Campeau et al. 2009

Campeau et al. (2009). PLoS ONE DOI: 10.1371/journal.pone.0006529 Citations: 1058

Viral vectors needed separate backbones for cDNA, shRNA, and miRNA, with few markers. This paper built 59 Gateway vectors spanning lentiviral/retroviral backbones and drug markers. Recombination reached over 95 percent efficiency in bacteria.

Parts used

CMV PromoterhPGK PromoterEF1α PromoterU6 PromoterH1 PromoterWPRE

Key findings

  • Campeau et al. developed a collection of 59 Gateway-compatible lentiviral and retroviral vectors for constitutive or inducible expression and depletion of proteins in mammalian cells. The Gateway LR recombination system achieved >95% cloning efficiency, eliminating instability issues common to viral vector cloning in bacteria. All lentiviral Destination vectors incorporated WPRE and cPPT to increase transduction efficiency, and the collection supported seven drug selection markers including puromycin, zeocin, and blasticidin.

  • The system integrated five promoter options—CMV, doxycycline-inducible CMV/TO, PGK, EF-1α (Pol II), and H1/U6 (Pol III)—into modular Entry and Destination vector pairs, enabling rapid exchange of promoters and selection markers without recloning cDNAs or shRNAs. Third-generation self-inactivating (SIN) lentiviral vectors were Tat-independent for improved biosafety. The pLenti X2 series exploited ΔU3 3′ LTR insertion to achieve shRNA cassette duplication upon integration, effectively doubling transgene copy number.

  • Three functional studies validated the system: the FKBP12 Destabilization Domain enabled faster conditional protein depletion than RNAi in mitotically arrested cells, primary fibroblasts were engineered to undergo inducible senescence with or without DNA damage, and overexpression of either ARID4B isoform induced G1 arrest, demonstrating the chromodomain was dispensable for this activity.

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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.