Alternative Gene Silencing Substrates
Nucleofection of Morpholino Oligos
Morpholino oligos provide a stable, specific and effective antisense activity. Developmental biologists have long since used these properties by microinjecting or electroporating Morpholinos into embryos to allow for splice-blocking or gene knockdown. However, efficient delivery of Morpholinos in mammalian cell cultures can be a challenge. While complex formation with lipid-based reagents is ineffective due to the non-ionic Morpholino backbone, nucleofection can be used as an efficient delivery method. As such, nucleofection of Morpholinos offers a straightforward alternative to siRNA-mediated gene knockdown and miRNA maturation inhibition.
Advantages of Morpholino oligos over DNA and RNA-based knockdown reagents ...
- Very robust and stable molecules due to non-biological backbone
- Exquisite specificity at low concentrations
- Largely free of off-taget effects
- Steric block mechanism allows for additional applications, e.g. block of miRNA maturation or alteration of mRNA splicing
... combined with Nucleofection allow for
- Efficient delivery of Morpholino oligos
- Morpholino-mediated gene silenicng in mammalian cell culture
Dose-dependent delivery of Morpholinos into ON 705 HeLa cells.
Cells were transfected with indicated concentrations of a splice-correcting Morpholino oligo. In the ON 705 HeLa cell line, the Morpholino corrects a splicing mutation splicing out a stop codon and putting luciferase in frame. After 24 hours, the cells were lysed and luciferase activity and protein amount were determined. Delivery success is proportional to the light output (normalized to total protein amount).
Data kindly provided by S. Knuth, GeneTools LLC, USA.
