non-expression vector - (Nov/16/2005 )
does anyone know of the existance of a vector that can be used to generate a stable mammalian cell line (i.e. a having an antibiotic resistance gene) and not having a promoter in front of MCS? (I'm trying to generate a cell line that has an integrated copy of a HIV genome with a frameshift in a gene and a luciferase reporter, but as HIV has an LTR and this one has funtional TAT it will be expressed as it is. I fear that having both LTR and for instance CMV will lead to such a high expression that it will kill my cells...).
Small problem, due to the lenght of the genome and reporter, I only have 2 blunt end cutting enzymes that I can use to get my genome out of a plasmid. (I can of course add A overhang with Taq, but that about it...)
Thanx in advance!
maybe a layzy answer. But i've done an exp in which i didn't want the promoter included in the vector. So i did just remove the promoter by restriction enzymes...
did you integrate the HIV LTR? Can't you inactivate it ?...
I was/am considering cutting an expression vector, I checked the sequence of some and have very few options, that's why I came here and asked.
I have both LTR's to insert (I want to generate pseudotyped virusses with these cells when transfection an expression vector for the gene with the frameshift, this system is working right now when doing co-transfection of the expression vector with the plasmid containing my reporter genome, but I want to generate higher titers...). I can inactivate my LTR's by inactivating HIV-tat, but that would mean that I constantly have a tat-inhibiting substance in my growth medium which would be rather expensive (and nothing's know yet about efficiency of these substances).
i've never titred my viruses preparations. But assuming the fact you have your on HIV ltr and the LTR of the retroviral plasmid that would increase th relative number of copies.
It's not linear because you may have perturbations for the inner LTR if you have laready a trnascription first-ltr-directed.
But you can titer the viruses.
Or, you can test your HIV in a plasmid that do not contain a promoter versus a plasmid that contain a promoter or LTR and test? Is it feasible ?
Wait a second, let me try to explain to you exactly what I'm trying to do here...
I have a "molecular clone" (that is, a plasmid that has an entire HIV-genome, including both ltr's) that has a frameshift mutation in one gene (the envelope gene) and has a luciferase inserted in the genome. The fact that it has the frameshift mutation in the envelope gene makes it impossible for this genome to create replication competent virusses (as the envelope proteins are the ones used for entry in target cells). I have a simple method to amplify envelope genes and clone them into an expression vector (with CMV-promoter). By co-transfecting 293T cells with the molecular clone and the expression vector, you can create virus that is capable of a single round of replication (it has the envelop protein, and can perform RT and integrate, then expressing luciferase which you can measure but it can not once again form virus because it lacks envelope).
This system works and is widely used. But, I want to try to get the molecular clone into the 293T cells, after which I would only have to transfect with my envelope expression vectors. I want to do this, because I believe (I have to try it of course) that in this way, I might be able to get higher viral titers (I wouldn't rely on succesfully transfecting cells with both plasmids and I would be able to transfect more expression plasmid DNA, so that more cells will take up this plasmid). Also, I wouldn't have to prep the molecular clone every so often.
Thanks for you reply!
and what about transfecting packaging cell lines? Like 293Pack (also called phoenix cells) they have the Env gene inserted?
I wasn't 100% complete in my last post, I'm using patient derived env genes... With this system, I can determine susceptibility of patient samples to entry inhibitors (substances that target the entry process that involves the env genes).
We currently have samples of 50 patients that are on a combination therapy that also has an entry inhibitor in it. I will use this system to evaluate susceptibility to the entry inhibitor in their therapy for subsequent samples (and also susceptibility to other entry inhibitors in clinical phase, or preclinical even).
But thanx for your suggestion!