- For quantification of the number of moving mRNAs analyze
about 30 cells. Calculation of the number of moving mRNA
per cell is depending on the length of the analyzed hyphae
(Fig.4c). In our case, most mRNAs can be detected simulta-
neously with the MS2 system and by either the PP7 or theλN
system.
4 Notes
- In order to adapt the system to other target mRNAs with for
example altered expression levels or to use the system in other
organisms it is advisable to fine tune the number of stem-loops
to obtain optimal results. Therefore, it might be necessary to
design the array of stem-loops de novo. In this case, we recom-
mend using the sequence information given in Fig.4a for the
stem-loops and separate the stem-loops by spacer sequences of
about 40 nucleotides. The spacer sequences should vary to
improve genetic stability and expression of the tagged mRNA
[38]. After optimization of the sequence we strongly recom-
mend to verify correct stem-loop folding in silico using the web
program mFold (http://unafold.rna.albany.edu/?q¼mfold).
The required plasmid DNA can be ordered by various compa-
nies offering gene synthesis. If then for example an array of 16
stem-loops is available, oligonucleotides for PCR reactions can
be easily directed against the spacer sequences to obtain PCR
products containing for example 6, 8, or 12 stem-loops. - To construct vectors encoding a C-terminal fusion of the RBP
fused to a fluorescent protein we recommend the use of the
versatile Golden-Gate cloning system recently established for
U. maydis[34]. In brief, the stop codon of the RBP of interest
is replaced by a sequence encoding a short linker amino acid
sequence AANAAT, which is fused to the start codon of the FP
such as GFP. This is achieved by using the DNA sequence
GCGGCCAACGCGGCCACC [33]. Thereby anSfiI restric-
tion enzyme site (underlined) is introduced that can also be
used for further standard cloning procedures, if for example the
FP should be replaced by a different color (e.g., replace GFP
with mCherry; [33]). - Switching the nitrogen source induces the hyphal growth ofU.
maydisdue to the expression of the responsible genes with the
nar1promoter [39]. - Swaying the microscope slide ensures an appropriate distribu-
tion of cells. It is highly recommended to let the cell suspension
dry on the slide to increase plane orientation of hyphae. - For better visualization the exposure time can be adjusted
ranging from 100 to 250 ms.
332 Sabrina Zander et al.