I have very inconsistent RNAi in vivo knockdown. I am looking for some alternative approaches. Hope I can use some of your expertise. Thank you.
Try any of the new genome engineering techniques that have blossomed in the past two years, such as CRISPR/cas, TALENs, etc.
As Alejandro Martin suggested, I would use CRISPR/cas and remove the whole gene. This will result in a better repression of your gene. I would suggest to replace your gene by the GFP cDNA using a donor plasmid (you can use 2 donor plasmid with 2 different resistant genes to improve the chance to get the KO at the homozygous stage). By this way, you would be able to sort the cells which are KO for your gene.
Hi Lewis,
RNAi on which cell/tissue type? How is your budget for this? Do you need a "close to 100%" silencing effect? Do you need a long-lasting silencing effect? Are you using dsRNA or siRNA? How is your delivery method?
I work with both methods in my lab (RNAi and Crispr) and disagree with our colleagues that you should skip RNAi and go to our most trendy topic Crispr. Of course CRISPR is awesome and most likely will take over any gene manipulation technique. But, if I got your question well, you don't want an approach for gene silencing as an alternative for RNAi, you want an approach to enhance your RNAi effect. Is that correct?
If so, I believe that by answering the above questions my help us approach your need.
Take care!
Henrique
Dear Lewis
If you plan to use an in vitro approach instead, maybe could you try working with some cellular model and transfect Sh or Si RNA of your gene of interest.
For sure, you won't get the same results, information and interpretation compared to what you might be observing by an in vivo KD, but those alternative approaches could be helpfull at some points.
Best.
Gilles
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques