I want see whether the naked siRNA can enter the V.dahliae spores or not? i mixed fungal spores and siRNA (flourescently labeled) and saw under microscope at various intervals, initially i couldn't find any spores that have uptake the siRNA, after a day i hardly found a spore or two. how can i increase the efficiency of this method? any suggestions please
Hi Latif
Spores can be sensitive to glucanases that effect the membrane. Novozymes once had a product called "Glucanex" that was designed to remove viscous Glucanes in the wine and juice production. A mild enzyme treatment with a Glucanase like that can make the membranes more permeable so you can transform Plasmids or DNA/RNA or increase the exchange between cytosol and the ambient media.
Has anybody tried a weak pulsed electric field? see for an example https://www.researchgate.net/publication/229691961_Influence_of_a_pulsed_electric_field_on_the_spores_and_oxygen_consumption_of_Aspergillus_niger_and_its_citric_acid_production
Article Influence of a pulsed electric field on the spores and oxyge...
Thanks Frank Hoger and Alex Ignatov for your answers. I tried to treat the spores with 0.01% tween 20, but it didn't work. any other chemical i can use?
Hi Latif
The cellwall of the spores is very stable, you need to activate it enzymatically, osmotically or electrically. There is a protocoll from Invitrogen Yeast cloning in Pichia pastoris pPICZ using a LiCl2 /PEG competent cell preparation from a fresh Yeast Culture
http://tools.lifetechnologies.com/content/sfs/manuals/ppicz_man.pdf (page 29)
similar to what you do with chemical competent E.colis, maybe you have a chance with this method.
The amount of Tween you used is to low to have an effect like the osmotic or electric impulse or the improved permeability of a digested cellwall (Protoplasts) have
http://en.wikipedia.org/wiki/Protoplast
Usually the Protoplasts of spores or fungal cells needed to get an additional electrical impuls (softer, because they are less stable than regular cells) to really be able to transfer DNA/RNA
Thank you so much for your suggestions, i will go through the suggested protocols and will let you know about the progress.
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