and details are as
follows:
1. Generate pdCas9-C1 by inserting the PCR product of dCas9
from pSLQ1658-dCas9-EGFP into AgeI/BamHI-digested
pEGFP-C1 vector (see Subheading 2.3, Primer pair 1).
2. For telomere labeling, generate a mammalian expression vector
termed SL2-sgTelo-MTSa/EGFP/pdCas9-C1, in which
sgRNA, EGFP, and dCas9 are expressed under the control of
362
Xiaotian Wu et al.