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Can Morpholino oligo be delivered in oocytes by lipofectin


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#1 vani

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Posted 06 December 2009 - 08:22 AM

Hey Guys

I am almost finishing my work as a Phd student. and many a time this forum and all of you have helped me alot. A big THANKS for that.
I am looking for a very quick answer if anyone knows
Can Morpholino oligos be delivered in oocytes in L-15 medium using lipofectin. Is there any protocol for that?
and how much concentration should I be using?
Please help me if any of you can. I really need this info urgently.
Waiting for your response.

cheers
Vani

#2 bob1

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Posted 06 December 2009 - 02:57 PM

It's technically possible, but usually it is done using microinjection, mostly because oocytes are precious/hard to come by and lipid based methods don't transfect 100% of the cells anyway. Also oocytes are large, so it is more difficult for the morpholino to get to the active sites.

#3 Jon Moulton

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Posted 07 December 2009 - 07:47 AM

Can Morpholino oligos be delivered in oocytes in L-15 medium using lipofectin. Is there any protocol for that?
and how much concentration should I be using?


Hi Vani,

It would help to know what species produced the eggs, as there are methods of transfection worked out for some embryos during development (for instance, chick).

As for lipofectin, the problem is that while lipofectin is a cationic lipid and electrostatically complexes with nucleic acids, Morpholino oligos are uncharged and will not form the electrostatic complexes. There is a work-around, which is to anneal a partially-complementary DNA strand to the Morpholino by Watson-Crick pairing. These heteroduplexes of DNA and Morpholino are called "Special Delivery" oligos and can be delivered using cationic lipids. Gene Tools has an ethoxylated polyenthylenimine (EPEI) optimized for that delivery. Special Delivery oligos haven't been commercially available for years, but a do-it-yourself protocol is on the web (section D in the link below).
http://www.gene-tool...SpecialDelivery

Other options are Endo-Porter (an endosomal release agent) or electroporation. Endo-Porter comes in DMSO solution and is poorly soluble in water. As the DMSO enters aqueous solution, the Endo-Porter peptides form aggregates. For cell culture, immediate swirling is recommended on adding the Endo-Porter to medium; this limits the size of the aggregates formed. For work in embryos, the clumping can contribute to needle clogging so you'll need to find a way to limit aggregate size when mixing the Endo-Porter with the aqueous Morpholino injection medium (assuming you will inject the embryos with the Morpholino & Endo-Porter solution). Electroporation is the usual method for introducing Morpholino into chick embryos and there are methods worked out for embryos of some other species. For links to protocols or abstracts, see the "Outside Resources" box in the right margin of this page:
http://www.gene-tools.com/node/17

Of course you are welcome to chat with folks at Gene Tools about your ideas. Good luck with the experiments!

- Jon

Edited by Jon Moulton, 07 December 2009 - 07:48 AM.

Jon D. Moulton
Gene Tools, LLC
www.gene-tools.com




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