Biology Reference
In-Depth Information
functional heteroduplex with mRNA, it must bind into the single-
strand-forming region of target sequence fold. To predict second-
ary structure of designated AODNs, several programs for secondary
structure prediction can be used, e.g., Soligo (
http://sfold.wad-
sworth.org/cgi-bin/soligo.pl
)
. As mentioned in the introduction,
only partial cDNA may be suffi cient for the proper design. In our
experience, 18-20-nt-long ODNs are optimal. A selectivity of pre-
dicted oligos to target gene has to be verifi ed by BLAST search
(
http://blast.ncbi.nlm.nih.gov/
)
. For each antisense oligo, cor-
responding sense ODN is used as a negative control. To achieve
the best results, more than one oligo pair should be tested; usually
three to four pairs are suffi cient. To protect ODNs against nucle-
ases, three bases at each end are synthesized with phosphorothio-
ate backbone. The HPLC purifi cation is usually necessary to reduce
nonspecifi c cytotoxicity.
2.2 Pollen
To obtain pollen, fl owers of outdoor- or glasshouse-grown tobacco
plants (
Nicotiana tabacum
cv.
Samsun
) are collected in warm and
dry weather conditions before opening; anthers are taken out and
kept in laboratory conditions on a fi lter paper for one day to let
anthers open and dehydrate (anthers might be surface sterilized
and dried in the laminar box to harvest sterile pollen). Dried pollen
grains are sifted through to remove anthers. Harvested pollen can
be kept frozen at −20 °C without apparent loss of the germination
capacity for several years.
2.3 Cultivation
Media
To grow tobacco pollen tubes in vitro, several different media
could be used [
20
]. For cultivation times spanning up to 4 h that
are required for the AODN experiments, simple liquid growth
media are suffi cient, such as the following PEG and sucrose media
(
see
Notes 2
and
3
):
1. PEG medium: 20 % w/v polyethyleneglycol 3350, 1.6 mM
boric acid.
2. Sucrose medium: 10 % w/v sucrose, 1.6 mM boric acid.
1. To obtain working solutions of antisense and sense ODNs,
lyophilized oligos from manufacturer are rehydrated with ster-
ile double-distilled water resulting in 1 mM stocks. 20
2.4 Reagents
for AODN Treatment
μ
L ali-
quots are kept at −80 °C.
2. Suspension of cationic delivery reagent (cytofectin, Gene
Therapy Systems, cat. Nr T610001) is divided into 10
L ali-
quots and stored at −80 °C. After thawing, the cytofectin ali-
quot can be kept at 4 °C for several weeks.
μ
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