Patent Grant
7494813
The invention relates to a VAC-BAC shuttle vector system for creation of recombinant poxviruses from DNA cloned in a bacterial artificial chromosome.
Poxviruses, which comprise a large family of double-stranded DNA viruses that infect vertebrate and invertebrate hosts, are distinguished by their large size, complex morphology, cytoplasmic site of replication, and encoding of proteins for viral transcription, replication, and immune defense (Moss, B. 2001 in: Fields Virology, eds. Knipe, D. M. & Howley, P. M. Lippincott Williams & Wilkins, Philadelphia, Vol. 2, pp. 2849-2883). Vaccinia virus (VAC), the best-studied member of the poxvirus family, was used for immunization against smallpox (Fenner, F. et al. 1988 Smallpox and its Eradication World Health Organization, Geneva) and subsequently as an expression vector for laboratory investigations and recombinant vaccines (Moss, B. 1996 PNAS USA 93:11341-11348). The introduction of new genes into the VAC genome is usually carried out by homologous recombination in infected cells. Typically, a foreign gene is cloned into a plasmid transfer vector downstream of a VAC promoter and flanked by VAC DNA from a non-essential site (Mackett, M. et al 1984 J Virol 49:857-864). The plasmid is then transfected into cells that are infected by VAC. Because recombination is inefficient, a variety of selection and screening methods have been devised (Earl, P. L. et al. 1998 in: Current Protocols in Molecular Biology, eds. Ausubel, F. M. et al. Greene Publishing Associates & Wiley Interscience, New York, Vol. 2, pp. 16.17.1-16.17.19). The process usually requires three to five plaque purifications over a period of several weeks. Alternative methods, involving the purification and cleavage of the VAC genome and either ligation in vitro or three-way recombination in vivo, have been described (Merchlinsky, M. & Moss, B. 1992 Virology 190:522-526; Scheiflinger, F. et al. 1992 PNAS USA 89:9977-9981; Merchlinsky, M. et al. 1997 Virology 238:444-451; Smith, E. S. et al. 2001 Nat Med 7:967-972; Timiryasova, T. M. et al. 2001 Biotechniques 31:534-540) but are cumbersome, depend on the presence of a unique restriction endonuclease site within the nearly 200,000 base pair genome, and still require plaque purification. The mutagenesis of VAC genes is even more difficult, and is usually carried out by a laborious transient dominant selection recombination scheme (Falkner, F. G. & Moss, B. 1990 J Virol 64:3108-3111).
Recently, the large DNA genomes of a baculovirus (Luckow, V. A. et al. 1993 J Virol 67:4566-4579) and several herpesviruses (Messerle, M. et al. 1997 PNAS USA 94:14759-14763; Borst, E. M. et al. 1999 J Virol 73:8320-8329; Delecluse, H. J. et al. 1998 PNAS USA 95:8245-8250; Horsburgh, B. C. et al. 1999 Gene Ther 6:922-930; Saeki, Y. et al. 1998 Hum Gene Ther 9:2787-2794; Smith, G. A. & Enquist, L. W. 1999 J Virol 73:6405-6414) have been cloned as bacterial artificial chromosomes (BACs). These circular mini-F BAC plasmids allow viral genomes to be stably maintained at low copy number and manipulated in Escherichia coli and then reconstituted as infectious virus by transfection of eukaryotic cells. The construction of baculovirus and herpesvirus BACs was relatively straightforward: plasmid sequences were inserted by recombination into the circular mature or replicating viral genomes and the latter were propagated in E. coli. Poxvirus genomes, however, are composed of linear double-stranded DNA molecules with covalently closed hairpin ends (Baroudy, B. M. et al. 1982 Cell 28:315-324) that are resolved from transient head-to-head or tail-to-tail concatemers during replication (Moyer, R. W. & Graves, R. L. 1981 Cell 27:391-401; Baroudy, B. M. et al. 1982 Cold Spring Harbor Symp Quant Biol 47:723-729; Merchlinsky, M. et al. 1988 J Mol Biol 199:399-413). Therefore, the method used for cloning baculovirus and herpesvirus genomes seemed inapplicable.
Circular bacterial plasmids that contain the poxvirus concatemer junction sequence are resolved into linear minichromosomes when transfected into poxvirus-infected cells (Merchlinsky, M. & Moss, B. 1986 Cell 45:879-884; Delange, A. M. et al. 1986 J Virol 59:249-259). It seemed likely to us that a much larger plasmid containing the entire poxvirus genome and concatemer junction would also be resolved. Although it may be possible to assemble such a 200,000 bp plasmid by in vitro methods, we chose an alternative approach that depended on an observation made several years ago. It was found that poxvirus DNA concatemers accumulated under conditions in which viral late protein synthesis was specifically inhibited and that recombination occurred so that about half of the concatemers were in the head-to-tail configuration (Merchlinsky, M. & Moss, B. 1989 J Virol 63:1595-1603; DeLange, A. M. 1989 J Virol 63:2437-2444). The latter configuration was crucial because a circle formed from a head-to-tail concatemer would contain the entire genome, whereas a circle formed from head-to-head or tail-to-tail concatemers would not. In principal, head-to-tail concatemers might undergo further recombination to form circles, although the latter have not been described. Alternatively, the highly efficient bacteriophage Cre-loxP recombinase system (Hoess, R. et al. 1984 Cold Spring Harb Symp Quant Biol 49:761-768) could be used to specifically enhance circularization. A loxP site is composed of a 34 bp sequence consisting of a core spacer of 8 bp and two 13 bp palindromic flanking sequences. When two cis loxP sites are in the same orientation, the DNA between them can be excised and circularized by Cre. One strategy was to clone the loxP sites so that they would be in the same orientation in head-to-tail concatemers but opposite for head-to-head or tail-to-tail concatemers. Here, we describe the formation and isolation of circular plasmids containing the full length VAC genome with an intact concatemer junction sequence. These VAC-BACs were stably propagated in E. coli and converted into infectious virus in mammalian cells. VAC-BACs allow the generation of mutant or recombinant viral genomes in bacteria, without need for recombination or plaque purification in mammalian cells.
The invention relates to a VAC-BAC shuttle vector system for creation of recombinant poxviruses from DNA cloned in a bacterial artificial chromosome.
A VAC-BAC is a bacterial artificial chromosome (BAC) containing a vaccinia virus genome (VAC) that can replicate in bacteria and produce infectious virus in mammalian cells.
What are the uses of a VAC-BAC?
What are the advantages of a VAC-BAC?
The ability to manipulate the vaccinia virus (VAC) genome, as a plasmid in bacteria, would greatly facilitate genetic studies and provide a powerful alternative method of making recombinant viruses. VAC, like other poxviruses, has a linear, double-stranded DNA genome with covalently closed hairpin ends that are resolved from transient head-to-head and tail-to-tail concatemers during replication in the cytoplasm of infected cells. Our strategy to construct a nearly 200,000 base pair VAC-bacterial artificial chromosome (BAC) was based on circularization of head-to-tail concatemers of VAC DNA. Cells were infected with a recombinant VAC containing inserted sequences for plasmid replication and maintenance in Escherichia coli; DNA concatemer resolution was inhibited leading to formation and accumulation of head-to-tail concatemers, in addition to the usual head-to-head and tail-to-tail forms; the concatemers were circularized by homologous or Cre-loxP mediated recombination; and E. coli were transformed with DNA from the infected cell lysates. Stable plasmids containing the entire VAC genome, with an intact concatemer junction sequence, were identified. Rescue of infectious VAC was consistently achieved by transfecting the VAC-BAC plasmids into mammalian cells that were infected with a helper non-replicating fowlpox virus.
VAC-BAC shuttle vectors are envisioned as supplanting other methods of making genetically engineered or recombinant poxviruses, which are used in hundreds of academic and commercial laboratories. Moreover the simplicity and rapidity of the new procedure is envisioned as greatly expanding the use of poxvirus expression vectors. The VAC-BAC vector is envisioned as being modified to allow expression by vaccinia virus of foreign genes inserted into the VAC-BAC by site-specific recombination or transposition. In one embodiment, the foreign gene is envisioned as encoding a therapeutic or immunogenic gene product. In another embodiment, the therapeutic or immunogenic gene product is envisioned as being a member selected from the group consisting of a growth factor, hormone, enzyme, vaccine antigen, cytotoxin, immunomodulatory protein, antisense RNA molecule, small interfering RNA, and ribozyme. The efficiency of the system is envisioned as allowing the screening of entire cDNA libraries for use in proteomics and to identify cancer-specific CTL epitopes. In addition, because genetic engineering can be done entirely in bacteria and no selection or screening is required for isolation of recombinant viruses, the procedure is well adapted for commercial companies to make clinical grade materials under good manufacturing practices. We anticipate a commercial kit including competent E. coli containing the VAC-BAC shuttle vector, a donor plasmid, and a helper avian poxvirus.
The process consisted of (i) inserting mini-F plasmid, loxP and GFP sequences into the VAC genome by homologous recombination, (ii) infecting cells with the recombinant VAC and promoting the accumulation of head-to-tail concatemers by inhibiting viral late protein synthesis (Merchlinsky, M. & Moss, B. 1989 J Virol 63:1595-1603; DeLange, A. M. 1989 J Virol 63:2437-2444) using ts21, a temperature-sensitive VAC with a mutation in the RPO18 subunit of RNA polymerase (Seto, J. et al. 1987 Virology 160:110-119; Ahn, B. -Y. et al. 1990 J Virol 64:3019-3024), or by adding isatin-β-thiosemicarbazone (IBT) (Woodson, B. & Joklik, W. K. 1965 PNAS USA 54:946-953; Pennington, T. H. 1977 J Gen Virol 35:567-571), (iii) allowing genome circularization to occur by homologous or Cre-mediated recombination, and (iv) transforming E. coli. The TK site was chosen for insertion of DNA sequences because it is non-essential and has been used extensively for this purpose (Mackett, M. et al. 1984 J Virol 49:857-864).
The terms “sequence-specific recombinase” and “site-specific recombinase” refer to enzymes that recognize and bind to a short nucleic acid site or sequence and catalyze the recombination of nucleic acid in relation to these sites.
The term “recombination site” refers to a short nucleic acid site or sequence which is recognized by a sequence- or site-specific recombinase and which become the crossover regions during the site-specific recombination event. Examples of such recombination sites include, but are not limited to, lox sites, frt sites, att sites and dif sites.
The term “lox site” as used herein refers to a nucleotide sequence at which the product of the cre gene of bacteriophage P1, Cre recombinase, can catalyze a site-specific recombination. A variety of lox sites are known to the art including the naturally occurring loxP (the sequence found in the P1 genome), loxB, loxL and loxR (these are found in the E. coli chromosome) as well as a number of mutant or variant lox sites such as loxP511, loxΔ86, loxΔ117, loxC2, loxP2, loxP3 and loxP23.
A variety of other site-specific recombinases may be employed in the methods of the present invention in place of the Cre recombinase. Alternative site-specific recombinases include:
A multi-step procedure was used for construction of the plasmid transfer vector pMBO1374-loxP-GFP-loxP-TKRL, which contains a split VAC TK ORF with the two halves in reverse orientation and a GFP expression cassette flanked by loxP sites (
Initial efforts to make a VAC-BAC were carried out using ts21-loxP-GFP-BAC. The accumulation of concatemeric DNA in cells infected with ts21-loxP-GFP-BAC at the non-permissive temperature of 40° C. was demonstrated by pulsed field gel electrophoresis and by detection of concatemer junctions by restriction enzyme analysis and DNA hybridization. To enhance circularization of the VAC genome, a Cre expression plasmid was transfected into cells prior to infecting them with ts21-loxP-GFP-BAC. The GFP cassette, which is flanked by similarly oriented loxP sites should be efficiently excised leaving a single loxP site in each VAC unit genome. A unit length genome flanked by loxP sites in the same orientation would also be excised from the concatemer, whereas DNA inversion would occur between oppositely oriented loxP sites. As seen in
E. coli were electroporated with DNA from the lysates of cells that had been infected with ts21-loxP-GFP-BAC and then plated on chloramphenicol selective medium. Bacterial colonies were picked, transferred to duplicate membranes, and hybridized with 32P-labeled probes containing sequences from the terminal HindIII C or B fragments of the VAC genome. Of 117 colonies analyzed, DNA from six (#5, 13, 22, 34, 51, and 59) reacted with both probes, one (#16) reacted with the C probe alone, and none reacted with the B probe alone (
As diagrammed in
Evidence that the concatemer junction links the C and B sequences was obtained by analyzing the digestion products of BstEII, a restriction enzyme that cuts near the hairpin ends of the mature genome. As depicted in
To determine whether the VAC-BACs contained full-length viral genomes, five were digested with ApaI restriction enzyme, which only cuts within the BAC sequence, and analyzed by pulsed-field gel electrophoresis. As seen in
DNA stability was determined for five clones during four successive overnight cultures. The HindIII restriction patterns of DNA isolated from the first and the fourth culture were identical in each case (
The next step was reconstitution of infectious virus from the VAC-BACs. Poxviruses replicate and transcribe their genomes in the cytoplasm, rather than the nucleus of infected cells. To accomplish this, poxviruses encode a unique transcription system that includes a multi-subunit DNA-dependent RNA polymerase, stage-specific promoters and cognate transcription factors (Moss, B. 2001 in: Fields Virology, eds. Knipe, D. M. & Howley, P. M. Lippincott Williams & Wilkins, Philadelphia, Vol. 2, pp. 2849-2883). The viral RNA polymerase and early transcription factors are packaged within infectious particles in order to begin a new infection. Therefore, an infection cannot be initiated with viral DNA alone and consequently a helper virus that provides early functions must be provided. For example, rabbitpox virus was recovered from rabbitpox DNA when the transfected cells were infected with a temperature-sensitive mutant of ectromelia virus (Sam, C. K. & Dumbell, K. R. 1981 Ann Virol 132E:135-150). Similarly, wild-type VAC was recovered when VAC DNA was transfected into cells that were infected with a temperature-sensitive mutant of VAC (Merchlinsky, M. & Moss, B. 1992 Virology 190:522-526) or the genetically distant fowlpox virus, which cannot replicate in mammalian cells or undergo recombination with VAC (Scheiflinger, F. et al. 1992 PNAS USA 89:9977-9981).
The previous rescue experiments were done with linear viral genomes with hairpin ends. Based on studies with cloned concatemer junctions (Merchlinsky, M. & Moss, B. 1986 Cell 45:879-884; Delange, A. M. et al. 1986 J Virol 59:249-259), however, we thought that the VAC-BACs would be replicated and resolved into linear genomes. CV-1 cells were infected with a low multiplicity of fowlpox virus, transfected with VAC-BAC DNA, and incubated for 4 to 8 days at 31° C. The recovery of infectious virus was determined by plaque assay on BS-C-1 cells, which like CV-1 cells are non-permissive for fowlpox virus. Comparable amounts of infectious virus were recovered from each of the VAC-BAC clones, whereas no virus was detected when either fowlpox virus or VAC-BAC was omitted (Table 1). As expected from the analysis of the parental plasmid, virus derived from clone 22 expressed GFP. The Hind III restriction enzyme digestion patterns of the virus clones were similar to each other and no fowlpox virus DNA was detected by Southern blotting. Thus, a poxvirus genome was successfully cloned and replicated as a bacterial plasmid and rescued as infectious virus.
In principal, circular genomes could also arise by homologous recombination of head-tail-concatemers either in the infected cells or in the bacteria after transfection. To evaluate this possibility, cells were transfected with ts21-loxP-GFP-BAC without the Cre-expressing plasmid. Under these conditions, one of 100 plasmids analyzed contained a complete VAC genome capable of producing infectious virus.
The VAC-BACs described thus far contain the ts21 mutation. Although this mutation could be eliminated by homologous recombination, we succeeded in producing VAC-BACs starting from the wild-type WR genome. A similar strategy was employed except that isatin-β-thiosemicarbazone (IBT) was used to accumulate head-to-tail concatemers.
We also attempted to make VAC-BACs without preventing concatemer resolution, with the thought that low amounts of head-to-tail concatemers might occur naturally. Several hundred plasmid clones were analyzed and some with nearly complete genomes were identified, leading us to believe that complete genomes might be recovered after a more extensive search. Thus, while Cre-mediated recombination and inhibition of concatemer resolution increase the efficiency of VAC genome circularization, they are not essential for producing VAC-BACs.
Poxvirus genomes consist of linear, double-stranded DNA molecules with covalently closed hairpin ends that do not undergo circularization during replication. Therefore, the straightforward method successfully used for cloning baculovirus and herpesvirus genomes as BACs (Borst, E. M. et al. 1999 J Virol 73:8320-8329; Delecluse, H. J. et al. 1998 PNAS USA 95:8245-8250; Horsburgh, B. C. et al. 1999 Gene Ther 6:922-930; Messerle, M. et al. 1997 PNAS USA 94:14759-14763; Saeki, Y. et al. 1998 Hum Gene Ther 9:2787-2794; Smith, G. A. and L. W. Enquist 1999 J Virol 73:6405-6414) was inapplicable. We knew, however, that circular bacterial plasmids that contain the poxvirus concatemer junction sequence are resolved into linear minichromosomes when transfected into poxvirus-infected cells (Delange, A. M. et al. 1986 J Virol 59:249-259; Merchlinsky, M. and B. Moss 1986 Cell 45:879-884). Therefore, we predicted that if an entire poxvirus genome were cloned as a circular plasmid with the concatemer junction sequence preserved, it would be resolved into a complete linear genome. The question, then, was how to construct such a 200,000 bp plasmid. Although it seemed possible to assemble this by in vitro methods starting with a plasmid containing a small concatemer junction, we decided on an alternative approach that depended on a curious observation made many years ago (DeLange, A. M. 1989 J Virol 63:2437-2444; Merchlinsky, M. and B. Moss 1989 J Virol 63:1595-1603). It was found that poxvirus DNA concatemers, which form as transient head-to-head and tail-to-tail genomes, accumulated under conditions in which viral late protein synthesis was inhibited (by use of specific temperature-sensitive mutants or with the drug isatin-β-thiosemicarbazone) and that the concatemers underwent recombination so that about half were in the head-to-tail configuration. This observation was crucial, because a circle formed from a head-to-tail concatemer would contain the entire genome, whereas a circle formed from head-to-head or tail-to-tail concatemers would not. The next question, then, was how to circularize the concatemer. One way would be to cleave the concatemer at a unique restriction site in vitro and ligate the ends together. However, we devised an alternative that specifically circularized head-to-tail recombinants and could be performed in vivo. This method took advantage of the efficiency and directionality of the Cre-loxP recombinase system. A loxP site is composed of a 34 bp sequence consisting of a core spacer of 8 bp and two 13 bp palindromic flanking sequences. When two cis loxP sites are in the same orientation, the DNA between them is excised and circularized; when the two sites are opposite, inversion occurs instead of excision. Our strategy then, was to clone the loxP sites so that they would be in the same orientation in head-to-tail concatemers but opposite for head-to-head or tail-to-tail concatemers. The system worked as planned and several clones containing the entire VAC genome were isolated and characterized. Importantly, the VAC-BACs were stable in E. coli after repeated cultures.
The next step was to reconstitute infectious virus from the VAC-BACs. Poxviruses replicate and transcribe their genomes in the cytoplasm, rather than the nucleus of infected cells. To accomplish this, poxviruses encode a unique transcription system that includes a multisubunit DNA-dependent RNA polymerase, stage-specific promoters and cognate transcription factors (Moss, B. 2001 in: D. M. Knipe and P. M. Howley (ed.), Fields Virology 4th ed., vol. 2 Lippincott Williams & Wilkins, Philadelphia p. 2849-2883). The viral RNA polymerase and early transcription factors are packaged within infectious particles in order to initiate a new infection. Therefore, an infection cannot be initiated with naked viral DNA. However, Sam and Dumbell (Sam, C. K., and K. R. Dumbell 1981 Ann Virol 132E:135-150) demonstrated that when DNA from rabbitpox virus was introduced into cells infected with a temperature-sensitive mutant of ectromelia virus, rabbitpox virus was recovered. Similarly, when VAC DNA was transfected into cells that were infected with a temperature-sensitive mutant of VAC, wild type virus was recovered (Merchlinsky, M. and B. Moss 1992 Virology 190:522-526). Scheiflinger et al. (Scheiflinger, F. et al. 1992 PNAS USA 89:9977-998) extended this technique by demonstrating that fowlpox virus rescued VAC DNA in mammalian cells that are non-permissive for fowlpox virus. Only VAC was recovered and recombination did not occur between the fowlpox virus and VAC genomes because of their sequence diversity. Here we showed that fowlpox virus could also rescue a VAC-BAC. The procedure consists of simply infecting mammalian cells with fowlpox virus (or another poxvirus that has restricted growth in mammalian cells) and transfecting the cells with the VAC-BAC. After several days, the cells can be harvested and infectious virus recovered without need for plaque purification.
The plasmid sequences can be inserted at any non-essential site in the VAC genome. Here we used the TK locus because it has been extensively used as a recombination site, disruption does not impair virus replication in tissue culture, and homologous recombination with a TK DNA fragment could be used to remove the insert. However, disruption of the TK gene does reduce the pathogenicity of VAC (Buller, R. M. L. et al. 1985 Nature 317:813-815) and alternative versions of the VAC-BAC system can use other sites of insertion and allow substitute procedures for excision of BAC sequences.
The ability to clone and propagate the entire VAC genome as a BAC makes it possible to modify or delete VAC genes or add foreign DNA using a variety of methods developed for bacterial systems. After completion of the DNA manipulation, infectious virus can be isolated without plaque isolation, allowing high throughput procedures. We envision the procedures used to construct VAC-BACs as being applied with minor modifications to other poxviruses, African swine virus, and additional linear double-stranded genomes with hairpin termini.
The ability to propagate the entire VAC genome as a BAC, makes it possible to modify or delete VAC genes or add foreign DNA using a variety of methods developed for bacterial systems (Lee, E. C. et al. 2001 Genomics 73:56-65). Moreover, by eliminating the need for plaque purification, production of recombinant viruses for library screening or other high throughput purposes should be facilitated.
Monolayers of BS-C-1 and CV-1 African green monkey cells, and human A543 cells were grown in Dulbecco's modified Eagle medium or Eagle minimal medium supplemented with 5 or 10% fetal calf serum (GIBCO). The VAC strain WR ts21 mutant (Seto, J. et al. 1987 Virology 160:110-119) was prepared by infecting confluent BS-C-1 monolayers at 31° C. with 0.01 plaque-forming units (PFU) of virus per cell. The attenuated fowlpox virus HP1.441 (Mayr, A. & Malicki, K. 1966 Zentralbl Veterinarmed [B] 13:1-13) was provided by G. Sutter and grown in chick embryo fibroblasts.
The mini-F BAC plasmid pMBO1374 (O'Connor, M. et al. 1989 Science 244:1307-1312), a derivative of pMBO131 (O'Gorman, S. et al. 1997 PNAS USA 94:14602-14607), was provided by G. A. Smith. A DNA fragment containing NotI-loxP-BglII-HaeIII-SacII sites was formed by annealing synthetic oligonucleotides 5′ataggcatGCGGCCGCATAACTTCGTATAATGTATGCTATACGAAGTTATAGATCT GGCCCCGCGGgga (SEQ ID NO: 1) and 5′tccCCGCGGGGCCAGATCTATAACTTCGTATAGCATACATTATACGAAGTTATG CGGCCGCatgcctat (SEQ ID NO: 2) (sequences in bold and lower case letters are restriction endonuclease sites and additional bases, respectively), digested with SacII and NotI, and ligated to SacII and NotI cleaved pMBO1374 resulting in pMBO1374-loxP.
Left (531 bp) and right (263 bp) segments of the vaccinia virus thymidine kinase (TK) gene were amplified by polymerase chain reaction (PCR) using the primer pairs 5′acatGCATGCATGAACGGCGGACATATTCAGTTGATAATCGGCCCC (SEQ ID NO: 3) (L1-SphI) and 5′cgcGGATCCCAACAATGTCTGGAAAGAACTGTCCTTCATCGATACCTATC (SEQ ID NO: 4) (L2-BamHI) and the primer pairs 5′agcatGGATCCAATTCTGTGAGCGTATGGCAAACGAAGGAAAAATAG (SEQ ID NO: 5) (R1-BamHI) and 5′gggGCATGCTGAGTCGATGTAACACTTTCTACACACCGAT (SEQ ID NO: 6) (R2-SphI), respectively. The two fragments were digested with SphI and ligated together to form a 543 bp DNA in which the orientation of the left and right TK segments was reversed. This TKRL fragment was gel purified, digested with BamHI and then ligated to BamHI-digested and shrimp alkaline phosphatase-treated pMBO1374-loxP to form pMBO1374-loxP-TKRL (
The ORF encoding enhanced green fluorescent protein (GFP) was amplified by PCR from pEGFP-N1 (Clontech) using 5′tgaGTCGACATGGTGAGCAAGGGCGAGGAGCTGTTCA (SEQ ID NO: 7) and 5′ataggcatGCGGCCGCCCGCGGTTACTTGTACAGCTCGTCCATGCCGAGAGTGATC C (SEQ ID NO: 8) primers, digested with SalI and NotI and gel purified. A three-way ligation was carried out with the latter fragment, the SacII and NotI digested NotI-loxP-BglII-HaeIII-SacII DNA, and SalI and NotI digested pSC11-S-B-A-K-N (Earl, P. L. et al 1998 in: Current Protocols in Molecular Biology, eds. Ausubel, F. M. et al. Greene Publishing Associates & Wiley Interscience, New York, Vol. 2, pp. 16.17.1-16.17.19) to form pSC11-GFP-loxP in which the VAC P7.5 promoter regulates GFP. The P7.5-GFP-loxP fragment was amplified by PCR from the latter plasmid using the primers: 5′ataggcatGCGGCCGCCACTAATTCCAAACCCACCCGCTTTTTATAGTAAGT (SEQ ID NO: 9) and 5′ataggcatGCGGCCGCATAACTTCGTATAATGTATG (SEQ ID NO: 10). The PCR product was digested with NotI and inserted in the unique NotI site of pMBO1374-loxP-TKRL to form pMBO1374-loxP-GFP-loxP-TKRL (
The pOG231 expression plasmid, containing Cre fused to a nuclear localization signal (O'Gorman, S. et al. 1997 PNAS USA 94:14602-14607), was provided by G. A. Smith. The Cre-expression cassette, excluding the nuclear localization signal, was subcloned by PCR using the following primers: 5′ccgCTCGAGGCCACCATGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTC (SEQ ID NO: 11) and 5′tccCCCGGGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCC (SEQ ID NO: 12). The Cre cassette was inserted in XhoI- and SmaI-digested pCIneo (PROMEGA) to form pCI-Cre in which the Cre gene was under the control of a cytomegalovirus promoter.
All constructs were analyzed by restriction enzyme digestions and the inserted fragments and junctions were sequenced.
BS-C-1 cells in a six-well plate were infected with 0.01 plaque forming units (PFU) of VAC WR or ts21 per cell. At 2 h after infection, the cells were transfected with 10 μl of LIPOFECTAMINE (Life Technology) and 2-3 μg of pMBO1374-loxP-GFP-loxP-TKRL that had been cut once with SphI. After 48 h at 37° C. for WR or the permissive temperature of 31° C. for the ts mutant, the cells were harvested and the virus was released by three freeze-thaw cycles. TK− recombinant VAC was selected in TK− A543 cells in the presence of 25 μg of bromodeoxyuridine per ml of medium. Plaques that exhibited green fluorescence were picked at 48 h after infection and purified by 5 cycles of plaque-isolation. The DNA of the recombinant VAC was analyzed by PCR and by digestion with the HindIII restriction enzyme.
BS-C-1 cells in a six-well plate were infected at a multiplicity of 3 PFU of ts21 or ts21-loxP-GFP-BAC and incubated for 24 h at 31° C. or 40° C. The cells were harvested, recovered by low-speed centrifugation (1,000×g for 5 min at 4° C.) and 2 million cells were embedded per agarose plug. The DNA was extracted using CHEF Genomic DNA Plug Kit (BIO-RAD). Slices from the agarose plugs were sealed in 1% agarose and analyzed by pulsed-field electrophoresis in 1% agarose (pulsed-field certified agarose, BIO-RAD) in 0.5×TBE (BIO-RAD) at 4-5 V/cm for 20-22 h, with a switch time of 50 and 90 s.
In some experiments, the plugs were incubated with 50 units of HindIII in 0.5 ml of digestion buffer for 12 h at 37° C. The buffer was replaced with 0.5 ml of fresh digestion buffer and 50 units of HindIII for 8 h more at 37° C. After electrophoresis, the gel was stained with ethidium bromide, photographed, exposed to UV light for 5 min, transferred to a nylon membrane and hybridized to 32P labeled probes containing part of the VAC HindIII B, C or A fragment.
Infected cells from an individual well of a 6-well plate were harvested, collected by low-speed centrifugation, rinsed 2-3 times with phosphate buffered saline, suspended in 50 μl of 0.15 M NaCl-0.02 M Tris-HCl (pH 8.0)-0.01 M EDTA buffer, followed by addition of 250 μl of 0.02 M Tris-HCl (pH 8.0)-0.01 M EDTA-0.75% SDS containing 0.65 mg of proteinase K per ml. After incubation for 10-12 h at 37° C., the samples were extracted twice with 1 volume of phenol, once with phenol-chloroform and once with chloroform, precipitated with ethanol and the DNA was dissolved in 20 μl of Tris-EDTA.
DNA was digested with BstEII, subjected to electrophoresis through a 1% agarose gel, and analyzed by Southern blotting using a 32P-labeled probe containing the terminal 70 bp repeat sequence.
BS-C-1 cells in individual wells of a six-well plate were transfected with 2 μg of pCI-Cre and 10 μl of LIPOFECTAMINE (Life Technology) and incubated at 37° C. At 24 h after transfection, the cells were infected with 5 PFU per cell of ts21-loxP-GFP-BAC and incubated for 24 h at 40° C. Alternatively, cells were infected with WR-loxP-GFP-BAC and incubated at 37° C. in the presence of 45 μM of isatin-β-thiosemicarbazone (IBT) per ml. The cells were harvested by scraping and DNA was extracted as described above, dissolved in 20 μl of Tris-EDTA, and used to transform E. coli DH10B electrocompetent cells (GIBCO BRL) in 0.1 cm cuvettes with the Gene Pulser electroporation system (BIO-RAD) at 1.8 kV, 200Ω and 25 μF. Bacteria were recovered in 1 ml of SOC medium (GIBCO) at 37° C. for 1 h, plated on LB-agar containing 50 μg of chloramphenicol and incubated overnight at 37° C. DNA was isolated by the alkaline lysis method and in some cases was purified by polyethylene glycol precipitation.
CV-1 cells in a 12-well plate were infected with fowlpox virus (0.005 PFU per cell), transfected with 2 μg of VAC-BAC DNA, and incubated for 4 to 8 days at 31° C.
In some circumstances, it may be advantageous to construct a BAC without the DNA sequences corresponding to the ends of the poxvirus genome. One way in which this can be accomplished is by constructing a recombinant poxvirus that has sequences for plasmid replication in E. coli in a non-essential site and a lox site introduced near each end of the poxvirus genome. Cells that express the Cre recombinase are infected with the recombinant poxvirus and the DNA is isolated and used to transfect E. coli. The circular BAC will then lack the concatemer junction sequence. Therefore, a helper plasmid is also constructed that contains the concatemer junction and additional flanking sequences. The procedure for making the infectious poxvirus using a helper non-replicating virus is then modified by co-transfecting the helper plasmid and the VAC-BAC into mammalian cells where homologous recombination between sequences of the helper plasmid and VAC-BAC can occur. When appropriately designed, the lox sites will be removed during the recombination.
While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incorporated by reference.
This application is a continuation and claims the benefit of priority of International Application No. PCT/US2003/011183 filed Apr. 10, 2003, designating the United States of America and published in English on Oct. 23, 2003, as WO 03/087330, which claims the benefit of priority of U.S. Provisional Application No. 60/371,840 filed Apr. 10, 2002 and U.S. Provisional Application No. 60/402,824 filed Aug. 9, 2002, all of which are hereby expressly incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 20050124070 A1 | Jun 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60402824 | Aug 2002 | US | |
| 60371840 | Apr 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US03/11183 | Apr 2003 | US |
| Child | 10959392 | US |
