If this works what kind of tranfection reagent is recommended for the islets?
While I personally haven't tried siRNAs in mouse islets, several of my colleagues have via Lipofectamine 3000, and they haven't had much success with them – the efficiency was very low. They're now, like I am, using adenoviral shRNA which has worked better. If you're set on siRNAs you could perhaps try Invitrogen's Lipofectamine RNAiMAX though I can't say for sure whether it'll be much better. Also, islets in our experience seem too fragile for electroporation.
Hope this helps, and good luck!
While I personally haven't tried siRNAs in mouse islets, several of my colleagues have via Lipofectamine 3000, and they haven't had much success with them – the efficiency was very low. They're now, like I am, using adenoviral shRNA which has worked better. If you're set on siRNAs you could perhaps try Invitrogen's Lipofectamine RNAiMAX though I can't say for sure whether it'll be much better. Also, islets in our experience seem too fragile for electroporation.
Hope this helps, and good luck!
Because of the structure of islets (in terms of arrangement of various cells with respect to each other) and the necessity of physical contact of any transfection method, it is impossible to get all cells transfected equally well.
Since adenovirus is very infective, one can get outer layer of cells to express gene of one's interest quite readily. If I recall my personal experience from ~ 25 years correctly, a contact time of about 10 min with beta-galactosidase containing adenovirus showed robust expression of the indicator enzyme in outer cells of islets. Compared to this outcome, chemical methods (including liposomal complexes), were far less effective.
if you try to increase the proportion of your transduction agent (including the titer of adenovirus), as you can imagine, you will kill more cells. Regardless of which method you chose for transduction, you will have to do some optimization yourself.
Thanks Jason and Dr. tausif for your reply. I wonder if you can provide me with the adenovirus infection protocol you are using?
we have achieved transfection of isolated islets using our self-deliverable siRNAs (sdRNAs). Will be happy to discuss
www.advirna.com
Dina,
Adenovirus tends to be very effective in infecting most cells (including cells in islets with the caveat discussed previously). You do not need any special protocols. Use the normal medium that you routinely use for keeping islets in culture and expose them to adenovirus at desired MOI. If you have limited amount of virus, reducing the volume of medium will be helpful. If need be, you can even let islets settle in a conical tube so you can aspirate majority of culture medium and replace it with prewarmed medium (equilibrated in your incubator) containing the amount of virus you wish to test. If this is the method you wish to try, keep the cap lose and tip the tube at a shallow angle after gently mixing to increase the surface area so the islets do not end up in hypoxic medium. About 30-60min is more than enough. Of course, if islets are kept in a way that it does not effect them too much, you can easily leave them overnight with adenovirus. Relatively speaking, you will see some improvement in transduction but far from directly proportional to the time of exposure.
Best,
Addendum:
About the MOI - if you try to exceed way beyond 100 infectious adenovirus particles/cell, you will begin to see significant cell damage and death. In case of islets, you also need to consider an additional point. You cannot use a conventional measure of total cell number, such as the total DNA contents etc. to calculate as to how much Ad to add to get your target MOI, simply because you will be preferentially infecting outer layer of cells.
- Badges
- Science method