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| Looking for "perfect" vector that must feature several biological capabilities: 1. when transfected into helper cell line, must signal or contain code for producing virus with broad mammalian cell host range, and viral capsid must contain gene of interest; helper cell line may already contain complementary episomal elements for viral replication/production 2. does not contain any elements for uncontrolled (constitutive) transcription in mammalian cells 3. contains high efficiency replication origin and selection for mammalian cells (i.e., neomycin/G418) 4. contains replication origin and selection in prokaryotes (for maintaining/archiving constructs) 5. contains sequences for host chromosome integration (specific or random integration targets okay, but specific targets preferred) I want to take advantage of the high efficiencies of gene delivery in mammalian cells offered by transduction, especially since these are not continuous cell lines (and don't need to be). Thus the vector needs to code or signal for its packaging into mammalian viruses. I would also like this vector to be stably integrated, although that is not critical. The problem with retroviral sequences providing integration is that they also cause constitutive (unregulated) transcription. I want to be able to insert into the multiple cloning site both the transcriptional control elements (promoters/enhancers) as well as the genes themselves (these will be lacZ or GFP reporters), and so vectors that feature unregulated transcriptional control sites are unwanted. (Although I suppose I could insert 5' to my promoter/gene triple/quadruple polyA signalling sites....not sure if they work?) I have looked into the Clontech pMSCV vector and some commercial adenoviral vectors. A drawback to the adenoviral vectors is (1) possible narrow host specificity and (2) do they contain sequences to integrate into host chromosomes? The promoterless and constitutive-promoter LacZ/GFP-reporter constructs available have the drawback that it would take considerable effort to make the transducible. Am I missing something? |
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