6. Adenoviral overexpression and antisense technology
Genes are over- or under-expressed to determine effects on physiological activity.
Over-expression ( up-regulation)
Adenoviral expression systems
- Adenoviral expression systems permit the transfer of genetic material into both dividing and non-dividing cells in vitro and in vivo
- Recombinant adenoviruses expressing the genes of interest are constructed
- An infected cell line will produce the protein of interest in its native state enabling the study of its effects on cell function
Retroviral expression systems
- The retroviral expression permits a highly efficient introduction of stable, heritable genes into the genome of dividing cells
- Retroviral expression vectors allow the expression and packaging of target genes following their transfection into a packaging cell line
- Following infection of the target cells, they become stably transduced containing only a single copy of the gene that is inheritable
Under-expression (down-regulation)
Antisense technology
- Antisense oligodeoxynucleotides (ODNs) are used to inhibit endogenous gene expression
- Phosphorothioate ODNs are utilised and tested in tissue culture to determine their effectiveness in down-regulating the expression of the target gene
- Real time Taqman PCR is used to monitor changes in gene expression
- ODNs can be fluorescently tagged to monitor transfection efficiency and cell localisation
- Results are then validated in in vivo studies in particular for neuronal genes where the antisense ODNs are administered ICV
siRNA
- RNA interference (siRNA) is a method of suppressing specific gene expression by complementary doublestranded RNA in non-mammalian, and mammalian systems
- Highly efficient in reducing target mRNA expression
- Changes in gene expression are monitored by real time PCR or by immunoblotting
- Various metabolic endpoints may then be measured to determine the effect of down-regulating the target mRNA
