A simple and efficient method for constructing an adenoviral cDNA expression library

Abstract

cDNA expression cloning is a powerful method for the identification of genes that are able to confer a selectable phenotype on specific cell types. An adenovirus vector is characterized by several advantages over plasmid DNA and retroviral vector-mediated gene transfer, such as broad host range and high infectivity. However, an expression cloning protocol using the adenovirus vector has not been reported. We describe here a simple and efficient method for constructing adenovirus cDNA expression libraries based on Cre-lox-mediated in vitro recombination between adenoviral shuttle plasmid cDNA libraries and adenoviral genomic DNA tagged with terminal protein. In a model experiment, EGFP clones present at the frequency of 0.003% in the shuttle plasmid library could be efficiently converted to adenoviral vector in a 6-cm dish under optimized conditions, indicating that high-complexity libraries harboring low-abundance cDNAs can be produced. The efficiency of this system was demonstrated by the isolation of cDNA for CD2 (frequency less than 1 in 0.3 x 10(4) transcripts in T cells) from the human T cells. This effective and versatile method can facilitate the functional identification of genes for a variety of purposes.

Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob

“…The virus production efficiency was highly improved by optimizing several factors such as utilization of the adenoviral terminal protein in this library construction method, 25 and in fact, GFP-expressing foci in a 60-mm dish were too numerous to count (more than 1000) following the transfection of recombined DNA. Therefore, to estimate how many different peptides were displayed on these adenoviruses, we set up dilution experiments with two shuttle plasmid libraries as described: 25 the pBHIDCAR-GFP-lib were mixed with pBHIDCAR-lib at various ratios (1:300, 1:3 Â 10 3 , 1:1 Â 10 4 , 1:3 Â 10 4 , 1:3 Â 10 5 ), recombined with left hand of digested DNA-TPC and transfected into 293-38.HissFv.rec cells.…”

“…24,25 Based on that technology, we have developed a novel system for producing adenoviral libraries displaying a variety of peptides on the HI-loop of the fiber knob ( Figure 1a), and used the libraries to select an adenoviral vector with the high infectivity in target cells (Figure 1b). Our system may allow the selection of targeted vectors to any cell type of interest, and may have broad implications for the development of targeted therapies.…”

Robust functional gene validation by adenoviral vectors: one-step Escherichia coli-Derived Recombinant Adenoviral Genome construction

“…Many approaches have been described for generating recombinant adenoviruses (rAV), principally involving direct plasmid construction of recombinant adenoviral genomes, [4][5][6][7][8] or indirect construction (two-plasmid systems). 3,[9][10][11][12][13][14][15][16] In general, one-plasmid systems are limited by choice of suitable restriction sites and the difficulties encountered in manipulating such large plasmids (B50 kbp). Two-plasmid systems include approaches whereby a shuttle plasmid (or DNA fragment), containing an expression cassette, is combined with a second (larger) plasmid (or viral DNA fragment) containing the majority of the adenoviral coding regions by homologous recombination in Escherichia coli, 9-11 or in mammalian packaging cell lines.…”

“…3,[12][13][14][15] Cre-lox-mediated rAV plasmid assembly has also been described, in both mammalian cell lines 13 and E. coli. 16 Additionally, methods for construction of adenoviral genomes by homologous recombination in yeast and their subsequent modification by two-step gene replacement have been described. 17,18 Existing methods involving rAV plasmid construction in E. coli generally entail some form of screening to identify the correct rAV-containing plasmid.…”

“…3,14,19 Many of these approaches have been applied to the construction of adenoviral libraries containing populations of transgenes of interest. 3,7,8,[14][15][16] However, such approaches may require specific cell lines, 3,7,14 screening to identify the correct rAV, 7,15,16 or plasmid DNA modification by techniques not amenable to high-throughput formatting. 16 Additionally, such approaches may entail pooling rAV plasmids, and as such are not clonal, 8,16 and give rise to the possibility of undesirable rAV species arising from inter-rAV recombination.…”