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Looking for Vector(s)

Looking for Vector(s) - Protocols and Methods Forum

Looking for Vector(s) - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 11-24-2003, 09:57 PM
Winston Smith
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Default Looking for Vector(s)



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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