Advice on Cloning Strategy - (Aug/02/2010 )

Hello

I am planing a cloning experiment, but I have a hard time to see how I could manage this.

I have a reporter vector from Clontech (pLVX-DD-ZsGreen1 , ), which has a PGK-puromycin cassette.

I want to replace puro by RFP. In recipient vector pLVX PGK-puro cassette could be excised by digestion with SphI and SexAI.

I have a vector (pBluscriptKS) which contains PGK-RFP, but in a 3' to 5' orientation ( ):
XbaI - RFP - BamHI - PGK-promoter - (SphI) - EcoRI. (Why does this nevertheless lead to fluorescent cells when transfected?)

If was planning to add a SexAI site 5' to XbaI site via PCR. Then I would have SexAI(- RFP - PGK-Prom -)SphI which would be compatible to pLVX digested with SphI and SexAI.

The problem is now that the orientation of the insert is inverted. So as far as I managed to imagine it, this would lead to the interchange of sense and antisense strand, and therefore no transcription.

Is that correct? If yes I have to find another strategy, which I am not sure I will :-)

Thanks for answers.
Greets
Lucas

The orientation of genes is irrelevant. What counts is the relative orientation of the promoter and coding regions. If your DNA fragment has both the promoter and the coding region, the orientation of the fragment in your construct should matter little. If you replace your antibiotic selection gene with a reporter, how are you selecting your constructs?

But when strands are interchanged, eg:

SexAI-GATCGG-SphI excised and inserted in a vector digested ---SphI SexAI----


would then lead to

5'-----GCATGCCCGATCACCWGGT-----3'
3'-----CGTACGGGCTAGTGGWCCA-----5'

(green is original insert sequence, red shows sequence of donor plasmid, black is new backbone.)

meaning that the frormer coding strand is now the template strand (lower, 3'-->5') and vice versa, which would result in antisense RNA, right?

To your question: We plan to produce lentiviral particles for in vivo experiments and we will FACS-sort infected cells to enrich for those containing the construct.

Your DNA strand does not know which is the template strand. It is symmetric -- there is a 5' and a 3' base at both ends. Only the promoter orientation establishes which is the template strand. If you change the orientation of the promoter, the identity of the template strand also changes.

You might want to rethink your strategy -- the transformation/transfection efficiency is usually quite low, and you might be running the flow machine quite a while to find a transformed call.

Now that you say it.. Makes sense. I was a bit confused after juggling around with RE sequences the whole day.

Transduction efficiency of lentivirus should be quite high, as far as I know (20% and higher). Or am I misinformed?

Greets
Lucas