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PMID: 19657394 Published · epublish English Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S.

A versatile viral system for expression and depletion of proteins in mammalian cells.

PloS one ·Vol. 4 ·No. 8 ·2009-08-06 ·Pages e6529

Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, Campisi J, Yaswen P, Cooper PK, Kaufman PD

Abstract

The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.

MeSH Terms
Animals Base Sequence Cell Line DNA Primers DNA, Complementary/genetics Electrophoresis, Polyacrylamide Gel Flow Cytometry Fluorescent Antibody Technique Genetic Vectors Humans Proteins/genetics RNA/genetics RNA Interference Recombination, Genetic Retroviridae/genetics
Chemicals
DNA Primers DNA, Complementary Proteins RNA
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Campeau Eric
Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America. Eric.Campeau@umassmed.edu
Ruhl Victoria E
Rodier Francis
Smith Corey L
Rahmberg Brittany L
Fuss Jill O
Campisi Judith
Yaswen Paul
Cooper Priscilla K
Kaufman Paul D
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Article Info
Journal
PloS one
Abbr.
PLoS One
ISSN
1932-6203
DOI
10.1371/journal.pone.0006529
Email
eric.campeau@umassmed.edu
Published
2009-08-06
Epub
2009-00-06
Pages
e6529
Language
English
Region
United States
NLM ID
101285081
PMCID
PMC2717805
Subset
IM
Grants
NIA NIH HHS · AG017242 · United States
NIGMS NIH HHS · F32 GM076863 · United States
NCI NIH HHS · U54 CA112970 · United States
NIA NIH HHS · P01 AG017242 · United States
NCI NIH HHS · R01 CA063503 · United States
NIGMS NIH HHS · 5 F32 GM076863-03 · United States
NIGMS NIH HHS · R01 GM055712-11 · United States
NCI NIH HHS · 1F32CA108393 · United States
NIGMS NIH HHS · R01 GM055712 · United States
NIGMS NIH HHS · R01 GM557 · United States
NCI NIH HHS · P01 CA092584 · United States
NCI NIH HHS · F32 CA108393 · United States
External Links
PubMed source DOI: 10.1371/journal.pone.0006529 PMC Full Text View journal details
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