Methods of directly selecting cells expressing inserts of interest Number:7,067,251 from the United States Patent and Trademark Office (PTO) owispatent The present invention relates to a high efficiency method of introducing DNA into linear DNA viruses such as poxvirus, a method of producing libraries in linear DNA viruses such as poxvirus, and methods of selecting polynucleotides of interest based on cell nonviability or other phenotypes.<H expressing, selecting, Methods, of, dire, 7067251.html, Patent, pto, 7067251

Title: Methods of directly selecting cells expressing inserts of interest

Abstract: The present invention relates to a high efficiency method of introducing DNA into linear DNA viruses such as poxvirus, a method of producing libraries in linear DNA viruses such as poxvirus, and methods of selecting polynucleotides of interest based on cell nonviability or other phenotypes.

Patent Number: 7,067,251 Issued on 06/27/2006 to Zauderer, et al.

Inventors: Zauderer; Maurice (Pittsford, NY); Smith; Ernest S. (Ontario, NY)
Assignee: University of Rochester (Rochester, NY)

Appl. No.: 818991

Filed: March 28, 2001

Current U.S. Class: 435/6 ; 435/320.1; 435/5; 435/69.1; 435/DIG.1; 435/DIG.14; 435/DIG.17; 435/DIG.4; 536/23.1; 536/23.5

Current International Class: C12Q 1/68 (20060101); C07H 21/04 (20060101); C12N 15/86 (20060101); C12N 15/87 (20060101)

Field of Search: 435/6,5,456,69.1,320.1,DIG.1,DIG.4,DIG.14,DIG.17,69.7,457,463,325 536/23.1,23.5,24.1,24.2 424/93.2,93.21,93.1

References Cited [Referenced By]

U.S. Patent Documents


4690915	September 1987	Rosenberg
5166057	November 1992	Palese et al.
5494807	February 1996	Paoletti et al.
5530096	June 1996	Wolfel et al.
5578473	November 1996	Palese et al.
5712115	January 1998	Hawkins et al.
5804382	September 1998	Sytkowski et al.
5843648	December 1998	Robbins et al.
5866383	February 1999	Moss et al.
5874560	February 1999	Kawakami et al.
6706477	March 2004	Zauderer
6800442	October 2004	Zauderer
6872518	March 2005	Zauderer
2002/0110543	August 2002	Chiocca et al.
2003/0124128	July 2003	Lillie et al.
2003/0133917	July 2003	Zauderer
2003/0194696	October 2003	Zauderer et al.

Foreign Patent Documents


19950385	Aug., 2000	DE
WO 95/33835	Dec., 1995	WO
WO 97/24438	Jul., 1997	WO
WO 97/26328	Jul., 1997	WO
WO 97/34143	Sep., 1997	WO
WO 99/30151	Jun., 1999	WO
WO 99/47643	Sep., 1999	WO
WO 99/55910	Nov., 1999	WO
WO 00/28016	May., 2000	WO
WO 01/72995	Oct., 2001	WO
WO 01/72995	Oct., 2001	WO
WO 02/053576	Jul., 2002	WO
WO 2004/037993	May., 2004	WO

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Primary Examiner: Ponnaluri; Padmashri
Attorney, Agent or Firm: Sterne, Kessler, Goldstein & Fox P.L.L.C.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Appl. No. 60/192,586, filed Mar. 28, 2000; U.S. Provisional Appl. No. 60/203,343, filed May 10, 2000; U.S. Provisional Appl. No. 60/263,226, filed Jan. 23, 2001; and U.S. Provisional Appl. No. 60/271,426, filed Feb. 27, 2001; each disclosure of which is herein incorporated by reference in its entirety.

Claims

What is claimed is:

1. A method of selecting a target polynucleotide, comprising: (a) introducing into a population of mammalian host cells a library of insert polynucleotides; wherein at least one of said insert polynucleotides comprises the target polynucleotide; wherein said library is constructed in a vaccinia virus vector using trimolecular recombination and wherein expression of said target polynucleotide directly or indirectly promotes host cell death; (b) culturing said host cells under conditions such that said insert polynucleotides are expressed; and (c) collecting insert polynucleotides from those host cells which undergo cell death; wherein said cell death is not the result of a cytotoxic T lymphocyte-induced lytic event.

2. The method of claim 1, further comprising: (d) introducing said collected polynucleotides into a population of host cells, wherein expression of said target polynucleotide directly or indirectly promotes host cell death; (e) culturing said host cells under conditions such that said insert polynucleotides are expressed; and (f) collecting insert polynucleotides from those host cells which undergo cell death.

3. The method of claim 2, further comprising repeating steps (d) (f) one or more times, thereby enriching for said target polynucleotide.

4. The method claim 3, further comprising purifying said collected polynucleotides.

5. The method of claim 1, wherein said cell death is the result of a cellular effect selected from the group consisting of cell lysis, expression of a suicide gene product, apoptosis, loss of viability, loss of membrane integrity, loss of structural stability, cell disruption, disruption of cytoskeletal elements, inability to maintain membrane potential, arrest of cell cycle, inability to generate energy, growth arrest, cytotoxic effects, cytostatic effects, genotoxic effects, and growth suppressive effects.

6. The method of claim 1, wherein said population of host cells is selected from the group consisting of: tumor cells, metastatic tumor cells, primary cells, transformed primary cells, immortalized primary cells, dividing cells, non dividing cells, terminally differentiated cells, pluripotent stem cells, committed progenitor cells, uncommitted stem cells, progenitor cells, muscle cells, epithelial cells, nervous system cells, circulatory system cells, respiratory system cells, endocrine cells, endocrine-associated cells, skeletal system cells, connective tissue cells, musculoskeletal cells, chondrocytes, osteoblasts, osteoclasts, myocytes, fully differentiated blood cells, fully differentiated epidennal cells, neurons, glial cells, kidney cells, liver cells, muscle cell progenitors, epithelial cell progenitors, nervous system cell progenitors, circulatory system cell progenitors, respiratory system cell progenitors, endocrine cell progenitors, endocrine associated cell progenitors, skeletal system cell progenitors, connective tissue cell progenitors, musculoskeletal cell progenitors, chondrocyte progenitors, osteoblast progemtors, osteoclast progenitors, myocyte progenitors, blood cell progenitors, epidermal cell progenitors, neuron progenitors, glial cell progenitors, kidney cell progenitors, liver cell progenitors and any combination thereof.

7. The method of claim 1, wherein said host cells are adherent to a solid support.

8. The method of claim 7, wherein said solid support is selected from the group consisting of: tissue culture plastic, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, magnetite, soluble material, partially soluble material, insoluble material, magnetic material, and nonmagnetic material.

9. The method of claim 7, wherein said solid support has a structure selected from the group consisting of: spherical, bead-like, bead, cylindrical, test tube-like, tube-like, tube, rod-like, rod, flat, sheet-like, sheet, test strip, strip-like, strip, bead, microbead, well, plate, tissue culture plate, petri plate, microplate, microtiter plate, flask, stick, vial, and paddle.

10. The method of claim 1, wherein said library of insert polynucleotides is selected from the group consisting of: a cDNA library, a genomic library, a combinatorial polynucleotide library, a library of natural polynucleotides, a library of artificial polynucleotides, a library of polynucleotides endogenous to said host cells, a library of polynucleotides exogenous to said host cells, an antisense library, and any combination thereof.

11. The method of claim 5, wherein said cell death is the result of apoptosis.

12. The method of claim 11, wherein apoptosis is induced through expression of a apoptosis-related gene product which directly promotes apoptosis.

13. The method of claim 11, wherein apoptosis is induced through expression of an apoptosis related gene product which indirectly promotes apoptosis.

14. The method of claim 13, wherein said apoptosis related gene product comprises a death domain containing receptor expressed on the surface of said host cells, and wherein said host cells are contacted with a ligand for said death domain containing receptor.

15. The method of claim 11, wherein said host cells are adherent to a solid support.

16. The method of claim 15, wherein those cells which have undergone apoptosis are released from said support.

17. The method of claim 16, wherein said released host cells, or contents thereof, are collected by removing the liquid medium in which said host cells are cultured.

18. The method of claim 15, wherein those host cells which have undergone apoptosis are fully or partially lysed, thereby releasing their cytoplasmic contents into the liquid medium in which said host cells are cultured.

19. The method of claim 18, wherein said released host cell contents are collected by removing the liquid medium in which said host cells are cultured.

20. The method of claim 5, wherein said cell death is the result of expression of a suicide gene product.

21. The method of claim 20, wherein said suicide gene product is selected from the group consisting of a diphtheria toxin A chain polypeptide, a Pseudomonas exotoxin A chain polypeptide, a ricin A chain polypeptide, an abrin A chain polypeptide, a modeccin A chain polypeptide, and an alpha-sarcin polypeptide.

22. The method of claim 20, wherein said host cells are progenitor cells comprising a suicide gene operably associated with a tissue-restricted promoter; wherein expression of said target polynucleotide directly or indirectly induces transcription of said tissue restricted-promoter, resulting in expression of said suicide gene; and wherein expression of said suicide gene promotes death of those progenitor cells comprising said target polynucleotide.

23. The method of claim 22, wherein said host cell is a RAW cell, and wherein said suicide gene is operably associated with the TRAP promoter.

24. The method of claim 23, wherein said target polynucleotide directly or indirectly regulates osteoclast differentiation.

25. The method of claim 23, wherein said suicide gene encodes the Diphtheria toxin A subunit.

26. The method of claim 22, wherein said tissue-restricted promoter is identified by gene expression profiling of said host cells under different physical conditions in microarrays of ordered cDNA libraries.

27. The method of claim 26, wherein said expression profiling compares gene expression under different physical conditions in host cells infected with a eukaryotic virus expression vector, wherein said eukaryotic virus expression vector is the vector used to construct said library of polynucleotides.

28. The method of claim 20, wherein said host cells are non-dividing cells comprising a suicide gene operably associated with a proliferation-specific promoter; wherein expression of said target polynucleotide directly or indirectly induces transcription of said proliferation-specific promoter, resulting in expression of said suicide gene; and wherein expression of said suicide gene promotes death of those non-dividing host cells comprising said target polynucleotide.

29. The method of claim 28, wherein said proliferation-specific promoter is identified by gene expression profiling of said host cells under different physical conditions in microarrays of ordered cDNA libraries.

30. The method of claim 29, wherein said expression profiling compares gene expression under different physical conditions in host cells infected with a eukaryotic virus expression vector, wherein said eukaryotic virus expression vector is the vector used to construct said library of polynucleotides.

31. The method of claim 20, wherein said host cells are adherent to a solid support.

32. The method of claim 31, wherein those host cells expressing said suicide gene product are released from said support.

33. The method of claim 32, wherein said released host cells, or contents thereof, are collected by removing the liquid medium in which said host cells are cultured.

34. The method of claim 31, wherein those host cells expressing said suicide gene product are fully or partially lysed, thereby releasing their cytoplasmic contents into the liquid medium in which said host cells are cultured.

35. The method of claim 34, wherein said released host cell contents are collected by removing the liquid medium in which said host cells are cultured.

36. The method claim 5, wherein cell death occurs within a period selected from the group consisting of: 48 hours after expression of said insert polynucleotide, 24 hours after expression of said insert polynucleotide, and 12 hours after expression of said insert polynucleotide.

37. The method of claim 1, wherein said host cells are infected with said library at an MOI selected from the group consisting of: from about 1 to about 10, about 1 to about 5, and about 1.

38. The method of claim 1, wherein said host cells are permissive for the production of infectious viral particles of said virus.

39. The method of claim 1, wherein said vaccinia virus is attenuated.

40. The method of claim 39, wherein said attenuation is by genetic mutation.

41. The method of claim 39, wherein said attenuation is by reversible inhibition of virus replication.

42. The method of claim 39, wherein said vaccinia virus vector is derived from strain MVA.

43. The method of claim 39, wherein said vaccinia virus vector is derived from strain D4R.

44. The method of claim 1, wherein said insert polynucleotide is in operably associated with a transcriptional control sequence.

45. The method of claim 44, wherein said transcriptional control sequence functions in the cytoplasm of a vaccinia virus-infected cell.

46. The method of claim 44, wherein said transcriptional control sequence comprises a promoter.

47. The method of claim 46, wherein said promoter is constitutive.

48. The method of claim 47, wherein said promoter is a vaccinia virus p7.5 promoter.

49. The method of claim 47, wherein said promoter is a synthetic early/late promoter.

50. The method of claim 44, wherein said transcriptional control sequence comprises a transcriptional termination region.

51. The method of claim 39, wherein said vaccinia virus vector is derived from strain WR.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a high efficiency method of introducing DNA into poxvirus, a method of producing libraries in poxvirus, and methods of isolating polynucleotides of interest based on cell nonviability or screening methods.

2. Background Art

Identification of Disease Genes. In the past decade it has become apparent that many diseases result from genetic alterations in signaling pathways. These include diseases related to unregulated cell proliferation such as cancers, atherosclerosis and psoriasis, as well as inflammatory conditions such as sepsis, rheumatoid arthritis and tissue rejection. The finding that these proliferative diseases are based on genetic defects is the basis of new approaches for disease management by designing drugs which modulate cell signaling. In order to develop highly specific drugs, i.e., drugs which potently interfere with uncontrolled cell proliferation but which have low toxicity or side effects, it is important to identify the genes encoding polypeptides involved in the cellular signal transduction pathways whose aberrant function may result in the loss of growth control.

Although tremendous progress in understanding relevant signal transduction pathways has been made in recent years, it is clear that many of the genes involved in the development of proliferative disorders remain to be discovered.

Toxic Sequences. Several approaches have been employed for the identification and isolation of cell proliferation genes such as oncogenes and tumor suppressor genes. Traditional approaches include detection of cytogenetic abnormalities in tumor cells, kindred analysis of familial forms of cancer, and loss of heterozygosity analysis in tumor cells. Each of these classical genetic approaches is limited in the type of gene which can be isolated or in the extensive time and labor required. A faster approach would be to identify disease genes using in vitro techniques. However, a major technical limitation to the cloning of many disease genes is their negative or toxic effect on cell proliferation when present in multiple copies, such as when carried on a vector.

One approach for identifying toxic sequences involves the selection of variants that have lost certain malignancy traits, namely "revertants." In this method, cells transformed by a variety of oncogenes are subsequently treated with a cytotoxic agent which kills dividing cells. "Revertants" that have lost the ability to rapidly divide are thus selected. However, revertant lines typically are difficult to identify and separate from the majority of rapidly growing transformed parental cells. In addition, the method may preclude the isolation of certain classes of revertants. The selection procedure may itself induce epigenetic or cytogenetic changes, thus further complicating the identification of genes responsible for the revertant phenotype.

Zarbl et al. developed an alternative assay for the selection of revertant tumor cells (Zarbl et al., 1991, Environmental Health Perspectives 93:83 89). This selection protocol is based on the prolonged retention of a fluorescent molecule within the mitochondria of a number of transformed cells relative to non-transformed cells. However, the approach is limited to particular transformation mechanisms because the prolonged dye retention phenotype is neither essential nor sufficient for cell transformation.

Other methods used to identify cell proliferation genes involve biochemical approaches for analyzing cell cycle regulators (Serrano et al., 1993, Nature 366:704 707; Xiong et al., 1993, Nature 366:701 704), random sequencing of expressed sequence tags (ESTs) and homology comparison (Lennon et al., 1996, Genomics 33:151 152), and methods for identifying differentially expressed genes, such as differential display (Liang et al., 1995, Methods Enzymol. 254:304 321). None of these approaches, however, offers a way to directly assess gene function as a method of identifying genes of interest, especially negative regulators of proliferation. Instead, candidates are identified based on a presumed (or identifiable) biochemical function or an abnormal pattern of expression. These candidates are then tested further for involvement in cancer. Such tests include mutation detection in primary cancers or cell lines, experiments using somatic cells (for example, to determine the effect of ectopic expression), or experiments in transgenic mice or knockout mice containing inactivated genes.

A more recent method for identifying cell proliferation genes involves the isolation of variants of transformed cells to identify a cell proliferation promoting activity. See U.S. Pat. No. 5,998,136. This selection system comprises the creation of growth arrested tumor cell lines or cells which undergo apoptosis by, for example, the expression of a gene encoding a growth suppressor or apoptosis-inducing gene product under the control of an inducible promoter, and selection of revertants that allow the cells to survive. Induction of the suppressor or apoptosis-inducing product causes suppression of tumor cell growth and/or cell death. Growth-proficient revertants cells are identified by virtue of their continued proliferation.

The identification of toxic molecules such as tumor suppressor genes and other inhibitors of cell proliferation to screen for potential new drugs is difficult using current technology. For example, it would be of great value to identify dominant negative mutations of signaling molecules that might be used to inhibit the unregulated growth of transformed cells. Those negative or toxic mutations that result in inhibition of cell growth or in cell death may be masked in a library or other population of cells due to the low efficiency of transfection. Additionally, such negative or toxic mutations cannot be selected for or screened using current technology because cells expressing such variants are lost from the population of transformants. These limitations may have been addressed to a limited extent by the use of inducible promoter systems, see, for example, those described in Levinson, A. D., "Gene Expression Technology," In D. V. Goeddel (Ed.), Methods in Enzymology, Academic Press, p. 497 (1991). However, this approach is labor-intensive, is not applicable to certain situations, and has met with varied success depending on the cell type and origin of the promoter utilized.

As alluded to above, there are methods to identify positive regulators of cell growth such as oncogenes, but approaches to isolate toxic genes such as tumor suppressor genes are limited. In addition to those described above, methods for isolating negative regulators include genetic analysis based on anti-sense RNA technologies.

Another approach is a method of selection subtraction by tagging a clone in an expression library, cloning the tagged clone into a vector, delivering the tagged clone to a target cell, and comparing tags before and after selection whereby toxic genes and the attached tags disappear. See WO 99/47643.

Yet another approach selects all transformants in a population of cells before those transformants expressing negative or toxic variants are lost from the population. See WO 97/08186. This method comprises use of a cloning vector encoding a recombinant immunoglobulin molecule (rAb) that is specific for a particular hapten and expressed on the cell surface. Cells receiving the vector express the rAb early after transfection, and are separated from the non-recipient cells by the ability to bind the cognate hapten conjugated to a solid surface, such as beads. This method does not distinguish recipients expressing a gene or cDNA of interest, e.g., a negative or toxic variant, from the remaining recipients.

Differentially Expressed Sequences. Cloning, sequencing, and identification of function of mammalian genes is a first priority in a genomic based drug discovery. In particular, it is important to identify and make use of genes which are spatially and/or temporally regulated in an organism, for example, genes involved in differentiation and growth regulation.

Animal model systems such as the fruit fly and the worm are often used in gene identification because of ease of manipulation of the genome and ability to screen for mutants. While these systems have their limitations, large numbers of developmental mutations have been identified in those organisms either by monitoring the phenotypic effects of mutations or by screening for expression of reporter genes incorporated into developmentally regulated genes.

Many features of the mouse make it the best animal model system to study gene function. However, the mouse has not been used for large scale classical genetic mutational analysis because random mutational screening and analysis is very cumbersome and expensive due to long generation times and maintenance costs.

A disadvantage in using animal models for the identification of genes is the need to establish a transgenic animal line for each mutational event. This disadvantage is alleviated in part by using embryonic stem (ES) cell lines because mutational events may be screened in vitro prior to generating an animal. ES cells are totipotent cells isolated from the inner cell mass of the blastocyst. Methods are well known for obtaining ES cells, incorporating genetic material into ES cells, and promotion of differentiation of ES cells. ES cells may be caused to differentiate in vitro or the cells may be incorporated into a developing blastocyst in which the ES cells will contribute to all differentiated tissues of the resulting animal. Vectors for transforming ES cells and suitable genes for use as reporters and selectors are also well known.

Gene entrapment strategies also have been employed to identify developmentally regulated genes. One type of entrapment vector is called a "promoter trap," which consists of a reporter gene sequence lacking a promoter. Its integration is detected when the reporter is integrated "in-frame" into an exon. In contrast, a "gene trap" vector targets the more prevalent introns of the eucaryotic genome. The latter vector consists of a splice-acceptor site upstream from a reporter gene. Integration of the reporter into an intron results in a fusion transcript containing RNA from the endogenous gene and from the reporter gene sequence.

Gene trap vectors may be made more efficient by incorporation of an internal ribosomal entry site (IRES) such as that derived from the 5' non-translated region of encephalomyocarditis virus (EMCV). Placement of a IRES site between the splice acceptor and the reporter gene of a gene trap vector means the reporter gene product need not be translated as a fusion product with the endogenous gene product, thereby increasing the likelihood that integration of the vector will result in expression of the reporter gene product.

Gossler, A., et al. Science 244:463 465 (1989) describe the use of enhancer trap gene trap vectors for use in identifying developmentally regulated genes. The gene trap vector consists of the mouse En-2 splice acceptor upstream from lacZ (reporter) and a selector gene (hBa-neo). This and other current methods requires elaborate screening procedures for linking a mutation to a particular spacial/temporal scheme or event whereby the mutation is detected in the relevant tissue.

A more recently developed method is complementation trapping. See WO 99/02719. This method makes use of known genes whose expression is restricted to specific tissue, tissues or specialized cells ("restricted expression") to facilitate identification and manipulation of new genes and their associated transcription control elements which have similar patterns of expression. The method comprises (i) transforming a eucaryotic cell with a DNA sequence encoding a first indicator component under the control of a promoter having restricted expression; (ii) transforming the cell of (i) or a descendent of the cell of step (i), by operably integrating into the cell's genome DNA lacking a promoter but which comprises a sequence encoding a second indicator component; (iii) producing tissue or specialized cells from the cell of (ii); and (iv) monitoring the tissue or specialized cells of (iii) for a detectable indicator resulting from both the first and second indicator components.

Expression Libraries. A basic tool in the field of recombinant genetics is the conversion of poly(A).sup.+ mRNA to double-stranded (ds) cDNA, which then can be inserted into a cloning vector and expressed in an appropriate host cell. A substantial number of variables affect the successful cloning of a gene of interest and cDNA cloning strategy thus must be chosen with care. A method common to many cDNA cloning strategies involves the construction of a "cDNA library" which is a collection of cDNA clones derived from the poly(A).sup.+ mRNA derived from a cell of the organism of interest.

A mammalian cell may contain up to 30,000 different mRNA sequences, and the number of clones required to obtain low-abundance mRNAs, for example, may be much greater. Methods of constructing genomic eukaryotic DNA libraries in different expression vectors, including bacteriophage lambda, cosmids, and viral vectors, are known. Some commonly used methods are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, N.Y. (1982).

Once a genomic cDNA library has been constructed and expressed in host cells, it is necessary to isolate from the thousands of host cells the particular cell or cells which contain the particular gene of interest. Many different methods of isolating target genes from cDNA libraries have been utilized, with varying success. These include, for example, the use of nucleic acid probes, which are labeled mRNA fragments having nucleic acid sequences complementary to the DNA sequence of the target gene. When this method is applied to cDNA clones of abundant mRNAs in transformed bacterial hosts, colonies hybridizing strongly to the probe are likely to contain the target DNA sequences. The identity of the clone then may be proven, for example, by in situ hybridization/selection (Goldberg et al., Methods Enzymol., 68:206 (1979)) hybrid-arrested translation (Paterson et al., Proceedings of the National Academy of Sciences, 74:4370 (1977)), or direct DNA sequencing (Maxam and Gilbert, Proceedings of the National Academy of Sciences, 74:560 (1977); Maat and Smith, Nucleic Acids Res., 5:4537 (1978)).

Such methods, however, have major drawbacks when the object is to clone mRNAs of relatively low abundance from cDNA libraries. For example, using direct in situ colony hybridization, it is very difficult to detect clones containing cDNA complementary to mRNA species present in the initial library population at less than one part in 200. As a result, various methods for enriching mRNA in the total population (e.g. size fractionation, use of synthetic oligodeoxynucleotides, differential hybridization, or immunopurification) have been developed and are often used when low abundance mRNAs are cloned. Such methods are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, supra.

Use of mammalian expression libraries to isolate cDNAs encoding mammalian proteins such as those described above would offer several advantages. For example, the protein expressed in a mammalian host cell should be functional and should undergo any normal posttranslational modification. A protein ordinarily transported through the intracellular membrane system to the cell surface should undergo the complete transport process. A mammalian expression system also would allow the study of intracellular transport mechanisms and of the mechanism that insert and anchor cell surface proteins to membranes. Further, use of a mammalian system would make it possible to isolate polynucleotides based on functional expression of mammalian RNA or protein.

One common mammalian host cell, called a "COS" cell, is formed by infecting monkey kidney cells with a mutant viral vector, designated simian virus strain 40 (SV40), which has functional early and late genes, but lacks a functional origin of replication. In COS cells, any foreign DNA cloned on a vector containing the SV40 origin of replication will replicate because SV40 T antigen is present in COS cells. The foreign DNA will replicate transiently, independently of the cellular DNA.

With the exception of some recent lymphokine cDNAs isolated by expression in COS cells (Wong, G. G., et al., Science 228:810 815 (1985); Lee, F. et al., Proc. Natl. Acad. Sci. USA 83.2061 2065 (1986); Yokota, T., et al., Proc. Natl. Acad. Sci. USA 83:5894 5898 (1986); Yang, Y., et al., Cell 47:3 10 (1986)), however, few cDNAs in general are isolated from mammalian expression libraries. There appear to be two principal reasons for this: First, the existing technology (Okayama, H. et al., Mol. Cell. Biol. 2:161 170 (1982)) for construction of large plasmid libraries is difficult to master, and library size rarely approaches that accessible by phage cloning techniques. (Huynh, T. et al., In: DNA Cloning Vol. I, A Practical Approach, Glover, D. M. (ed.), IRL Press, Oxford (1985), pp. 49 78). Second, the existing vectors are, with one exception (Wong, G. G., et al., Science 228:810 815 (1985)), poorly adapted for high level expression, particularly in COS cells. The reported successes with lymphokine cDNAs do not imply a general fitness of the methods used, since these cDNAs are particularly easy to isolate from expression libraries: Lymphokine bioassays are very sensitive ((Wong, G. G., et al., Science 228:810 815 (1985); Lee, F. et al., Proc. Natl. Acad. Sci. USA 83:2061 2065 (1986); Yokota, T. et al., Proc. Natl. Acad. Sci. USA 83:5894 5898 (1986); Yang, Y. et al., Cell 47:3 10 (1986)) and the mRNAs are typically both abundant and short (Wong, G. G. et al., Science 228:810 815 (1985); Lee, F., et al., Proc. Natl. Acad. Sci. USA 83:2061 2065 (1986); Yokota, T., et al., Proc. Natl. Acad. Sci. USA 83:5894 5898 (1986); Yang, Y., et al., Cell 47:3 10 (1986)).

Thus, expression in mammalian hosts previously has been most frequently employed solely as a means of verifying the identity of the protein encoded by a gene isolated by more traditional cloning methods. For example, Stuve et al., J. Virol. 61(2):327 335 (1987), cloned the gene for glycoprotein gB2 of herpes simplex type II strain 333 by plaque hybridization of M13-based recombinant phage vectors used to transform competent E. coli JM101. The identity of the protein encoded by the clone thus isolated was verified by transfection of mammalian COS and Chinese hamster ovary (CHO) cells. Expression was demonstrated by immunofluorescence and radioimmunoprecipitation.

Oshima et al. used plaque hybridization to screen a phage lambda gt11 cDNA library for the gene encoding human placental beta-glucuronidase. Oshima et al., Proceedings of the National Academy of Sciences, U.S.A. 84:685 689 (1987). The identity of isolated cDNA clones was verified by immunoprecipitation of the protein expressed by COS-7 cells transfected with cloned inserts using the SV40 late promoter.

Transient expression in mammalian cells has been employed as a means of confirming the identity of genes previously isolated by other screening methods. Gerald et al., Journal of General Virology 67:2695 2703(1986). Mackenzie, Journal of Biological Chemistry 261:14112 14117 (1986); Seif et al., Gene 43:1111 1121 (1986); Orkin et al., Molecular and Cellular Biology 5(4):762 767 (1985). These methods often are inefficient and tedious and require multiple rounds of screening to identify full-length or overlapping clones. Prior screening methods based upon expression of fusion proteins are inefficient and require large quantities of monoclonal antibodies. Such drawbacks are compounded by use of inefficient expression vectors, which result in protein expression levels that are inadequate to enable efficient selection.

Seed et al., U.S. Pat. No. 5,506,126 developed a cloning technique based upon transient expression of antigen in eukaryotic cells and physical selection of cells expressing the antigen by adhesion to an antibody-coated substrate, such as a culture dish. This method for cloning cDNA encoding a cell surface antigen comprises preparing a cDNA library; introducing this cDNA library into eukaryotic mammalian cells; culturing the cells under conditions allowing expression of the cell surface antigen; exposing the cells to a first antibody or antibodies directed against the cell surface antigen, thereby allowing the formation of a cell surface antigen-first antibody complex; subsequently exposing the cells to a substrate coated with a second antibody directed against the first antibody, thereby causing cells expressing the cell surface antigen to adhere to the substrate via the formation of a cell surface antigen-first antibody-second antibody complex; and separating adherent from non-adherent cells. However, this method is limited to the isolation and cloning of proteins which are expressed and transported to the cell surface, whose expression does not adversely affect cell viability, and for which specific antibody has been isolated.

Poxvirus Vectors. Poxvirus vectors are used extensively as expression vehicles for protein and antigen expression in eukaryotic cells. The ease of cloning and propagating vaccinia in a variety of host cells has led to the widespread use of poxvirus vectors for expression of foreign protein and as vaccine delivery vehicles (Moss, B. 1991, Science 252:1662 7).

Customarily, a foreign protein coding sequence is introduced into the poxvirus genome by homologous recombination. In this method, a previously isolated foreign DNA is cloned in a transfer plasmid behind a vaccinia promoter flanked by sequences homologous to a region in vaccinia which is non-essential for viral replication. The transfer plasmid is introduced into vaccinia virus-infected cells to allow the transfer plasmid and vaccinia virus genome to recombine in vivo via homologous recombination. As a result of the homologous recombination, the foreign DNA is transferred to the viral genome.

Although homologous recombination is efficient for transferring previously isolated foreign DNA of relatively small size into vaccinia virus, the method is much less efficient for transferring large inserts, for constructing libraries, and for transferring foreign DNA which is deleterious to bacteria.

Alternative methods using direct ligation vectors have been developed to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination (Merchlinsky, M. et al., 1992, Virology 190:522 526; Scheiflinger, F. et al., 1992, Proc. Natl. Acad. Sci. USA. 89:9977 9981). In such protocols, the DNA from the genome is digested, ligated to insert DNA in vitro, and transfected into cells infected with a helper virus (Merchlinsky, M. et al., 1992, Virology 190:522 526, Scheiflinger, F. et al., 1992, Proc. Natl. Acad. Sci. USA 89:9977 9981). In one protocol, the genome was digested at a unique NotI site and a DNA insert containing elements for selection or detection of the chimeric genome was ligated to the genomic arms (Scheiflinger, F. et al., 1992, Proc. Natl. Acad. Sci. USA. 89:9977 9981). This direct ligation method was described for the insertion of foreign DNA into the vaccinia virus genome (Pfleiderer et al., 1995, J. General Virology 76:2957 2962). Alternatively, the vaccinia WR genome was modified by removing the NotI site in the HindIII F fragment and reintroducing a NotI site proximal to the thymidine kinase gene such that insertion of a sequence at this locus disrupts the thymidine kinase gene, allowing isolation of chimeric genomes via use of drug selection (Merchlinsky, M. et al., 1992, Virology 190:522 526).

The direct ligation vector vNotI/tk allows one to efficiently clone and propagate previously isolated DNA inserts at least 26 kilobase pairs in length (Merchlinsky, M. et al., 1992, Virology, 190:522 526). Although large DNA fragments are efficiently cloned into the genome, proteins encoded by the DNA insert will only be expressed at the low level corresponding to the thymidine kinase gene, a relatively weakly expressed early class gene in vaccinia. In addition, the DNA will be inserted in both orientations at the NotI site.

The cloning methods and the selection methods above have a number of drawbacks and limitations. Therefore it is desirable, and the objective of the present invention, to develop cloning and selection methods that would permit the identification and isolation of novel genes based on functional analysis.

BRIEF SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there is provided a method of high efficiency cloning using a linear DNA virus vector such as vaccinia virus vector, comprising tri-molecular recombination.

In accordance with another aspect of the present invention, there is provided a method of producing a library using a linear DNA virus vector such as vaccinia virus vector.

In accordance with yet another aspect of the present invention, there is provided a method of cloning a polynucleotide which negatively affects cell viability.

In accordance with yet another aspect of the present invention, there is provided a method of cloning a polynucleotide in a nondividing cell.

In accordance with yet another aspect of the present invention, there is provided a method of directly or indirectly selecting a polynucleotide which negatively affects cell viability from a plurality of polynucleotides.

In accordance with yet another aspect of the present invention, there is provided a method of directly or indirectly selecting a polynucleotide which encodes an epitope from a plurality of polynucleotides.

In accordance with another aspect of the present invention, there is provided a method of directly or indirectly selecting a polynucleotide which alters a phenotype of a cell.

In accordance with yet another aspect of the present invention, there is provided a method of modifying a linear DNA virus vector such as vaccinia virus.

In accordance with a further aspect of the present invention, there is provided a kit for producing a library using tri-molecular recombination. In one embodiment, the invention provides a kit for producing an antisense expression library comprising a linear DNA viral genome such as vaccinia virus or two fragments thereof, and two vectors for producing a transfer plasmid containing a polynucleotide insert in each of two orientations. In another embodiment, the invention provides a kit for producing a protein expression library comprising a linear DNA genome such as vaccinia virus or two fragments thereof, and three vectors for producing a transfer plasmid containing a polynucleotide insert in each of three translation reading frames.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. Nucleotide Sequence of p7.5/tk (SEQ ID NO:1) and pEL/tk (SEQ ID NO:3). The nucleotide sequence of the promoter and beginning of the thymidine kinase gene for v7.5/tk and vEL/tk. The partial thymidine kinase amino acid sequence is also shown (SEQ ID NO:2).

FIG. 2. Southern Blot Analysis of Viral Genomes p7.5/tk and pEL/tk. The viruses v7.5/tk and vEL/tk were used to infect a well of a 6 well dish of BSC-1 cells at high multiplicity of infection (moi) and after 48 hours the cells were harvested and the DNA was isolated using DNAzol (Gibco). The final DNA product was resuspended in 50 microliters of TE 8.0 and 2.5 microliters were digested with HindIII, HindIII and ApaI, or HindIII and NotI, electrophoresed through a 1.0% agarose gel, and transferred to Nytran (Schleicher and Schuell) using a Turboblotter (Schleicher and Schuell). The samples were probed with p7.5/tk (A) or pEL/tk (B) labeled with .sup.32P using Random Primer DNA Labeling Kit (Bio-Rad) in QuickHyb (Stratagene). The lower portion of the figure denotes a map of the HindIII J fragment with the positions of the HindIII, NotI, and ApaI sites illustrated. The leftmost 0.5 kilobase fragment has electrophoresed off the bottom of the gel.

FIG. 3. Restriction Enzyme Analysis of Virus Genomes Using CHEF Gel. BSC-1 cells were infected at high multiplicity of infection (moi) by vaccinia WR, vEL/tk, v7.5/tk, or vNotI/tk. After 24 hours the cells were harvested and formed into agarose plugs. The plugs were equilibrated in the appropriate restriction enzyme buffer and 1 mM PMSF for 16 hours at room temperature, incubated with restriction enzyme buffer, 100 ng/ml Bovine Serum Albumin and 50 units NotI or ApaI for two hours at 37.degree. C. (NotI) or room temperature (ApaI) and electrophoresed in a 1.0% agarose gel on a Bio-Rad CHEFII apparatus for 15 hours at 6 V/cm with a switching time of 15 seconds. The leftmost sample contains lambda DNA, the second sample contains undigested vaccinia DNA, and the remainder of the samples contain the DNA samples described above each well digested with ApaI or NotI where vEL refers to vEL/tk and v7.5 refers to v7.5/tk. The lower portion of the figure is a schematic map showing the location of the NotI and ApaI sites in each virus.

FIG. 4. Analysis of v7.5/tk and vEL/tk by PCR. One well of a 6 well dish of BSC-1 cells was infected with v7.5/tk, vEL/tk, vNotI/tk, vpNotI, vNotI/lacZ/tk, or wild type vaccinia WR at high multiplicity of infection (moi) and after 48 hours the cells were harvested, and the DNA was isolated using DNAzol (Gibco). The final DNA product was resuspended in