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research and clinical applications [ 5 ], whereas this technique is still
not commonly used in the plant research. The AODN method is
especially useful in species where stable transformation is hard to
achieve. Furthermore, AODNs are particularly benefi cial for those
species with only partially covered genome sequence, because only
hundreds of base pairs are usually needed for successful design. On
the other hand, the AODN technique has also drawbacks, the most
crucial one being only a partial reduction of the target gene expres-
sion, where the reduction varies from 40 to 80 % [ 6 , 7 ].
Single-stranded DNA antisense oligodeoxynucleotides share
their underlying basic principle with the widely utilized RNA inter-
ference technique: an oligo(deoxy)nucleotide must bind a target
sequence through Watson-Crick base pairing ultimately resulting
in impaired gene expression. Single-stranded DNA could be very
quickly degraded by exonucleases, which are especially abundant in
plant cells. For successful gene knockdown, AODNs have to be
protected by modifi cations of its backbone. Phosphorothioate,
where one of the nonbridging oxygen is replaced by sulfur, is by far
the most frequently used modifi cation. Phosphorothioate AODNs
bind to the target sequence and formed hetero-RNA-DNA duplex
is recognized and cleaved by RNAse H [ 8 ]. Since phosphorodi-
amidate AODNs are charged molecules, they cannot simply pass
through the lipid bilayer by diffusion and one has to use cationic
delivery molecules, such as cytofectin. In contrast to phosphoro-
thioate AODNs, phosphorodiamidate morpholino oligomers
(morpholinos) are uncharged DNA analogues, although their
delivery into intact plant cells is not well studied [ 9 , 10 ].
Interestingly, morpholinos function through steric hindrance
imposed on the ribosome during mRNA translation.
The fi rst report on the usage of AODNs in the plant research
is dated to 1992 [ 11 ], and the technique was successfully employed
in pollen tube biology in 1994 [ 12 ]. During the last decade, several
reports using phosphorothioate and morpholino AODN strategy
in a variety of plant species and cell types have been published [ 3 ,
7 , 10 , 13 - 19 ]. Intriguingly, the alternative mechanism of AODN
uptake into plant cells through active transport via mono- or disac-
charide translocators aided by sucrose itself was proposed [ 15 ].
Here we present a detailed description of the gene suppression
in tobacco male gametophyte mediated by short AODNs compris-
ing the design, the cultivation procedure, and the analysis. Also the
usage of sense ODNs as a negative control is stressed.
2
Materials
2.1 Antisense
Oligonucleotides
One of the most crucial part of the successful application of AODN
strategy is careful oligo design. The good oligo must be specifi c,
must not form secondary structures, and in order to create
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