Antisense Approaches in Analyzing the Functional Role of Proteins in the Central Nervous System - Drugs of Abuse: Neurological Reviews and Protocols - page 365

at www.ncbi.nlm.nih.gov/BLAST by selecting standard nucleotide-nucleotide
BLAST (blastn).
3. In addition to aCSF as controls, three different types of oligonucleotides were
used in our study, which is considered to be more stringent than just using
one of them. The sense oligonucleotide that has a different base composition
to the antisense oligonucleotide could be potentially problematic because the
former may cause different base concentrations extracellularly and, especially,
intracellularly after the degradation. Different base concentrations may provoke
different metabolic effects on cellular functions. Accordingly, mismatched and
scrambled oligonucleotides that have the same base composition to the antisense
oligos seem to be better than sense oligos.
4. Frequency and dose of antisense delivery are determined by expression patterns
of genes of interest and can be best worked out by trial and error. If the gene has
a low basal expression, and turns over rapidly, one injection 6-8 h prior to study
may be suffi cient, considering that exogenous oligonucleotides usually reach a
maximal intracellular concentration after 2-16 h in most cases (2) . If the basal
expression is high with a low turnover, longer and repeated pretreatments are
needed (29) . Empirically, 2-3 d are suffi cient for a substantial effect on receptors,
and injections twice daily seem to work well (2 , 28 ; this study) . An alternative
delivery of antisense is the use of osmotic minipumps loaded with antisense
oligonucleotides. The implanted pump allows a continuous infusion for at least
7 d, which may be particularly preferable for inhibiting certain proteins (30) .
A detailed protocol for delivering antisense oligonucleotides with an osmotic
pump was described elsewhere (18) .
5. High doses of oligonucleotides may cause nonspecifi c effects and loss of neural
cells surrounding the infusion site at varying extents. Sequence containing
four consecutive G bases seems more toxic than other sequences (31) . Thus, a
conventional histological examination of infusion site after the end of the study
is required to evaluate extent of possible neurotoxicity. Reversibility of antisense
effect will also be a valid reassurance of lack of toxicity.
6. Mechanisms underlying the uptake of oligonucleotides into cells are not explicitly
understood. It may be processed by a simple event similar to fluid-phase
endocytosis (pinocytosis) given a water-soluble nature of oligonucleotides.
However, the uptake of oligos into the cells appears to be cell-type specifi c. Thus,
surface receptors or channels may likely participate in the uptake.
7. There is a simple way to assess the uptake and intracellular concentration of the
oligonucleotide. Application of the oligonucleotide conjugated with fl uorochromes
(fluoroscein isothiocyanate [FITC] or tetramethylrhodamine isothiocyanate
[TRITC]) will allow detection of kinetics and intracellular distribution of the
oligo and diffusion area of the labeled oligo in the injected tissue. However,
the intracellular level of oligos with conjugated fl urochromes may not necessar-
ily represent the real level of intact oligos because oligos may undergo rapid
degradation after uptaken into the cells. Intracellular accumulation of fl uorescent
oligos may genuinely mean an effective uptaking process.