1 GenVec Inc., 65 West Watkins Mill Road, Gaithersburg, MD 20878, USA
2 KILA Consultants, LLC, 7713 Warbler Lane, Rockville, MD 20855-1033, USA
Correspondence
Duncan McVey
dmcvey{at}genvec.com
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ABSTRACT |
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Published ahead of print on 23 September 2003 as DOI 10.1099/vir.0.19446-0.
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INTRODUCTION |
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Adenovirus gene transfer methods provide advantages for the assay of gene function. Adenovirus vectors can efficiently transduce both quiescent and cycling cells, which, along with their broad tropism, make adenoviruses well suited as gene transfer vectors. The adenovirus genome can be readily modified and propagated in bacteria. The genomes produced can then be used in complementing cell lines to create virus (Berkner & Sharp, 1983; Hanahan & Gluzman, 1984
). In this study, we adapted the adenovirus genome construction and transfer methods to the challenge of generating gene libraries for the identification of gene function.
Human adenovirus type 5 (Ad5) is a 36 kb, double-stranded DNA virus which is widely used to express heterologous genes replacing the Ad5 E1 region. Deletion of the E1 region renders the virus replication deficient. The E1 region comprises the E1A and E1B transcription units (Berk & Sharp, 1977, 1978
; Chow et al., 1979
; Kitchingman et al., 1977
). E1A is the first region transcribed after virus infection to yield the alternatively spliced 12S and 13S transcripts (Berk et al., 1979
; Jones & Shenk, 1979
; Nevins, 1981
; Nevins et al., 1979
). Analysis of various mutant viruses revealed the 13S gene product is required and sufficient to complement all E1A functions necessary for productive virus infection in cell culture (Berk et al., 1979
; Jones & Shenk, 1979
; Montell et al., 1982
; Nevins, 1981
; Nevins et al., 1979
). In this study, we demonstrated that adenovirus libraries can be generated with a complexity of >1x105. Using a library screening strategy, this level of complexity is sufficient to confirm the complementing function of E1A 13S. This proof of principle experiment demonstrates that adenovirus vector libraries can be generated for the identification of genes with specific biological function.
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METHODS |
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Cosmid cDNA library construction.
A total of 20 150 mm plates with 50 000 plaques from the first expansion of the lambda cDNA library were eluted in SM buffer and DNA was isolated with a lambda genomic purification kit from Promega, following the manufacturer's instructions. Phage DNA was restricted with SwaI/SpeI, passed through a plasmid purification cartridge from Qiagen and ligated into XbaI/SwaI-restricted pAdRSV.XS, which had been pretreated with shrimp alkaline phosphatase (Boehringer Mannheim). The ligation mixture was packaged into phage heads using GIGA Pack Gold extract. XL-1 Blue cells were then transduced and plated on 150 mm agarose plates. Colonies were harvested after overnight incubation with LB and brought to 30 % glycerol before being snapfrozen on dry ice and stored at -80 °C. After freezing, the cosmid library contained 4x107 kanamycin-resistant c.f.u. ml-1. Next, 200 µg cosmid library DNA was isolated from an overnight culture grown in the presence of kanamycin at 30 °C after being inoculated with 1 ml of the cosmid library and purified using a Qiagen endotoxin-free DNA column.
Creation of the AdLibrary.
Cosmid library DNA was restricted with PacI, extracted with phenol and phenol/chloroform/isoamyl alcohol (25 : 25 : 1) before being precipitated with ethanol, dried and resuspended in pyrogen-free H2O. The DNA (5 µg) was brought to 100 mM CaCl2 with a final volume of 250 µl. Next, 250 µl 2xHBS buffer (250 mM NaCl, 50 mM HEPES, pH 7·4, and 1·5 mM NaHPO4) was quickly added, mixed and incubated at room temperature for 12 min before being added to a 60 mm plate containing 1x106 HEK 293 cells that had been incubated in DMEM with 5 % FCS 2 h before. At 4 h post-transfection, the cells were washed once with 1 ml 1 mM EGTA in HBS buffer (125 mM NaCl, 25 mM HEPES, pH 7·4, and 0·75 mM NaHPO4) and twice with 2ml DMEM containing 2 % FCS. Cells were then incubated in 5 ml DMEM containing 5 % FCS. After 5 days, the cells were freezethawed three times and 1 ml of lysate was used to infect a 10 cm plate containing HEK 293 cells (70 % confluent) for 1 h before being incubated with 5 ml DMEM supplemented with 5 % FCS. Four days later, CPE was observed and the cells were freezethawed three times to create the AdLibrary.
Selection of AdLibrary.
A 10 cm plate of A549 cells (70 % confluent) was infected with 1 ml AdLibrary lysate. At 5 days p.i., cells were harvested and freezethawed three times to create passage 1. Two additional passages were carried out to create passage 2 and 3 lysates, except that passage 3 was harvested at 4 days p.i.
Detection of active virus particles.
Six-well plates were seeded with 1·2x106 HEK 293 cells per well in DMEM supplemented with 5 % FCS. At 2026 h later, the cells were washed with 1x PBS, infected with 200 µl of serial dilutions from the virus lysates made with DMEM containing 5 % FCS. Virus was removed 1 h later and the cells were fed with 3 ml DMEM containing 5 % FCS. At 2224 h p.i., cells were washed with PBS and fixed with 2 ml icecold methanol for 15 min at room temperature and washed once more with PBS. A mouse antibody specific for adenovirus DNA-binding protein in 500 µl PBS was incubated with the cells for 1 h before being removed. Cells were subsequently washed with PBS before the addition of an FITC-conjugated anti-mouse antibody and incubation for 1 h in the dark. The number of UV-induced green fluorescent cells was determined under a microscope.
PCR analysis.
A 5 µl sample of lysate in a 100 µl reaction mixture was heated at 94 °C for 5 min. PCR was carried out using Taq polymerase, buffers and conditions (30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min) as suggested by the manufacturer (Boehringer Mannheim). The sequences for oligonucleotides 1 (Ad5 nt 278297), 2 (RSV promoter) and 3 (Ad5 nt 33493368) are 5'-CGCGGGAAAACTGAATAAGA-3', 5'-CGATTGGTGGAAGTAAGG-3' and 5'-CCTGGTGCGGGTCTCATCGTA-3', respectively.
Southern blot analysis for E1 sequences.
DNA was resolved on a 0·8 % agarose gel in 1x TBE buffer, transferred to a nylon membrane (Boehringer Mannheim) and UV cross-linked in a Stratagene Stratalinker 2400. Membranes were prehybridized with DIG Easy Hybridization buffer (Boehringer Mannheim) at 55 °C for 1 h. The membrane was then probed with a PCR product of Ad5 (nt 559798) labelled using the DIG Luminescent Detection kit (Boehringer Mannheim) (E1 probe) overnight at 55 °C. The membrane was washed three times at 55 °C in 0·1x SSC and 0·1 % SDS before being exposed to film and developed.
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RESULTS |
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AdLibrary construction
Construction of the cDNA AdLibrary proceeded through a three-step process. Initially, the cDNA generated from HeLa cells infected with wild-type Ad5 was cloned as a lambda library with a complexity of 2·5x105 p.f.u. Of these plaques, 0·4 % contained E1A sequences, as assessed by plaque-lift analysis. The cDNA used in creating the lambda cDNA library had a median size of 1·6 kb when resolved on an agarose gel (data not shown). The second step was to move the cDNA from the lambda library into the RSV expression cassette found in pAdRSV.XS to create the cosmid library. The resulting cosmid library had a total complexity of 4x105 c.f.u. Restriction analysis of cosmid DNA isolated from 18 random colonies showed them to all contain an insert. Finally, the cosmid library was converted to an AdLibrary in HEK 293 cells. This was accomplished by standard CaPO4 transfection techniques with Ad5 genomic DNA liberated from the cosmid backbone by PacI restriction. Analysis of the AdLibrary lysate revealed it to contain 4x107 active particles ml-1, as measured by a f.f.u. assay (Table 2).
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Characterization of E1 complementing activity
The DNA sequences with E1 complementing activity were identified by PCR isolation. Lysates from P1 and P3 were used as template with oligonucleotides designed to amplify all sequences present within the expression cassette. The primers used annealed to the Ad5 genome (Fig. 1, 1) and RSV promoter (Fig. 1, 2
). In the absence of a transgene, these two primers are 744 bp apart in the vector. As can be seen in Fig. 2
(A), a single prominent band of approximately 1·8 kb was generated from the P3 lysate, while no bands were observed from the P1 lysate. The insert is predicted to be near 1 kb in size. Although not quantitative, these data are consistent with the selection and expansion of Ad5 observed both phenotypically and by f.f.u. analysis. The PCR product was subjected to Southern blot analysis employing an E1A probe. The result identified the major band to have identity with the viral E1A region (Fig. 2B
) and was of a size predicted for the 13S splice variant. A second primer set using oligonucleotides 1 and 3 (positions shown in Fig. 1
) confirmed these results (data not shown). Direct sequencing of the PCR product identified it to be derived from the E1A 13S transcript (data not shown). The 5' and 3' junction of the cDNA mapped to Ad5 nt 522 and 1632, which compares favourably to previously mapped sites of transcription initiation and termination at positions 499 and 1633, respectively.
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DISCUSSION |
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Complementation of E1 function was the basis for selection in this study. A library in which the E1A and E1B regions of the virus were replaced with an expression cassette was assayed for virus growth on A549 cells. This cell line is known to contain an E1B complementing activity (Hay et al., 1999). The majority of isolates contained E1A 13S cDNA and a minority contained wild-type genomic E1 regions, presumably the result of recombination with the complementing cell during construction of the library. Plaque purification of the isolates confirmed E1A 13S to be sufficient to complement for all E1 functions in A549 cells.
When using screening methods, the probability of finding a gene that encodes a desired trait is directly related to the number of different genes or mutations available to test. The methods described in this study are applicable to the generation of highly complex libraries. Cosmid cloning was chosen as the method to generate the AdLibrary genomes before their conversion to virus because of its proven ability for use in library generation. The cosmid library used in this study had a complexity of 4x105 c.f.u. The cDNA used in constructing the cosmid library had E1A sequences with a representation of approximately 14x10-3. These results suggest that AdLibrary technology may be applicable to the isolation of genes that are represented at relatively low levels in a cDNA library.
Alternate genome configurations will be required for different AdLibrary gene discovery projects. To facilitate such constructions, we have generated two systems: a plasmid (D. E. Brough and others, unpublished data) and an ACE phage system (McVey et al., 2002) to modify the vector genome. In these systems and in those of other investigators (Chartier et al., 1996
; Crouzet et al., 1997
; Ketner et al., 1994
; Miyake et al., 1996
), the viral genome is modified and propagated in bacteria or yeast and genomes converted to virus when transduced into complementing cells. These construction technologies allow any region of the genome to be modified for library purposes. It is, therefore, possible to directly address in the vector design the particular attributes required for the biological assay.
AdLibraries may be applicable to many types of genetic selection, employing both gain and loss of function. In the field of virology and virus vector systems, it is anticipated that additional genes will be identified that will complement other virus growth defects, which will assist in cell line construction. Alteration of the virus coat for a novel tropism could be used to direct a therapeutic vector to a defined cell type. This would greatly enhance vector utility and safety for gene therapy. AdLibraries can also be applied to mammalian cell lines with well-defined loss of function mutations (Hollstein et al., 1991; Shapiro et al., 1995
). Such cell lines provide the opportunity to search for genes capable of complementing these defects. AdLibraries also have the potential for use in gain of function studies in which the generation of a biological phenotype is caused by gene expression. Examples of assays readily adaptable to AdLibrary screening are promoter activation assays, employing a marker gene such as
-lactamase, or a flow cytometry assay based on the detection of an extracellular receptor. All of these applications will result in the identification of genes by their function with no prior gene characterization required.
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NOTE ADDED IN PROOF |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Berk, A. J. & Sharp, P. A. (1978). Structure of the adenovirus 2 early mRNAs. Cell 14, 695711.[Medline]
Berk, A. J., Lee, F., Harrison, T., Williams, J. & Sharp, P. A. (1979). Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell 17, 935944.[Medline]
Berkner, K. L. & Sharp, P. A. (1983). Generation of adenovirus by transfection of plasmids. Nucleic Acids Res 11, 60036020.[Abstract]
Chartier, C., Degryse, E., Gantzer, M., Dieterle, A., Pavirani, A. & Mehtali, M. (1996). Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol 70, 48054810.[Abstract]
Chow, L. T., Broker, T. R. & Lewis, J. B. (1979). Complex splicing patterns of RNAs from the early regions of adenovirus-2. J Mol Biol 134, 265303.[Medline]
Crouzet, J., Naudin, L., Orsini, C. & 11 other authors (1997). Recombinational construction in Escherichia coli of infectious adenoviral genomes. Proc Natl Acad Sci U S A 94, 14141419.
Elahi, S. M., Oualikene, W., Naghdi, L., O'Connor-McCourt, M. & Massie, B. (2002). Adenovirus-based libraries: efficient generation of recombinant adenoviruses by positive selection with the adenovirus protease. Gene Ther 9, 12381246.[CrossRef][Medline]
Graham, F. L., Smiley, J., Russell, W. C. & Nairn, R. (1977). Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36, 5974.[Abstract]
Griffiths, A. D. & Duncan, A. R. (1998). Strategies for selection of antibodies by phage display. Curr Opin Biotechnol 9, 102108.[CrossRef][Medline]
Hanahan, D. & Gluzman, Y. (1984). Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome. Mol Cell Biol 4, 302309.[Medline]
Hatanaka, K., Ohnami, S., Yoshida, K. & 7 other authors (2003). A simple and efficient method for constructing an adenoviral cDNA expression library. Mol Ther 8, 158166.[CrossRef][Medline]
Hay, J. G., Shapiro, N., Sauthoff, H., Heitner, S., Phupakdi, W. & Rom, W. N. (1999). Targeting the replication of adenoviral gene therapy vectors to lung cancer cells: the importance of the adenoviral E1b-55kD gene. Hum Gene Ther 10, 579590.[CrossRef][Medline]
Hollstein, M., Sidransky, D., Vogelstein, B. & Harris, C. C. (1991). p53 mutations in human cancers. Science 253, 4953.[Medline]
Jones, N. & Shenk, T. (1979). An adenovirus type 5 early gene function regulates expression of other early viral genes. Proc Natl Acad Sci U S A 76, 36653669.[Abstract]
Ketner, G., Spencer, F., Tugendreich, S., Connelly, C. & Hieter, P. (1994). Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone. Proc Natl Acad Sci U S A 91, 61866190.[Abstract]
Kitamura, T. (1998). New experimental approaches in retrovirus-mediated expression screening. Int J Hematol 67, 351359.[CrossRef][Medline]
Kitchingman, G. R., Lai, S. P. & Westphal, H. (1977). Loop structures in hybrids of early RNA and the separated strands of adenovirus DNA. Proc Natl Acad Sci U S A 74, 43924395.[Abstract]
Lochmuller, H., Jani, A., Huard, J., Prescott, S., Simoneau, M., Massie, B., Karpati, G. & Acsadi, G. (1994). Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (E1+
E3) during multiple passages in 293 cells. Hum Gene Ther 5, 14851491.[Medline]
Mahlmann, S., McLaughlin, J., Afar, D. E., Mohr, R., Kay, R. J. & Witte, O. N. (1998). Dissection of signaling pathways and cloning of new signal transducers in tyrosine kinase-induced pathways by genetic selection. Leukemia 12, 18581865.[CrossRef][Medline]
McVey, D., Zuber, M., Ettyreddy, D., Brough, D. E. & Kovesdi, I. (2002). Rapid construction of adenoviral vectors by lambda phage genetics. J Virol 76, 36703677.
Michiels, F., van Es, H., van Rompaey, L. & 26 other authors (2002). Arrayed adenoviral expression libraries for functional screening. Nat Biotechnol 20, 11541157; erratum 21, 199.
Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Sato, Y., Takamori, K., Tokuda, C. & Saito, I. (1996). Efficient generation of recombinant adenoviruses using adenovirus DNAterminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci U S A 93, 13201324.
Montell, C., Fisher, E. F., Caruthers, M. H. & Berk, A. J. (1982). Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site. Nature 295, 380384.[Medline]
Nevins, J. R. (1981). Mechanism of activation of early viral transcription by the adenovirus E1A gene product. Cell 26, 213220.[Medline]
Nevins, J. R., Ginsberg, H. S., Blanchard, J. M., Wilson, M. C. & Darnell, J. E., Jr (1979). Regulation of the primary expression of the early adenovirus transcription units. J Virol 32, 727733.[Medline]
Shapiro, G. I., Edwards, C. D., Kobzik, L., Godleski, J., Richards, W., Sugarbaker, D. J. & Rollins, B. J. (1995). Reciprocal Rb inactivation and p16INK4 expression in primary lung cancers and cell lines. Cancer Res 55, 505509.[Abstract]
Silhavy, T. J., Berman, M. L. & Enquist, L. W. (1984). Experiments with Gene Fusions, pp. 303. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Toby, G. G. & Golemis, E. A. (2001). Using the yeast interaction trap and other two-hybrid-based approaches to study proteinprotein interactions. Methods 24, 201217.[CrossRef][Medline]
Walter, G., Konthur, Z. & Lehrach, H. (2001). High-throughput screening of surface displayed gene products. Comb Chem High Throughput Screen 4, 193205.[Medline]
Received 18 June 2003;
accepted 3 September 2003.
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