With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector ofPCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector. Vectors were optimized for high-throughput applications by making them “self-excising” through incorporating the I-SceI homing endonuclease into the vector, removing the need to linearize vectors prior to transfection into packaging cells. AdZvectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor, thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

Introduction

While replication-deficient adenovirus (Ad) vectors are used extensively as gene delivery vehicles in the research environment to analyze gene function, they are increasingly being adopted in large scale studies of gene function (1), drug discovery (www5.gelifesciences.com/aptrix/upp00919.nsf/Content/viralvectors_homepage), and as a delivery platform for RNAi (2). Clinically, Ad type 5 is the most commonly used vector in human gene therapy protocols. The human adenovirus 5 (Ad5) genome (∼36 kb) comprises a linear dsDNA, bracketed by inverted repeats. Genes are grouped into four early transcription units, numbered E1, E2, E3, and E4, as well as delayed early units and a major late unit (3). Ad vectors are rendered replication-deficient by deleting the essential E1 gene region, and must therefore be propagated on helper cells expressing E1 functions (4,5). The E3 region is nonessential for replication in vitro, and its additional excision allows the insertion of transgenes up to ∼8 kb. Additional sequences can be deleted from the Ad genomic backbone; however, this renders the vector substantially more difficult to produce and use in routine in vitro applications.

The Ad genome can readily be cloned and manipulated in Escherichia coli. A wide range of popular vector systems have been developed that are based on recombination between prokaryotic transfer vectors and the Ad genomic backbone in E. coli, or following plasmid co-transfection into E1-expressing cell lines (e.g., 293 or 911 cells) (6,7,8,9). Nevertheless, the procedure for generating Ad recombinants still remains relatively labor-intensive and poorly suited to cloning multiple genes simultaneously: sequential sub-cloning steps are required to configure transgenes into the transfer vector, while the recombination step required to insert the transgene into the Ad genomic backbone can be problematic. Finally, the recombinant virus will not be generated if transgene expression is not compatible with vector replication. We were motivated to radically redesign the Ad vector to overcome these shortcomings. To this end we have developed a vector compatible with recombineering technology.

The novel vector system, herein designated AdZ (Ad with zero cloning steps), utilizes recombination-mediated genetic engineering (recombineering) to allow for direct insertion of a transgene into the vector within E. coli with no requirement for a transfer vector. Recombineering makes use of temperature-controlled λ phage recombination functions in E. coli strain SW102 (10,11,12) to mediate homologous recombination between DNA elements with as few as 30 bp of homology. Thus, overlapping homology with vector sequences can readily be incorporated into PCR primers, annealed double-stranded oligonucleotides or custom synthesized genes such that the transgene is inserted directly and in the desired orientation into the vector. The DNA element encoding the transgene is introduced by electropo-ration into recombineering bacteria that already carrying a bacterial artificial chromosome (BAC) containing the AdZ vector. The AdZ vector was further optimized to provide an extremely robust technology suited to the simultaneous construction of large numbers of recombinant Ads (RAds).

Full protocols for the use of the AdZ system are provided on our web site (AdZ.cf.ac.uk).

Materials and Methods

Cells

Cells were grown in DMEM (Gibco, Paisley, UK) containing 10% FCS. Ad recombinants were propagated in 911, 293, or 293TREx cells (Invitrogen, Paisley, UK) that express the tet repressor (4,13). HFFF-htert-hCAR are human fetal foreskin fibroblasts (HFFFs) that have been immortalized using htert (14), and engineered to express the human Coxsackie-adenovirus receptor (hCAR) using retrovirus derived from LX5N-hCAR (15), provided by J. DeGregori (University of Colorado Health Sciences Center, Denver, USA).

PCR

PCR products were amplified using the Expand Hi-Fi PCR system (Roche Applied Science, East Sussex, UK) according to manufacturer's instructions with 100 pmol of each primer (desalted purity) (Invitrogen). PCR products were gel purified with GFX DNA purification kit (GE Healthcare, Bucks, UK) and eluted in 30 µl ddH2O.

Plasmids

Plasmid pGalK (11) was supplied by N. Copeland (National Cancer Institute, Frederick, MD, USA) and expresses GalK. Plasmid pBeloBAC1 1 (New England Biolabs, Ipswitch, MA, USA) is a single copy vector expressing chloramphenicol resistance. The LoxP and cos sites were removed by digesting with HpaI and ApaLI, overhanging sequences filled in with the Klenow polymerase and religated to generate pAL767. pMV100 contains the strain AD 169 HCMV MIE promoter (-299 to +69) and polyadenylation sequence from the same gene (+2757 to +3053) (16). pCAG–I-SceI was a gift from M. Jasin (Memorial Sloan-Kettering Cancer Center, New York, NY, USA) and expresses the I-SceI ORF with NLS and HA tag (17). pShuttle and pAdEasy-1 are components of the AdEasy vector construction system and were obtained from B. Vogelstein (Johns Hopkins University, Baltimore, MD,USA)(18).