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Antisense oligonucelotides (Limitations (Unable to achieve complete loss…
Antisense oligonucelotides
Mechanism
Bind to mRNA to prevent transcription
Watson-Crick base pairing
25 bases
Backbone imparts resistance to degradation
Enhanced by MOE
Effect depends on chemistry and target sequence position
Degradative- RnaH dependent
Non-degraditive RnaH independent
Limitations
Off-target effects
Unable to achieve complete loss of function
Added difficulty in tetraploid species
Can bind protein
Compensatory adaptation
Analogous to all genetic methods
Can be toxic
Due to off-target effects
Requires much titration
Only 10-50% produced are effective
Diluted during cell division
Useful in terms of avoiding toxicity- but difficult in terms of titrating doses
One needs to consider their rate of degradation
Long oligo more specific but can form secondary structure
Advantages
Treatment
Two recent nature papers attest
Conditional
Useful in Xenopus as recalcitrant to siRNA
Development paper
Ease of delivery
Ideal for monogenic conditions
:
Little pathogenic immune response as can occur with viral vectors
Xenbase has blast
Controls
Always test one mismatch
Always test one scrambled control
Rescue experiments
Protein and qtPCR of target
Activity assays
Screens with several potential targets
Potential issue with PRDX as they are similar