Looking for Vector(s)

Winston Smith FK at noplace.com
Mon Nov 24 16:57:19 EST 2003


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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