In vitro transcription

[Mega [Andreas Stuermer]]

I've been looking into in-vitro ttranscription. It looks like it could avoid GM regulation issues and be fine for some projects. These kits' prizes are horrible though. Does anyone know where to get the cheapest ones?

Also I was thinking about unifying the process. Nowadays you do:

T7-> RNA synthesis
5' capping
polyA tail addition

There are some viral RNA elements which substitute 5' cap and polyA tail, so maybe it would be xool to have a plasmid with this elements. All you need is to add T7 polymerase, and you get stable mRNA that can be translated (in vivo or in vitro). Any thoughts on that?

[Bryan Bishop]

On Tue, Jan 3, 2017 at 4:47 PM, Mega [Andreas Stuermer] <masters...@gmail.com> wrote:

I've been looking into in-vitro ttranscription. It looks like it could avoid GM regulation issues and be fine for some projects. These kits' prizes are horrible though. Does anyone know where to get the cheapest ones?

There is a freeze drying approach for in vitro transcription. Some folks have used this with filters/filter paper. For example,
http://www.cell.com/abstract/S0092-8674%2814%2901291-4

- Bryan
http://heybryan.org/
1 512 203 0507

[Koeng]

For the polyA tail, you can use 75 As (75 tested as best for yeast in vivo) followed by a HDV ribozyme to make it cleanly cut. Sequencing this flat out doesn't work, so you're going to have to do some small electrophoresis runs and get it synthesized by a company. Genescript works well.

There are no RNA elements that truly substitute a 5' cap in yeast, to my knowledge (which is purely in vivo in yeast). IRESs, (internal ribosomal entry sites) don't work properly in a non-nuclear context in yeast. From the data, it's inferred that perhaps they function as weak promoters in the nucleus. In any case, capping is not strictly necessary in yeast for expression, although it helps to a significant degree. Capping might just be easier in vitro. 

I have no experience in mammals, which is likely your desired host organism. If it is, then I think that IRESs would likely work for whatever your project might be. 

-Koeng

[Mega [Andreas Stuermer]]

Alright thanks!

I found some other papers, they did it with cultured mammalian cell extract. I guess the same procedure applies to animal primary cells