3. Following the standard procedure for the confocal laser scanning microscope in use search the growing hyphae for expression of GFP-tagged Csy4∗ associated to its cognate stem-loop
repeat of the target mRNA.
4 Notes
1. The ORF can be codon-optimized depending on the target
organism: see http://www.kazusa.or.jp/codon/ [26].
2. The restriction sites are chosen so that the one separating the
stem-loops does not occur or is unique in the target vRNA, to
allow easy identification of tagged constructs by restriction
digest. The flanking sites correspond to a unique site in the
vRNA where the insertion does not disrupt any open reading
frames or regulatory elements. If a different vRNA is to be
tagged, sites need to be chosen accordingly. If no suitable
insertion site is present, it can be engineered by PCR
mutagenesis.
3. Vector pAB261 and the resulting pAL13.Csy4∗-GFP are
based on the N. crassa expression vector pMF272 [27] which
contains flanking sequences for homologous integration at the
his-3 locus (NCU03139) of N. crassa. Expression of Csy4∗-GFP is under the control of the constitutive Ptef-1 promoter of
N. crassa.
4. N. crassa strain FGSC6103 (see www.fgsc.net strain list) is a
histidine auxotrophic mutant due to the truncation of the
essential his-3 locus. Integration of the transformation cassette
from pAL13.Csy4∗-GFP complements his-3 and allows selection of positive transformants on standard Vogel’s minimal
medium, i.e., on medium that is not supplemented with
0.5 mg/mL L-histidine.
5. Transforming bacteria with 2 μL of a 1:10 dilution of the
digested plasmid without re-ligation will provide an estimate
of what percentage of colonies is due to incompletely digested
plasmid, rather than re-ligation and, thus, approximately how
many colonies should be screened to identify clones without
the NLS.
6. Increasing the insert/vector ratio will increase, whereas reducing it will decrease, the number of tandem inserts obtained on
average, and can be optimized depending on the desired number of inserts. There is no control over the orientation of the
insertions, but we have found that the large majority of clones
obtained contain all inserts in the same, either sense or antisense orientation, though it remains unclear why that is
the case.
RNA Imaging with Csy4
175
repeat of the target mRNA.
4 Notes
1. The ORF can be codon-optimized depending on the target
organism: see http://www.kazusa.or.jp/codon/ [26].
2. The restriction sites are chosen so that the one separating the
stem-loops does not occur or is unique in the target vRNA, to
allow easy identification of tagged constructs by restriction
digest. The flanking sites correspond to a unique site in the
vRNA where the insertion does not disrupt any open reading
frames or regulatory elements. If a different vRNA is to be
tagged, sites need to be chosen accordingly. If no suitable
insertion site is present, it can be engineered by PCR
mutagenesis.
3. Vector pAB261 and the resulting pAL13.Csy4∗-GFP are
based on the N. crassa expression vector pMF272 [27] which
contains flanking sequences for homologous integration at the
his-3 locus (NCU03139) of N. crassa. Expression of Csy4∗-GFP is under the control of the constitutive Ptef-1 promoter of
N. crassa.
4. N. crassa strain FGSC6103 (see www.fgsc.net strain list) is a
histidine auxotrophic mutant due to the truncation of the
essential his-3 locus. Integration of the transformation cassette
from pAL13.Csy4∗-GFP complements his-3 and allows selection of positive transformants on standard Vogel’s minimal
medium, i.e., on medium that is not supplemented with
0.5 mg/mL L-histidine.
5. Transforming bacteria with 2 μL of a 1:10 dilution of the
digested plasmid without re-ligation will provide an estimate
of what percentage of colonies is due to incompletely digested
plasmid, rather than re-ligation and, thus, approximately how
many colonies should be screened to identify clones without
the NLS.
6. Increasing the insert/vector ratio will increase, whereas reducing it will decrease, the number of tandem inserts obtained on
average, and can be optimized depending on the desired number of inserts. There is no control over the orientation of the
insertions, but we have found that the large majority of clones
obtained contain all inserts in the same, either sense or antisense orientation, though it remains unclear why that is
the case.
RNA Imaging with Csy4
175