8. The appropriate overhangs are added to the 5
0 end of selected
protospacer for targeting telomeres in N. benthamiana:
(a) Forward sequence: 5
0 -attgGGGTTTAGGGTTTAGGG
TTT-3
0
(b) Reverse sequence: 5
0 -aaacAAACCCTAAACCCTAAA
CCC-3
0
9. To guarantee a proper annealing of the oligonucleotides, do
not decrease the incubation time at room temperature.
10. Mere heat inactivation of the digestion reaction to linearize the
gRNA expression vector is not recommended to prevent religation of the excised fragment. However, due to the small size
of the excised fragment, a PCR purification kit is sufficient for
the purification of the linearized gRNA expression vector.
11. To guarantee a proper ligation, an incubation time of at least
1 h is recommended.
12. The signals can be detected even up to 4 days after injection;
however, the number of nuclei showing signals decreases
over time.
13. Optimal fixation conditions may vary between species or tissue
types. The durations described here are working well for
N. benthamiana leaves. However, if the quality of the immunostaining is not as desired, an increase or decrease of fixation
time might have an impact on how well the structure of the
nuclei is preserved.
14. Chop by quickly moving the razor blade up and down in a
steady rhythm. It is important not to squeeze the leaf, as this
does not release nuclei. The LB01 buffer should only turn
green as a result of homogenization, not squeezing the leaf.
References
1. Meldi L, Brickner JH (2011) Compartmentalization of the nucleus. Trends Cell Biol
21:701–708
2. Hoshi Y, Yagi K, Matsuda M, Matoba H,
Tagashira N, Pla ˛der W, Malepszy S,
Nagano K, Morikawa A (2011) A comparative
study of the three cucumber cultivars using
fluorescent staining and fluorescence in situ
hybridization. Cytologia 76:3–10
3. Bystricky K (2015) Chromosome dynamics
and folding in eukaryotes: insights from live
cell microscopy. FEBS Lett 589:3014–3022
4. Kanda T, Sullivan KF, Wahl GM (1998)
Histone-GFP fusion protein enables sensitive
analysis of chromosome dynamics in living
mammalian cells. Curr Biol 8:377–385
5. Belmont AS, Straight AF (1998) In vivo visualization of chromosomes using lac operatorrepressor binding. Trends Cell Biol 8:121–124
6. Kato L (2001) Detection of chromosomes
tagged with green fluorescent protein in live
Arabidopsis thaliana plants. Genome Biol 2:
research0045
7. Jovtchev G, Borisova B, Kuhlmann M, Fuchs J,
Watanabe K, Schubert I, Mette MF (2011)
Pairing of lacO tandem repeats in Arabidopsis
thaliana nuclei requires the presence of hypermethylated, large arrays at two chromosomal
positions, but does not depend on H3-lysine9-dimethylation. Chromosoma 120:609–619
8. Lindhout BI, Fransz P, Tessadori F, Meckel T,
Hooykaas PJJ, Zaal BJ (2007) Live cell
Live-Cell Imaging with a Telomere-Specific Guide RNA
355
0 end of selected
protospacer for targeting telomeres in N. benthamiana:
(a) Forward sequence: 5
0 -attgGGGTTTAGGGTTTAGGG
TTT-3
0
(b) Reverse sequence: 5
0 -aaacAAACCCTAAACCCTAAA
CCC-3
0
9. To guarantee a proper annealing of the oligonucleotides, do
not decrease the incubation time at room temperature.
10. Mere heat inactivation of the digestion reaction to linearize the
gRNA expression vector is not recommended to prevent religation of the excised fragment. However, due to the small size
of the excised fragment, a PCR purification kit is sufficient for
the purification of the linearized gRNA expression vector.
11. To guarantee a proper ligation, an incubation time of at least
1 h is recommended.
12. The signals can be detected even up to 4 days after injection;
however, the number of nuclei showing signals decreases
over time.
13. Optimal fixation conditions may vary between species or tissue
types. The durations described here are working well for
N. benthamiana leaves. However, if the quality of the immunostaining is not as desired, an increase or decrease of fixation
time might have an impact on how well the structure of the
nuclei is preserved.
14. Chop by quickly moving the razor blade up and down in a
steady rhythm. It is important not to squeeze the leaf, as this
does not release nuclei. The LB01 buffer should only turn
green as a result of homogenization, not squeezing the leaf.
References
1. Meldi L, Brickner JH (2011) Compartmentalization of the nucleus. Trends Cell Biol
21:701–708
2. Hoshi Y, Yagi K, Matsuda M, Matoba H,
Tagashira N, Pla ˛der W, Malepszy S,
Nagano K, Morikawa A (2011) A comparative
study of the three cucumber cultivars using
fluorescent staining and fluorescence in situ
hybridization. Cytologia 76:3–10
3. Bystricky K (2015) Chromosome dynamics
and folding in eukaryotes: insights from live
cell microscopy. FEBS Lett 589:3014–3022
4. Kanda T, Sullivan KF, Wahl GM (1998)
Histone-GFP fusion protein enables sensitive
analysis of chromosome dynamics in living
mammalian cells. Curr Biol 8:377–385
5. Belmont AS, Straight AF (1998) In vivo visualization of chromosomes using lac operatorrepressor binding. Trends Cell Biol 8:121–124
6. Kato L (2001) Detection of chromosomes
tagged with green fluorescent protein in live
Arabidopsis thaliana plants. Genome Biol 2:
research0045
7. Jovtchev G, Borisova B, Kuhlmann M, Fuchs J,
Watanabe K, Schubert I, Mette MF (2011)
Pairing of lacO tandem repeats in Arabidopsis
thaliana nuclei requires the presence of hypermethylated, large arrays at two chromosomal
positions, but does not depend on H3-lysine9-dimethylation. Chromosoma 120:609–619
8. Lindhout BI, Fransz P, Tessadori F, Meckel T,
Hooykaas PJJ, Zaal BJ (2007) Live cell
Live-Cell Imaging with a Telomere-Specific Guide RNA
355