Dear Matteo,

To transfer these cassettes into a lentiviral vector you'll need to remove both polyA sequences, or the packaging will fail.

You can safely make LV with dual promoters, and they've been extensively used (e.g. pLVX or pLKO series amongst others). In your particular case however, I would advise against this approach.

If the main reason for not using an IRES or T2A is to save as much as you can from your original vector, please consider that ordering a synthetic cassette containing a PGK mCherry T2A GFP mAbi3 to clone it into a lentiviral vector would be faster and cheaper than moving your cassettes over multiple cloning rounds.

Additionally, P2A (but not IRES) will ensure stoichiometric abundance of your proteins in target cells. Something that dual promoters won't guarantee.

PS. while you can't use polyA, I'd still recommend adding a WPRE sequence at the end of your Abi3. This will boost mRNA stability level and also trigger some polyadenilation, but without interrupting the primary RNA (which is your lentiviral genome during packaging).

Hope it helps

Francesco

You can clone your cassette in the reverse orientation to the lentiviral gRNA transcription. In this case the polyA sequences will not interfere with virus production.

Dear Francesco and Yoav, thank you so much for the answers!

@Yoav Lubelsky: That's a very good suggestion! I thought terminators would act bidirectionally, but you're right that they don't.

@Francesco Aulicino: I've just taken a look at the pLVX/pLKO vectors (which I hadn't seen before) and you're right that the cassettes are not that different from what I would have in mind.

Regarding the rest of your suggestions, for brevity's sake I didn't expand on the details of my approach, but the idea would be to excide the central poly(A) and then amplify the whole cassette stopping short of the other poly(A), then move the amplified fragment to a lentiviral backbone (with WPRE), that way I would get rid of the 2nd poly(A) as well.

You're right that P2A/T2A alone ensure equimolar stoichiometry, but I would be perfectly fine with having the 2nd ORF expressed at lower levels than the first, though your point is very well taken. Right now I'm still brainstorming about how to handle these additional cloning steps, so let's see if it's advantageous to try and salvage how much I can of the current construct or if I should rather synthesize the two coding sequences intercalated by an IRES/P2A.

Hi Matteo,

Thanks for clarifying your cloning plan. It makes sense.

The best approach then (without having to synthesise anything), is to PCR amplify two cassettes from your original vector:

1) PGK mCherry

2) GFP mAbi3

You merge them using a T2A signal embedded in the oligonucleotides. The forward oligo of cassette 1 has to contain homology for the lenti-backbone, the same goes for the reverse oligo of cassette 2.

This way you can easily do a 3-parts gibson assembly and directly have your final vector with stoichiometric expression.

The alternative is to order 1 synthetic cassette, and choose whatever topology you like (IRES, T2A, 2 promoters etc). Given the price of the oligos, and the time required to PCR amplify, purify and assemble the reactions, gene synthesis is still competitive.

Both approaches should take roughly the same amount of time anyway.

Best wishes

Francesco