I am an user of TRIPZ as well, and it's true that is not the perfect system, but so far, the best I've tried. The % of knock down is not usually a problem of the vector itself but the sequence of the shRNA. With the same sequence you'll never get better than that.

2) I woudn't use lipofectamine with this vector. The efficiency of integration will be much less than with the generation of the lentivirus. I would suggest trying of improving that step.

I don't get your question 3. This vector is designed to have your cells (frozen and usual passages) without Doxycicline, and only induce the expression in your particular experiment. I would not recommend use of long term experiments or mantain the cells with Doxo.

4) I've also noticed the RFP background expression, but only in certain cell lines. However the increase of RFP expression with Doxo is always hundreds of time higher. So, If with experiments you can "survive" with this leaky expression, I woudn't worry more. I am afraid that you cannot do much more on that.

5) As far as I know, Doxy has a low half life. So, either you put more (up to 10ug/ml) or you change your media more often.

Hope it helps,

Miguel

Lentiviruses tend to integrate in regions of open, activated chromatin. These "position integration effects" can lead to constitutive expression of your RFP-shRNA cassette even in the absence of inducer. This problem is worsened by the fact that the rtTA transactivator is always a bit leaky (that is, it always is able to mediate some transcriptional activity even in the absence of any ligand).

If you need to get better control of shRNA expression, use a Tet-OFF system instead.

Have you selected your cells with puromycin to eliminate cells that failed to get transduced? A selection step might improve the efficiency of knockdown.

In reference to your second point, you cannot compare lentiviral transduction with transient transfection in this case. In transient transfections, the 5' LTR will act as a strong constitutive promoter that will drive expression of the RFP-shmir cassette independently of the tet response element. So naturally, the RFP will appear very strong compared to the levels you get in a transduction setting (where the 5' LTR is dead as a promoter). Furthermore, in a transient transfection, you can get thousands of copies of plasmid into your cells compared to tens of copies of virus in a transduction experiment.

@Miguel Segura thank you vry much fr your help ... What i meant by my question 3 is that.. If i transduce 1 plate of cells with virus,select it with puromycin..then split that into 4 plates subsequently when required...all the 4 plates should have equal RFP expression on Tet induction..right? but if I freeze 3 plates out of 4 and work with the remaining 1 for the time being...the RFP expression of the 3 freezed vials(when thawed later and induced with Tet) is low as compared to the 1 which I did not freeze.

I replace media everyday when I use 2 microgm/ml doxycycline...I never tried using high Doxycycline concentration

@Daniel Cohen thanks a lot ...

yes I select my cells with puromycin and also maintain it in puromycin containing media

Just a suggestion on your question 4) regarding leaky expression. You can sort out cells with leaky expression by FACS sorting for RFP + cells (without Dox). Or you can select for RFP -ve cells and then check for induction of these clonal lines with dox. I have used the same strategy on another vector that had GFP.

I am just starting to use the pTRIPZ to generate stable lines for expression of a 16KDa protein. Even though its designed for shRNAs, i hope it will work if i replace the tRFP or put an IRES after that. Esp. as it has a TRE-CMV promoter.

@Tenzin Gocha

What is your experience with the plasmids you designed for your protein with ptripz. Iam also busy in designing ptripz with IRES-GOI and also replacing RFP with GOI in other strategy. At the moment iam checking these constructs which i made. Just wondering how far you are and whats your opinion ?

Raju

I have been using TRIPZ system and so far it is working great in a few cell lines. Now I am trying to use in other cell line but it is puro resistant so do you know how I can clone other marker into TRIPZ plasmid or if it is possible to generate lentivirus with two resistant marker plasmids????

Thanks

Judit

Hi Judit. I work with pTRIPZ also and, I think it is possible but not the most straight-forward thing to do. There are no unique restriction sites flanking the Puro resistance gene, so you can't do a simple cut paste operation.

However, I noticed that there is one dual cutting enzyme, Acc65I, which is going to be your friend. It cuts in two places: right upstream of Puro, and then downstream of the WPRE. So, if you cut with this enzyme, you will cut out a ~1.5 kb segment containing the 3' end of IRES, Puro gene, and WPRE.

Next, to replace with your desired gene of interest (and put the WPRE back), I would suggest the easiest route would be to just order the desired DNA sequence (you can order linear DNA sequences up to 2 kb for an okay price...like $200 or so I think). Just make sure to include the matching restriction sites on the ends of the DNA that you design! So your designed DNA should look like:

extra base pairs----restriction site----3' end of IRES----new reporter gene-----WPRE----restriction site----extra base pairs

Note that there might be other solutions, this is a little pricey too, but this was the first I noticed, and I think is fairly straightforward to get to your desired result.

Hope it helps!

Miguel, how does pTRIPZ system compare to U6 based systems overall (with the same guiding sequence)?