Biology Reference
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3. pTH-Ubi-gate. Vector containing the Pp108 locus targeting
site, hygromycin resistance, and the maize ubiquitin promoter
for constitutive expression, followed by a Gateway cassette.
4. NLS4
Physcomitrella
cell line [
13
]. This line expresses a nuclear
GFP-GUS reporter that is silenced by expression of RNAi
constructs designed in the pUGGi vector or the control pUGi
plasmid.
5. NLS4-LA [
14
]. This line contains a Lifeact-mEGFP construct
in the NLS4 background and can be used to evaluate changes
in F-actin resulting from specifi cally silencing a gene. The
Lifeact-mEGFP is not silenced by the RNAi constructs because
it does not have the GUS sequences that cause the nuclear
GFP reporter to be silenced.
2.3 DNA
Purifi cation Kits
Commercial maxi-prep and midi-prep kits can be used for purifi ca-
tion of plasmid DNA as well as classic methods for large-scale plas-
mid preparations. Because of their simplicity and the high purity
produced by the existing commercial kits, they should be used
when starting (
see
Note 3
). Once a consistent routine for moss
transformation is established and a high number of transformants
are routinely attained, the classic “homemade” protocols that pres-
ent a considerably lower cost should be attempted. When an initial
phenotype is investigated, it may be advisable to use a midi-prep
size of purifi cation, because if after a few attempts a construct does
not produce a phenotype, the DNA obtained from a midi-prep will
be all that is needed. Once a phenotype is observed, it is advisable
to prepare large quantities of plasmid using a maxi-prep. This
becomes critical for the RNAi construct when performing tran-
sient complementation assays.
2.4 Software
1. ImageJ. Can be freely downloaded from the NIH: (
http://rsb.
info.nih.gov/ij/
). Additional plug-ins to read a variety of image
formats may be necessary; they are available from the Laboratory
for Optical and Computational Instrumentation (LOCI) at the
University of Wisconsin-Madison (
http://loci.wisc.edu/bio-
formats/downloads
). Alternatively, a distribution version con-
taining the plug-ins and additional materials are available from
the Fiji site: (
http://fi ji.sc/
). We provide an optimized ImageJ
macro for morphological analysis of small moss plants called
MorphologyMacroPublicV2.6.txt that can be downloaded
from
http://users.wpi.edu/~lvidali/MMB_moss/
.
2. Statistical analysis software. We routinely use Origin Pro
8.1(Origin Lab Corp.), but any other statistical software
can be used as long as it computes ANOVA and Tukey post
hoc tests.
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