Question

A researcher use morpholine method to transiently knockdown gene expression

how does this method work?

and what are the steps involved in this experiment? (4 marks)

Answer 1

. Morpholinos block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces of ribonucleic acid (RNA). Morpholinos are used as research tools for reverse genetics by knocking down gene function.

RNA Morpholino- Morpholino RNA Heteroduplex HO 0 HO Watson-Crick bonds Bases positioned for strong binding Usually 25 base Morpholino oligos are used O P-o Ho

steps involved in morpholine method:-

Translation Blocking: By sterically blocking the translation initiation complex, Morpholinos can knock down expression of many target sequences completely enough that after waiting for existing protein to degrade, the target protein band disappears from Western blots. Unlike many antisense types (e.g. siRNA, phosphorothioates), Morpholinos generally do not cause degradation of their RNA targets; instead, they block the biological activity of the target RNA until that RNA is degraded naturally, which releases the Morpholino.

Splice Blocking: By blocking sites involved in splicing pre-mRNA, Morpholinos can be used to modify and control normal splicing events. This activity can be conveniently assayed by RT-PCR, with successful splice-modification appearing as changes in the RT-PCR product band on an electrophoretic gel. The band might shift to a new mass or, if splice-modification triggers nonsense-mediated decay of the transcript, the wild-spliced band will lose intensity or disappear.