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product in plant cell growth or proliferation. When a single
construct is not anticipated to silence many genes, sequences
corresponding to all the genes to be investigated should be
strung together [ 5 - 7 ]. This can be easily achieved by the
incorporation of restriction sites into the primers and re-
amplifi cation of ligated constructs ( see Note 5 ). Details for the
construction of these types of constructs are available from the
Bezanilla laboratory website ( http://bezanillalab.com/moss-
methods.html ) and the publications indicated above.
2. For transient complementation coupled with RNAi, it is nec-
essary to use the untranslated regions (UTRs) of the gene for
silencing. Sometimes it is diffi cult to determine the precise
location of the 5
UTR due to lack of conservation between
species and between homologous genes, because the gene
models corresponding to 5 ' UTR regions are less accurate
than the coding sequence regions ( see Note 6 ). From the lim-
ited number of constructs we have designed [ 3 , 5 , 7 ], we
found that the 5
UTR regions work well for longer genes,
UTR regions are also appropriate for short genes.
3. Once a phenotype is obtained using coding sequence regions,
the phenotype should be replicated by the UTR constructs.
Using the quantitative approach presented here to measure
phenotypic changes (see below), it should be simple to
establish the degree to which both constructs cause a similar
phenotype. Furthermore, the capacity to silence highly
homologous genes separately allows one to determine if genes
can perform redundant functions.
while the 3
3.2 Transient RNAi
1. Transform 30
g ( see Note 7 ) of RNAi construct using
published protocols for transformation and plate protoplasts
on cellophane-covered protoplast regeneration medium-
bottom (PRM-B) plates using liquid plating medium ( see
Note 8 ; ref. [ 10 ]).
2. Four days after transformation, transfer plants to regular
growth medium (PPNH4) plates containing 15
μ
μ
g/ml hygro-
mycin ( see Note 9 ).
3. Grow for 3 more days.
4. Proceed to Image Acquisition step.
3.3 Transient RNAi
and Complementation
1. In the same transformation tube, add 30
μ
g of RNAi construct
g of complementing construct based
on the pTH-Ubi backbone.
2. The optimal level of plasmid may need to be determined by
titrating the amount of plasmid added and can vary from a few
μ
and as a fi rst attempt 30
μ
g to 60
μ
g ( see Note 10 ).
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