WO2011153440A2 - Transposon mutagenesis system and methods of use - Google Patents

Abstract

The present invention provides methods for altering a phenotype of an in vitro cell through the use of transposons. A method may include incubating a cell that includes at least two transposons and a polynucleotide encoding a transposase that causes transposition of the transposons to genomic DNA of the cell, and observing a phenotype in the resulting mutagenized cells. Also provided is a transposon mutagenesis system to introduce a polynucleotide into genomic DNA of an in vitro cell.

Description

TRANSPOSON MUTAGENESIS SYSTEM AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 61/396,957, filed June 4, 2010, which is incorporated by reference herein.

GOVERNMENT FUNDING

The present invention was made with government support under Grant No. ROl CAl 13636, awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND

During the last few decades investigators have begun to engineer specific genetic changes into primary or immortalized human cells in order to study phenotypes of interest, such as cell transformation, cancer metastasis, and drug resistance. These models are exciting because they offer the potential to discover primate-specific mechanisms that may be clinically relevant, but cannot be studied in rodent models.

Transposon mutagenesis has proven to be useful for functional genomic screens in organisms such as Drosophila melanogaster (Spradling et al., Proc. Natl. Acad. Sci. USA, 1995;92:10824-10830), Caenorhabditis elegans (Plasterk, Curr. Top. Microbiol. Immunol. 1996;204:125-143) and plants (Osborne et al, Curr. Opin. Cell Biol, 1995;7:406-413) but the lack of active elements in higher eukaryotes has precluded their use for mammalian functional genomics. Progress towards the development of transposons useful in mammalian studies was made possible when transposon systems such as Sleeping Beauty (SB) and piggyBac (PB) were developed. These transposon systems have proven useful as an in vivo mouse mutagen for phenotype gene discovery, especially cancer. However, their usefulness as a mutagen in vitro has been limited.

Other methods of mutagenesis in cultured cells have also been somewhat limited. Retroviral mutagenesis is skewed by the natural predisposition of viral insertions into the 5' end or promoter regions of genes and it can also be hard to track insertion of retroviruses due to the large amount of native viral DNA in the host genome. ENU mutagenesis is not easily trackable and requires genetic crosses, which are not even possible with cultured cells, to identify the location of mutations by this chemical mutagen. Other methods include RNAi or cDNA overexpression library screens. RNAi can only give phenotypes by knocking down genes and thus cannot identify genes that need to be over-expressed to induce the phenotype of interest. Additionally, the RNAi library needs to cover every gene and have a considerable knockdown to be useful, and few to no RNAi libraries have these attributes. cDNA overexpression screens are inversely problematic in that they can only produce a phenotype by overexpressing a gene/cDNA. Additionally, the cDNA overexpression library typically must contain all known genes and express them at high levels to be most useful, but again few to no cDNA libraries have these attributes. Further, it is more difficult to study phenotypes requiring a 'double hit' using these other methods.

SUMMARY OF THE INVE TION

The present invention represents an advance in the identification of genes responsible for a broad spectrum of phenotypes in cultured cells. This technology is the first platform to use episomal, self-replicating vectors to deliver mutagenic transposons for forward genetic screens in cell culture. The usefulness of transposons as a mutagen in vitro has been limited, mainly due to the lack of a large pool or source of mutagenic transposons in the in vitro setting; this difference between in vitro and in vivo studies stems from concatemerization phenomenon of transgenes upon mouse transgenesis by pro-nuclear injection. Thus, when a transgenic mouse is produced, using a linearized plasmid, it tends to concatemerize and integrate randomly into the genome, producing an animal with many copies of the transgene in a large array or concatemer. This integrated transposon concatemer serves as the pool of mutagenic transposons for forward genetic screens in mice. Thus, to adapt transposon mutagenesis to an in vitro cell culture based system, a pool of mutagenic transposons for use in vitro was developed.

The present invention provides methods for altering a phenotype of a cell. In one embodiment, a method may include incubating a cell that mcludes at least 2 transposons and a polynucleotide encoding a transposase that causes transposition of the transposons to genomic DNA of the cell. The incubating is under conditions suitable for growth and results in mutagenized cells having transposons that have transposed to genomic DNA. The cell may be, for instance, a human cell or a murine cell, such as a mouse cell.

In one embodiment, the cell may include integrated in its genome a vector having the at least 2 transposons and/or the polynucleotide encoding the transposase. In one embodiment, the method may further include introducing into the cell a first vector that includes at least 2 transposons. The first vector may replicate in the cell or it may integrate into the genomic DNA of the cell. In one embodiment, the method may further include introducing into the cell a second vector that includes a polynucleotide encoding a transposase. The second vector may replicate in the cell or it may integrate into the genomic DNA of the cell. In one embodiment, the method may further include introducing into the cell a vector that includes at least 2 transposons and a polynucleotide encoding a transposase, and the vector may replicate in the cell or may be integrated into genomic DNA of the cell. The transposons may include cis-acting elements that bind a Sleeping Beauty transposase or a PiggyBac transpoase. In one embodiment, a transposon includes cis-acting elements that bind a Sleeping Beauty transposase and cis acting elements that include a PiggyBac transpoase The transpoase may be a Sleeping Beauty transposase or a PiggyBac transposase.

The method may also include observing a phenotype in the mutagenized cells, wherein the phenotype is not present in control cells. In one embodiment, the cell is a tumorigenic cell, and in one embodiment, the cell is a non-tumorigenic cell. In one embodiment, the observing may include exposing the mutagenized cells to a compound at a concentration that kills the control cells, and identifying a mutagenized cell that is resistant to the compound. Non-limiting examples of compounds include alkylating agents, antimetabolites, anthracyclines, plant alkaloids, and topoisomerase inhibitors. In one embodiment where the cell is a non-tumorigenic cell before mutagenesis with the transposons, the observing may include administering the mutagenized cells to an animal, and identifying a tumor in the animal, wherein the tumor is derived from an administered mutagenized cell. The animal may be an irnmuno-incompetent animal. In one embodiment, a phenoype may be immortalization, tumorigenesis, drug resistance, or transformation.

The method may further include mapping the location of a transposon present in genomic DNA of a cell, and optionally determining the identity of the genomic DNA where a transposon has inserted. For example, a transposon may insert in a coding region or a regulatory region to give rise to an altered phenotype.

Also provided herein is a transposon mutagenesis system to introduce a

polynucleotide into genomic DNA of an in vitro cell. In one embodiment, the system includes a first vector that includes at least two transposons, and a second vector that includes a polynucleotide encoding a transposase, wherein the transposase causes transposition of the transposons from the first vector to genomic DNA of the cell. In one embodiment, the system includes a vector that has at least two transposons and a polynucleotide encoding a transposase, wherein the transposase causes transposition of the transposons from the first vector to genomic DNA of the cell.

Further provided herein is a kit. In one embodiment, a kit may include packaging materials, a first vector that includes at least two transposons, and a second vector that includes a polynucleotide encoding a transposase, wherein the transposase causes transposition of the transposons from the first vector to genomic DNA of the cell.

As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded RNA and DNA. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide may be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. A polynucleotide may include nucleotide sequences having different functions, including, for instance, coding regions, and non-coding regions such as regulatory regions.

As used herein, "gene" refers to a nucleotide sequence that encodes an mRNA. A gene has at its 5' end a transcription initiation site and a transcription terminator at its 3' end. As used herein, a "target gene" refers to a specific gene whose expression is inhibited by a polynucleotide as described herein. As used herein, a "target mRNA" is an mRNA encoded by a target gene. Unless noted otherwise, a target gene can result in multiple mRNAs distinguished by the use of different combinations of exons. Such related mRNAs are referred to as splice variants or transcript variants of a gene.

As used herein, the terms "coding region" and "coding sequence" are used interchangeably and refer to a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A "regulatory sequence" is a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators. The term "operably linked" refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.

An "enhancer" is a regulatory sequence that increases the rate of transcription initiation of a coding region. Enhancers usually exert their effect regardless of the distance, upstream or downstream location, or orientation of the enhancer relative to the start site of transcription. Without intending to be limiting, an enhancer is typically a nucleotide sequence where a polypeptide can bind and stabilize the association of RNA polymerase allowing initiation of transcription to proceed.

As used herein, the term "polypeptide" refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term "polypeptide" also includes molecules which contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, enzyme, and protein are all included within the definition of polypeptide and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.

Conditions that are "suitable" for an event to occur, such as introduction of a polynucleotide into a cell or growth of a cell, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event.

The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Unless otherwise specified, "a," "an," "the," and "at least one" are used

interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. The PB-SB-OncX Mutagenic Transposon. (a) The format and functional component of the mutagenic transposon (Tn) are shown. The Cagg promoter followed by splice donor (SD) has been included to overexpress genes or truncated transcripts if the Tn is inserted upstream or within a intron, respectfully. There are splice acceptors (SA) in both orientations and a bi-directional SV40 polyA to disrupt gene function if inserted into an intron or exon. This cargo is directly flanked by the Sleeping Beauty (SB) transposon Inverted Repeats and Direct Repeats (IR/DRs). The SB mutagenic transposon is further flanked by PiggyBac (PB) Inverted Terminal Repeats (ITRs), this format allows for mobilization of the mutagemc cargo by either the PB or SB

transposase. (b) PB-SB-OncX showing engineered restriction enzyme sites necessary for use in creating the Tn concatemer vectors, discussed below.

Figure 2. Adaption of the Gateway cloning system to engineer transposon concatemers. (a) Diagram of the Gateway systems L-R Clonase reaction to create expression clones. Any cargo between attLl-attL2 sites is exchanged for the cargo between the attRl-attR2 sites, this is accomplished by recombination carried out by lambda phage recombination enzymes: Integrase, IHF (Integration Host Factor), and Excisionase. (b) The Gateway system was adapted to create transposon concatemers by restriction enzyme cloning multiple copies of the PB-SB-OncX vector into both the pENTR 'Master clone' and pDEST 'Expression clone' vector followed by L-R Clonase reaction to produce the 'expression clone' pCEP4-40ncX. (c) Two sequential rounds of cloning the attRl -attR2 DEST cassette into the pCEP4-OncX vector followed by L-R Clonase reaction were then performed to further create pCEP4-60ncX and pCEP4- 80ncX. (d) Diagram of the all-in-one mutagenic vector, termed pCEP4-50ncX-PB7- ERt2, also generated using Gateway L-R Clonase reaction, (e) Diagram of the Tol2 deliverable all-in-one mutagenic vector, termed pTol2-50ncX-PB7-ERt2, also generated using Gateway L-R Clonase reaction, (f) Diagram of the Tol2 deliverable 60ncX mutagenic vector, termed pTol2-60ncX, also generated using Gateway L-R Clonase reaction.

Figure 3. Sleeping Beauty Transposition from pCEP4-OncX vectors. A PCR based transposon excision assay demonstrates SB mediated transposition of SB-OncX Tn from various pCEP4-OncX vectors when co-transfected into HE -293T cells. Primers amplify a 2.3kb product if transposons are present in the concatemer vector and a smaller 250bp product if transposons have been mobilized from the vector.

Figure 4. Engineering cultured cells to stably express the Transposon Mutagenesis System, (a) Diagram of the procedure used to engineer NIH-3T3 cells to stably express the transposon mutagenesis system. Briefly, cells were transduced with a lentiviral vector expressing SBlOO-E ES-eGFP and sorted for a pure population using the eGFP marker and then transfected with pCEP4-OncX vector and selected for using

Hygromycin. (b) Fluorescence microscopy of engineered cells demonstrating the presence of the transposase cassette and pCEP4-OncX vector by co-expression of eGFP and dsRED expression, respectively.

Figure 5. Linker Mediated-PCR identifies chromosomal transposon insertion sites, (a) Diagram of Linker Mediated-PCR (LM-PCR) procedure used to identify transposon insertion sites. Briefly, genomic DNA is digested with a common 4-base pair restriction enzyme and linkers containing the complementary sequence are ligated, followed by a primary and secondary nested PCR and high throughput sequencing. To be considered a 'true insertion' sequence reads must contain transposon sequence, barcode, mappable adjacent genomic sequence, and linker sequence (and a TA dinucleotide duplication for SB insertions), (b) Agarose gel of secondary PCR products of LM-PCR procedure. Canonical 'smears' demonstrate numerous and diverse PCR product that represent corresponding numerous and diverse transposon insertions into genomic DNA. (i) in vitro samples that have very few insertions (1-20 per sample) by transient transfection with SB Tn and transposase plasmids in MAPC cells, (ii) in vivo samples that have 1,000s of insertions in mouse somatic cells that induced osteosarcomagenesis over 8-12 months, (iii) in vitro samples that likely have lOO's-1000's of insertions in FTEK-293T and NIH-3T3 cells expressing the transposon mutagenesis system for only 2-3 weeks, (c) Example of sequence reads from LM-PCR performed on HEK-293T and NIH-3T3 cells expressing the transposon mutagenesis system using SB. Importantly, insertions contain all elements of 'true insertions.' Single underlined nucleotides, IR/DR sequence; double underlined nucleotides, linker sequence; triple underlined nucleotides, flanking TA dinucleotides; remaining nucleotides, mappable adjacent genomic sequence. Figure 6. Mice Injected with Mutagenized NIH-3T3 Cells Can Develop Lung Nodules.

(a) Image of nodules that developed in nude mice tail vein injected with the

constitutively active K-Ras V12G positive control cells. Numerous nodules were observed in these animals and they developed in approximately 1 -month post injection.

(b) Image of one small nodules identified in a nude mouse 5.6 months after tail vein injected with NIH-3T3 cells expressing SBlOO-IRES-eGFP and the pCEP-60ncX vector.

(c) Image of a larger nodule identified in a nude mouse 10 months after tail vein injected withNIH-3T3 cells expressing SBlOO-IRES-eGFP and the _PCEP-60ncXvector.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention includes vectors and methods for using the vectors. A vector is a replicating polynucleotide, such as a plasmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. The vector may include a coding sequence. A vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polypeptide encoded by the coding region, i.e., an expression vector. A vector can be both a cloning vector and an expression vector. The term vector includes, but is not limited to, plasmid vectors, cosmid vectors, artificial chromosome vectors, or, in some aspects of the invention, viral vectors. Examples of viral vectors include adenovirus, herpes simplex virus (HSV), alphavirus, simian virus 40, picornavirus, vaccinia virus, retrovirus, lentivirus, and adeno-associated virus. In one embodiment, the vector is a plasmid. In one embodiment, a vector is capable of replication in the cell to which it is introduced; in another embodiment the vector is not capable of replication. In one embodiment, the vector is capable of integrating into genomic DNA of a recipient cell. In one

embodiment, the vector is unable to mediate the integration of the vector sequences into the genomic DNA of a cell. An example of a vector that can mediate the integration of the vector sequences into the genomic DNA of a cell is a retroviral vector or a lentiviral vector, in which an integrase mediates integration of the retroviral vector sequences.

A vector may replicate in a eukaryotic cell. In such embodiments a vector includes an origin of replication. Examples of origins of replication are known in the art and are routinely used. In one embodiment, an origin is the OriP origin from an Epstein Barr virus. OriP is a polynucleotide sequence to which an EBNA-1 polypeptide binds to result in replication of a polynucleotide containing the OriP polynucleotide. The EBNA-1 polypeptide may be encoded by a coding region present in the vector and acting in cis, or may be encoded by a coding region not present in the vector and acting in trans.

Examples of nucleotide sequences that function as an OriP and encode an EBNA-1 polypeptide are readily available to the skilled person. Other origins of replication may also be used for replication of a vector in a eukaryotic cell.

A vector may include polynucleotide sequences that help to integrate the vector into genomic DNA of a cell. In one embodiment, a vector includes additional polynucleotides that result in integration of the vector, for instance a plasmid, into genomic DNA of a cell. Examples of suitable transposons for use in integrating a vector into a cell's genomic DNA include, but are not limited to, Tol2, Frog Prince, P-elements, and Passport. Examples of suitable viral polynucleotides for use in integrating a vector into a cell's genomic DNA include, but are not limited to, lentivirus (e.g., modified HIV- 1, SIN, FrV), retrovirus (e.g., ASV, ALV or MoMLV), and adenovirus (e.g., AAV). Examples of suitable polynucleotides that mediate site-specific integration for use in integrating a vector into a cell's genomic DNA include, but are not limited to, LoxP (for use with Cre recombinase) and FRT (for use with FLP recombinase).

A vector may include a coding sequence encoding a polypeptide that permits the eukaryotic cell containing the polypeptide to be identified. Selectable markers permit the selection of cells containing the selectable marker. An example of a type of selectable marker is drug resistance, including, for instance, resistance to hygromycin, puromycin, or the neomycin analog G418. Detectable markers may permit detection of cells containing the detectable marker. Examples of such detectable markers that can be used in this way include fluorescent proteins (e.g., green, yellow, blue, or red fluorescent proteins such as DsRed), luciferase, and chloramphenicol acetyl transferase, β- galactosidase, and other molecules detectable by their fluorescence, enzymatic activity or immunological properties, and are typically useful when detected in a cell.

A vector may include polynucleotides to permit replication in a prokaryotic cell and selection of prokaryotic cells containing a vector. Origins of replication and selectable markers useful for replication and selection in prokaryotic cells are known to the skilled person, are routinely used, and are readily available.

Construction of vectors employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) or Ausubel, R.M., Ed. Current Protocols in Molecular Biology (1994).

In one embodiment, a vector includes a transposable element. A transposable element, also referred to herein as a "transposon," is a polynucleotide that is able to excise from a donor polynucleotide, for instance, a vector, and integrate into a target site, for instance, a cell's genomic DNA. A transposon includes a polynucleotide that includes a nucleic acid sequence flanked by cis-acting nucleotide sequences located at the termini of the transposon.

A nucleic acid sequence is "flanked by" cis-acting nucleotide sequences if at least one cis-acting nucleotide sequence is positioned 5' to the nucleic acid sequence, and at least one cis-acting nucleotide sequence is positioned 3' to the nucleic acid sequence. A nucleic acid sequence flanked by cis-acting nucleotide sequences may be referred to herein as a "flanked sequence." Cis-acting nucleotide sequences include at least one inverted repeat at each end of the transposon, to which a transposase binds. Transposases is described in greater detail herein.

In one embodiment, a transposon present in a vector is a "Sleeping Beauty" transposon, also referred to herein as SB transposon. Various examples of SB

transposons are known to the skilled person and are routinely used (Ivies et al., 1997, Cell, 91:501-510; U.S. Patent 6,489,458 (Hackett et al.); U.S. Patent 7,160,682 (Hackett et al.), U.S. Appl. Serial No. 09/569,257 (Mclvor et al), U.S. Appl. No. 20050003542 (Kay et al.), U.S. Appl. No. 20060026699 (Largaespada et al.), U.S. Appl. No.

20040077572 (Hackett et al), Mates et al., 2009, Nat Genet, 41(6):753-761, Cui et al., 2002, J. Mol. Biol., 318:1221-1235, Geurts et al., 2003, Mol. Ther., 8:108-117, and Liu et al., 2004 J. Gene. Med., 6:574-583).

In one embodiment when the transposon present in a vector is an SB transposon, the vector includes specific nucleotide sequences that are juxtaposed to the transposon. For instance, a vector includes a "TAACCC" on one the right side of the transposon and a "GGGGA" on the left side of the transposon, or an "AAATA" on the right side of the transposon and a "TGTCT" on the left side of the transposon, or a "TTGAT" on one the right side of the transposon and a "CTCGG" on the left side of the transposon, or a "TGCCT" on one the right side of the transposon and a "ACGTA" on the left side of the transposon. In one embodiment, the vector includes specific nucleotide sequences which are juxtaposed to the transposon, and increase the frequency of transposition of the transposon compared to the frequency of transposition of the transposon when the vector includes, for instance, a "TAACCC on the right side of the transposon and a "GGGGA" on the left side of the transposon. In one embodiment, a vector includes a "TATA" nucleotide sequence that is present the left side of the transposon, or an "AT AT" on the right side of the transposon. In one embodiment, the vector includes a "TATA" nucleotide sequence that is present on the left side, and an "AT AT" on the right side of the transposon. Alternatively, the vector may include a "TGATA" on the right side of the transposon and a "CTGTA" on the left side of the transposon. In one embodiment, the vector does not include a "TTAAG" on one the right side of the transposon and an " AATAA" on the left side of the transposon, or an " AACTA" on one the right side of the transposon and a "TGGCT" on the left side of the transposon, or an "AGCCA" on one the right side of the transposon and a "TAGTT" on the left side of the transposon.

In one embodiment, a transposon present in a vector is a "PiggyBac" transposon, also referred to herein as PB transposon. Various examples of PB transposons are known to the skilled person and are routinely used (Wilson et al., 2007, Mol. Ther., 15:139-145; Rad et al., 2010, Science, 330:1104-1107, U.S. Appl. No. 20020173634 (Fraser et al.), U.S. Appl. No. 20070204356 (Fraser), U.S. Appl. No. 20100221824 (Fraser and Li), Yusa et al., 2011, Proc. Natl. Acad. Sci. USA, 108:1531-1536).

The flanked sequence may include one or more nucleic acid sequences that act as insertional mutagens. An insertional mutagen is a nucleic acid sequence whose insertion will affect the level of expression or the nature of the product expressed by a coding region near or in which the flanked sequence is inserted by transposition. When the nature of the product expressed is altered, the nucleic acid is referred to as a "disruptive sequence." When the level of expression is altered, the nucleic acid is referred to as an "affective sequence." Transposons of the present invention may include one or more insertional mutagens, which may be disruptive and/or affective sequences. In one embodiment, the flanked sequence includes a non-coding sequence that can alter the nature of the product expressed by a coding region when the transposon inserts the nucleic acid sequence in or near that coding region in a cell. Any nucleotide sequence that will alter the nature of the product expressed by a coding region present in the cell can be used. Examples of disruptive sequences include stop codons in each of the possible frames, transcription terminators, splice acceptor sites, splice donor sites, silencer elements, and the like. A disruptive sequence may, for example, lead to the formation of a truncated protein during protein expression.

In one embodiment, the disruptive sequence includes a splice acceptor (SA) site. A splice acceptor site is a nucleotide sequence that is generally involved in RNA splicing to remove intronic RNA sequences. While not intending to be bound by theory, the splice acceptor site is normally involved in the excision of introns, during which it is bound by an RNA-protein complex referred to as a spliceosome, cleaved, and then joined to a splice donor site that has already been cleaved, resulting in the excision of an mtervening portion of the nucleotide sequence in a lariat formation. Splice acceptor sequences are well known in the art, and can be readily obtained from genes at a position between the exon and intron where they mediate splicing. Alternately, SA sites may be chemically or enzymatically synthesized. Whether a polynucleotide functions as a splice acceptor can be easily determined using methods known in the art. Splice Acceptor (S A) sites typically end in AG dinucleotides that are highly conserved. The remaining nucleotides of the sequence are primarily cytidine or thymidine. Exemplary SA sites include the nucleotide sequences 5 ' CCCCCCCCCCCNCAG-3 ' and 5 ' TTTTTTTTTTTNTAG-3 ' , where N represents a nucleotide that can be either A, G, C, or T. In one embodiment an S A site is the engrailed-2 (En2) S A. En2 is a well-characterized splice acceptor used for gene trap mutagenesis in mouse embryonic cells (Skarnes et al., 1992, Genes Dev.,

6:903-918). In another embodiment, the flanked sequence includes two splice acceptor sites, with the second splice acceptor site being provided in an orientation opposite that of the first splice acceptor site. This allows a splice acceptor site to be properly read during transcription regardless of the orientation of the transposon. Splice acceptor sites, as well as splice donor sites, are described in further detail by Padgett et al. (1988, Ann. Rev. Biochem. J., 55:1119-1150). In one embodiment, the flanked sequence including a splice acceptor site also includes a transcription termination signal site operably linked to the SA site. A termination signal site may be, for example, a polyadenylation (pA) signal site. If there are two S A sites, this may result in the pA signal sites being positioned between the two SA sites, due to the opposite orientation of the SA sites. In one embodiment, the two pA sites, positioned between the two S A sites, may be replaced by a single, bidirectional pA site. Polyadenylation signal sites are well known by those skilled in the art, and can be readily obtained from genes where they are used to terminate transcription, or can be chemically or enzymatically synthesized. Whether a polynucleotide functions as a polyadenation signal can be easily determined using methods known in the art. While not intending to be bound by theory, the pA signal site provides a signal to polyadenylate a cleavage site that typically occurs about 15-30 nucleotides downstream from the pA signal site. Polyadenylation generally results in the addition of about 200 adenylate (AMP) residues to form a poly(A) tail on the mRNA formed. A polyadenylation (pA) signal site preferably includes the nucleotide sequence AAUAAA. The provision of two SA sites with downstream pA sites facilitates gene trapping that can terminate transcription when integrated in either orientation in a gene when the flanked sequence is inserted downstream from a coding sequence, as splicing will occur during transcription between the SA site of the flanked sequence and the SD site of an upstream gene.

In one embodiment, the disruptive sequence includes a splice donor (SD). Splice donors are described in further detail, herein. Splice donors may also result in truncation of an expressed protein by insertion within an intron region within a protein.

In another embodiment, the flanked sequence includes a non-coding sequence that can alter the level of expression of a coding region when the transposon inserts near that coding region in a cell, referred to herein as "affective sequences." The affective sequence may either increase or decrease the level of expression of a coding region. Any nucleotide sequence that will alter the level of expression of a coding region present in a cell can be used. Examples of affective sequences include enhancers, promoters, matrix attachment sequences, transcription binding sites, and the like. Matrix-attached regions (MARs) have been demonstrated to nest origins of replication and transcriptional enhancers, and rules have been proposed to facilitate the classification of a DNA sequence as a matrix attachment region (Boulikas, 1993, J. Cell Biochem., 52:14-22). A transcription binding site is a nucleotide region with an affinity for transcription factors that alters the expression of a coding region upon binding by a transcription factor. Transcription binding sites are generally found within promoters or enhancers.

Transcription factors include, for example, homeodomain proteins, POU transcription factors, DNA bending proteins, and zinc finger transcription factors. Useful promoters, enhancers, and transcription binding sites are readily available and known to those skilled in the art. Affective sequences may, for example, lead to the increased expression of a signal transduction protein produced by a coding region.

An example of an affective sequence is a promoter. Various promoters are readily available to the skilled person and are used routinely. Useful promoters include constitutive promoters, tissue specific promoters, and developmental stage specific promoters. For example, promoters include the human cytomegalovirus immediate early promoter and the EFl promoter, which are constitutive promoters, and the rat probascin 1 promoter and the Pax2 promoter, which are active in the prostate gland and the developing hindbrain, eye, and urogenital system, respectively. In one embodiment, the promoter is a strong promoter; e.g., it is able to cause a significant increase in expression of an operably linked coding region. Useful promoters include promoters that function in many different types of cells, for instance, lung cells, gastrointestinal tract cells, and brain cells. In one embodiment, this promoter sequence is a long terminal repeat (LTR) sequence. LTR sequences are preferred as they are strong and ubiquitous, and have been shown to be capable of activating oncogenes upon insertion. A particularly preferred LTR is the LTR of the Murine stem cell virus (MSCV). LTRs are retroviral

transcriptional control sequences that contain identical sequences that can be divided into three elements; U3, R, and U5. The U3 region of an LTR typically includes both a promoter and an enhancer. Further information on LTRs may be found in Retroviruses, eds. Coffin et al., p. 205-261 (1997).

In one embodiment, the flanked sequence including an affective sequence also includes a splice donor site operably linked to the affective sequence. A splice donor site is a nucleotide sequence that is generally involved in RNA splicing to remove intronic RNA sequences. While not intending to be bound by theory, the splice donor site typically is cleaved by nucleophilic attack at the 5' splice junction and is then bound to a splice acceptor site after cleavage at the 3' splice junction. The splicing mechanism that removes an intron utilizing the splice donor and splice acceptor sites is mediated by a spliceosome. Splice Donor sites typically end in GT (or GU) dinucleotides that are highly conserved. Splice donor sequences are well known in the art, and can be readily obtained from genes at a position between the exon and intron where they mediate splicing. Alternately, SD sites may be chemically or enzymatically synthesized.

Whether a polynucleotide functions as a splice donor can be easily determined using methods known in the art. Preferred SD sites include the nucleotide sequences

GTAAGT and GTGAGT. If two splice acceptor sites are provided, the splice donor site may be positioned between the two SA sites. This results in the upstream SA being in the improper orientation, thus avoiding mere excision of a portion of the flanked sequence.

A transposon of the present invention may include one or more disruptive sequences and one or more affective sequences, or a combination thereof. In combination with the different transposon cis-acting elements, many embodiments are possible. One embodiment is shown in Fig. 3 A, which provides a map of a

polynucleotide from a plasmid, where the polynucleotide includes an OncX transposon, which contains, going from the 5' to the 3' end, a piggyBac ITR sequence, a Sleeping Beauty IR/DR sequence, a first SA site, a first pA site, a promoter (Cagg), an SD site, and (in inverted orientation), a second pA site and a second SA, flanked at the end by a Sleeping Beauty IR/DR sequence and a piggyBac ITR sequence, marking the end of the transposon.

In one embodiment, a vector may include at least 1 transposon, at least 2 transposons, at least 3 transposons, at least 4 transposons, at least 5 transposons, at least 6 transposons, at least 7 transposons, at least 8 transposons, at least 9 transposons, at least 10 transposons, at least 11 transposons, or at least 12 transposons.

In one embodiment, a vector includes a coding region encoding a transposase. A transposase is a polypeptide that binds an inverted repeat or a direct repeat of a transposon and catalyzes the excision of a transposon from a donor polynucleotide (e.g., a vector) and subsequent integration of the transposon into the genomic DNA of a cell. The transposase may be present as a polypeptide. Alternatively, the transposase is present as a polynucleotide that includes a coding sequence encoding a transposase. The polynucleotide can be RNA, for instance an mRNA encoding the transposase, or DNA, for instance a coding sequence encoding the transposase. The polynucleotide encoding a transposase may be on a vector, or present in a chromosome. When the transposase is present as a coding sequence encoding the transposase, in some aspects of the invention the coding sequence may be present on the same polynucleotide (e.g., a vector) that includes the transposon, i.e., in cis. In other aspects of the invention, the transposase coding sequence may be present on a second polynucleotide (e.g., a vector), i.e., in trans.

In one embodiment, a transposase present in a vector is a "Sleeping Beauty" transposase, referred to herein as SB transposase. Various examples of SB transposase are known to the skilled person and are routinely used (Ivies et al, 1997, Cell, 91:501- 510; U.S. Patent 6,489,458 (Hackett et al.); U.S. Patent 7,160,682 (Hackett et al), U.S. Appl. Serial No. 09/569,257 (Mclvor et al.), U.S. Appl. No. 20050003542 (Kay et al.), U.S. Appl. No. 20060026699 (Largaespada et al), U.S. Appl. No. 20040077572

(Hackett et al.)). In one embodiment SB transposase is SB 11 (see Dupuy et al., 2009, Cancer Res., 69:8150, U.S. Appl. No. 20040077572 (Hackett et al.)), and hi one embodiment SB transposase is SB100 (see Mates et al., 2009, Nat Genet, 41 :753-761, U.S. Appl. No. 20110117072 (Izsvak et al.)).

In one embodiment, a transposase present in a vector is a "PiggyBac" transposase, referred to herein as PB transposase. Various examples of PB transposase are known to the skilled person and are routinely used (Wilson et al., 2007, Mol. Ther., 15:139-145; Rad et al., 2010, Science, 330:1104-1107, U.S. Appl. No. 20020173634 (Fraser et al), 20070204356 (Fraser), 20100221824 (Fraser and Li)). In one embodiment PB

transposase is PB7 (see Yusa et al, 2011, Proc. Nat. Acad. Sci. USA, 108:1531-1536).

A coding region encoding a transposase may be operably linked to a promoter. Useful promoters include, for example, constitutive promoters, tissue specific promoters, and developmental stage specific promoters. Useful promoters include, but are not limited to, the CAGGS promoter, the PGK promoter, the human cytomegalovirus immediate early promoter and the EFl promoter. Useful promoters also include inducible promoters such as, for instance, tet operator sequences (see, for instance, Bujard et al. (WO 96/01313)). Another example of a promoter is provided by the Rosa26 locus. The ubiquitous Rosa promoter is provided by the ROSA26 mutant cell line, produced by the combination of embryonic stem cells with the ROSA geo retrovirus (see U.S. Appl. No. 20060026699 (Largaespada et al.)). The coding region encoding a transposase may also be operably linked to an enhancer. Enhancers are further defined herein. Enhancers are readily available, and are well known to those skilled in the art. See, for example, Blackwood et al., Science, 281, 60 (1998). In one embodiment, an enhancer is used in combination with a promoter. For example one embodiment of the invention uses the CAGGS combined promoter/enhancer, which is a chimeric promoter containing a chicken β-actin promoter and a cytomegalovirus enhancer. The transposase coding region can be operably linked to the promoters and/or enhancers through use of a poly(A) trap vector, or by other means known to those skilled in the art.

In one embodiment, a vector may include one or more transposons and one or more coding regions encoding a transposase.

The present invention provides methods for using the vectors described herein. The invention allows efficient insertion of genetic material into the genomic DNA of a cell for the mutation, evaluation of function, and subsequent cloning of genomic DNA, such as coding sequences and/or genomic regulatory sequences. The methods may be used to identify genomic DNA sequences involved in any cell phenotype that can be induced by the overexpression or decreased expression of a coding region and observed.

The type of cell used in the method is not intended to be limiting, and essentially any cell may be used. For instance, the cell may be a primary cell (e.g., a cell that have recently been removed from a subject and is capable of limited growth in tissue culture medium), or a cultured cell (e.g., a cell that is capable of long term culture in tissue culture medium). A cell may be tumorigenic (e.g., able to form tumors when

administered to an immuno-incompetent animal, such as a nude mouse), or non- tumorigenic. Primary cells and cultured cells are referred to herein as "in vitro" cells. As used herein, the term "in vivo" refers to a cell that is within the body of an ariimal. The cell can be from any animal, including, but not limited to, a vertebrate, including mice, rats, humans, non-human primates, livestock (e.g., pig, horse, cow, goat, sheep), fish (e.g., zebrafish), etc. Cells explanted from an animal may from any location and/or any tissue including, but not limited to, lymphocytes, hepatocytes, neural cells, muscle cells, a variety of blood cells, stem cells, tumor cells (metastatic and benign), pluripotent cells, and totipotent cells. In one embodiment the method includes providing a cell that includes one or more of the vectors described herein. In one embodiment the method includes introducing into a cell one or more of the vectors described herein. The vector having the transposons may replicate in the cell or may be integrated into genomic DNA of the cell. The vector encoding the transposase may replicate in the cell or may be integrated into genomic DNA of the cell. In one embodiment, a vector may include both transposons and a polynucleotide encoding a transposase, and such a vector may replicate in the cell or may be integrated in genomic DNA of the cell.

The method by which the transposon and/or transposase are introduced to the cell is not intended to be a limiting aspect of the present invention. For instance, the transposon and/or transposase can be introduced by any suitable method, and such methods are known to the skilled person and routinely used. Methods include, but are not limited to, anionic or cationic lipid, or other standard transfection mechanisms including liposomes, electroporation, particle bombardment, hydrodynamic injection, or microinjection used for eukaryotic cells.

The cells may be incubated under conditions suitable for transposition of the transposon from a vector into genomic DNA of the cells. This can be allowed to continue to increase the number of insertions present in a single cell.

The method may further include observing the cells to determine if a phenotype has changed. Examples of altered phenotypes include, but are not limited to, drug resistance, tumor formation, cancer metastasis, and cell transformation. For instance, in one embodiment, the methods include detecting a cell having characteristics of a tumor after transposition has been allowed to occur. For instance, in one embodiment, after transposition has been allowed to occur the cells may be exposed to a compound at a concentration that inhibits, including kills, control cells that were not exposed to the transposon. The presence of cells resistant to the compound suggests that a transposon has inserted into or near a coding region that is involved in resistance to the compound. Any compound that is toxic to cells may be used, and examples include alkylating agents, antimetabolites, anthracyclines, plant alkaloids, or topoisomerase inhibitors. In one embodiment, expression of the transposase is continued until a phenotype is observed. The method optionally includes mapping the location of the transposons present in a cell having an observed phenotype. The locations of the transposons can then be used to identify the genomic coding sequences and or/genomic regulatory sequences altered by insertion of the transposons. By identifying genomic coding sequences and/or regulatory sequences whose alteration is commonly associated with a tumor or other phenotype, the function of these sequences may be characterized. For example, a genomic coding sequence and/or regulatory sequence that has been commonly altered in tumor tissue may be characterized as a tumor associated gene. Examples of tumor- associated genes include proto-oncogenes, oncogenes, and tumor suppressor genes.

Transposons of the present invention preferably include an insertional mutagen which increases the ability of the transposon to induce a tumor or other phenotypic change upon insertion into a genomic coding sequence and/or regulatory sequence of a cell. Alteration of the nucleic acid sequence will, in turn, affect the level of expression or the nature of the product expressed. When the nature of the product expressed is altered, the nucleic acid is referred to as a disruptive sequence. When the level of expression is altered, the nucleic acid is referred to as an affective sequence. A disruptive sequence can induce various types of mutations, including, for example, C-terminal truncations, N- terminal truncations, and insertion of promoters and/or enhancers.

A disruptive sequence may include a splice acceptor (S A) and a transcription termination signal site such as a polyadenylation (pA) signal site. Without intending to be limited by theory, if a transposon containing a disruptive sequence containing these sequences is inserted downstream from a gene, the S A and pA site combination will splice to the nearby gene and end transcription. Preferably, by providing a disruptive sequence with splice acceptor and transcription termination signals in both orientations, the transposon can act as a disruptive sequence when inserted in either orientation.

Truncation of a protein, such as a kinase, for example, may result in the removal of regulatory regions of the kinase, converting the kinase from a proto-oncogene to an oncogene.

An affective sequence may include a splice donor (SD) and a promoter. Without intending to be limited by theory, if an affective sequence is inserted upstream from a gene, the SD and promoter combination will splice to a nearby gene and enhance the production of that gene via the promoter activity. Should the gene affected be an oncogene or other gene that stimulates cell proliferation or other tumor-related activity, the resultant increased expression will also tend to encourage tumor formation.

Tumor formation is generally stimulated either by activation of an oncogene or proto-oncogene, or through inhibition of a tumor suppressor gene. Tumor formation may also be stimulated by genetic changes that result in a variety of subsequent cellular changes including, but not limited to, angiogenesis upregulation, growth factor independence, cell cycle progression, metastasis, invasiveness, inhibition of apoptosis, suppression of differentiation, and evasion of immune surveillance.

An oncogene, as defined herein, is a gene that can cause a cell to develop into a tumor cell. Oncogenes typically encode growth factors or protein kinases such as, for example, tyrosine kinases and GTPases. A proto-oncogene, as defined herein, is a gene whose protein product has the capacity to induce cellular transformation if it sustains a genetic insult. Activation to convert a proto-oncogene to an oncogene generally involves either a mutation of the proto-oncogene or an increased concentration of the product of the proto-oncogene, through an increase in product expression, stability, or gene duplication. A tumor suppressor gene, as defined herein, is a gene that reduces the probability that a cell will turn into a tumor cell. A mutation or deletion of such a gene will increase the probability of the cell containing the damaged gene to become a tumor cell. On the other hand, increased production of a tumor suppressor gene through, for example, increased promoter activity, can decrease tumor formation. Tumor suppressor genes also include growth suppressors, recessive oncogenes, and anti-oncogenes.

Examples of proto-oncogenes, that can become oncogenes when mutated, include Erbb2, Kras, Src, Bcl2, and telomerase-encoding genes. Examples of oncogenes include erbB- 2, Ras, Src, Bcl-2, and telomerase-encoding genes. Examples of genes that encode important tumor suppressors include p53, Rb, APC, and BRCA.

A cell into which a transposon-containing vector has been introduced may be assayed for the presence of a phenotype that is not present, or present to a different degree, in the same cell that does not include the transposon integrated in its genome. In one embodiment, an altered phenotype can be observed visually by eye, for instance with the naked eye. In one embodiment, an altered phenotype can be observed by detecting or measuring using other methods, such as, but not limited to, histological analysis, or by other methods appropriate to the phenotype being evaluated. Transposons may also result in the expression of multiple phenotypes by the cell.

Methods for mapping the location of a particular polynucleotide sequence such as a transposon are known in the art and are routinely used. Examples include, for instance, in situ hybridization, such as fluorescence in situ hybridization. In situ hybridization methods typically use a polynucleotide probe that is complementary to and will hybridize with nucleotides of the transposon. The conditions for hybridizing a polynucleotide probe to a transposon vary depending upon the polynucleotide sequence of the probe, and methods for determining such conditions are known in the art.

A preferred method for mapping the location of a particular polynucleotide sequence is determining the sequence of the cell's genomic DNA that flanks the transposon. Several methods are known in the art for determining the sequence of the cell's genomic DNA that flanks the transposon, and include, for mstance, polymerase chain reaction (PCR) based methods. PCR based methods include, for instance, inverse PCR and various linker-mediated PCR techniques. Linkers are used in ligation-mediated (LM) PCR, for example, a cloning strategy that is used in a preferred embodiment of the invention to determine the location of transposon insertions. LM-PCR cloning is described in greater detail in Example 2, herein. Chromosomal flanking sequences can also be recovered using a plasmid rescue technique when the transposon includes sequences that support plasmid replication when introduced into E. coli.

Inverse PCR typically includes digesting the genomic DNA containing the transposon with a restriction endonuclease that does not cut the transposon, ligating the polynucleotides at a low concentration to promote intramolecular ligation, and then using primers that hybridize to different strands of the transposon and point outward from the transposon. The amplification takes place between the two primers and across the ligation junction, including both upstream and downstream chromosomal flanking sequences. The polynucleotide sequence of the amplified polynucleotide can then be determined, and compared to the known and publicly available databases containing genomic sequences of mammals such as, for instance, mouse, rat, or primate (e.g., monkey). Such methods are known to the art (see, for example, Hackett et al., WO

99/25817). The nucleotide sequence of primers useful for a PCR based method, and the conditions for amplifying a polynucleotide, will vary depending upon the nucleotide sequence of the transposon. Methods for determining useful primers and amplification conditions are routine in the art. A primer typically has at least 15 nucleotides, preferably, at least 20 nucleotides, most preferably, at least 25 nucleotides.

The location of the transposon can also be determined using a restriction endonuclease capable of cleaving a restriction site within the transposon. This yields at least one restriction fragment containing at least a portion of the integrated transposon, which portion includes at least a portion of an inverted repeat sequence along with an amount of genomic DNA of the cell that is adjacent to the inverted repeat sequence. The specificities of numerous endonucleases are well known and can be found in a variety of publications, e.g. Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: New York (1989). The polynucleotide of the transposon thus preferably includes a restriction endonuclease recognition site, preferably a 6-base recognition sequence. Following insertion of the transposon into the genomic DNA of the cell, the DNA is isolated and digested with the restriction endonuclease. Where a restriction endonuclease is used that employs a 6-base recognition sequence, the cell DNA is cut into about 4000- base pair restriction fragments on average. In those embodiments using SB transposon, since the site of DNA insertion mediated by the SB transposase generally occurs at TA base pairs and the TA base pairs are typically duplicated such that an integrated nucleic acid fragment is flanked by TA base pairs, TA base pairs will be immediately adjacent to an integrated transposon. The genomic DNA of the genomic fragment is typically immediately adjacent to the TA base pairs on either side of the integrated polynucleotide.

After the DNA of the cell is digested, the resulting fragments can be cloned in a vector using methods well known to the art, thereby allowing the identification of individual clones containing genomic fragments that include at least a portion of the inserted transposon and genomic DNA of the cell adjacent to the integrated transposon. A non-limiting example of identifying the desired genomic fragments is hybridization with a probe complementary to the sequence of the inverted repeats. Alternatively, linkers can be added to the ends of the digested fragments to provide complementary sequence for PCR primers. Where linkers are added, PCR reactions are used to amplify fragments using primers from the linkers and primers binding to a nucleotide sequence within the inverted repeats. Methods may be used to determine the identity of genes associated with changes in phenotype. The use of transposons with flanked sequences that include insertional mutagens may lead to phenotypic changes such as tumor formation as a result of a variety of genetic changes in the cells into which the vectors of the present invention have been introduced. The degree to which cells into which vectors have been introduced and integrated are prone to tumor development depends, in part, on the number of transposons integrated into genomic DNA in the cells. In one embodiment, the method includes the use of a vector having multiple transposons and a transposase operably linked to an inducible promoter. The polynucleotide encoding the transposase may be present on the same vector as the transposons or a separate vector. Expression of the transposase may be continued until a desired phenotype is observed.

Once a change in phenotype has been induced in a cell, the cells displaying the phenotype may be isolated and their genetic makeup characterized. The nature of the genetic changes associated with the transposition of transposons from vectors of the present invention within a cell can be evaluated using the methods of genomic analysis described herein. This can be done to evaluate, for example, the genetic changes involved in a phenotype, and identify groups of genes that are involved in a phenotype. For instance, the present invention can be used to identify genes associated with an increase or decrease in the ability to form a tumor, an increase or a decrease in drug resistance, a change in the transformation state of a cell, or a change in the metastatic potential of a cell. Changes in the transformation state of a cell include, for instance, a change in anchorage dependent growth of a cell (where increased anchorage independent growth correlates with increased transformation), or a change in contact inhibition (wherein decreased contact inhibition correlates with increased transformation). Changes in the metastatic potential of a cell include, for instance, increased migration of a cell (where increased migration of a cell correlates with increased metastatic potential).

Methods for measuring these phenotypes are known to the skilled person and are routine. For example, an increase in the ability to form a tumor may be determined by evaluating the ability of a cell line to replicate long term in tissue culture medium, or by

administrating cells to an immunoincompetent animal, such as a nude mouse.

In one embodiment, cooperating genes associated with a change in phenotype, such as tumor formation, can be identified in cells in which transposons have been introduced. Cooperating genes are a plurality of genes that have an additive or synergistic effect in causing a cell to become a tumor cell when these genes have been altered by insertion of transposons of the present invention in or near the genes. For example, cooperating genes may be a plurality of genes that function to provide proteins involved in a particular signaling pathway. Cooperating genes include coding sequences that express proteins that interact, as well as regulatory sequences that may regulate the expression of coding regions.

The present invention also provides a kit for detecting a coding region involved in a phenotype. The kit includes one or more of the vectors described herein in a suitable packaging material in an amount sufficient for transformation into cells to permit replication of the vectors. Optionally, other reagents such as buffers and solutions needed to practice the invention are also included. Instructions for use of the packaged polypeptide or primer pair are also typically included.

As used herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the vectors can be used for the intended purpose. In addition, the packaging material contains instructions indicating how the materials within the kit are employed. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the vectors. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of vector and cell to be admixed, temperature, buffer conditions, and the like.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. EXAMPLE 1

Development and Evaluation of a Highly Versatile, Unbiased Transposon Mutagenesis System for use in Cultured Cells

We set out to develop a highly versatile unbiased transposon mutagenesis system for use in a wide variety of cell types to identify genes involved with a broad spectrum of phenotypes, including: drug resistance, cancer metastasis, cell transformation, and essentially any observable phenotype that is induced by overexpressing or inactivating genes. To this end, we have engineered an Epstien Bar Virus (EBV) based self- replicating vector harboring a concatemers of mutagenic transposons. These vectors are reportedly maintained at high copy numbers, up to 20 per cell, over time under drug selection. EBV vectors are also known to replicate only once per cell cycle and segregate evenly along with endogenous chromosomes. The mutagenic transposon used to create these concatemers has been designed with both Sleeping Beauty (SB) and

PiggyBac (PB) Inverted terrninal repeats allowing for screens utilizing either SB or PB transposase. The transposon cargo contains a Cagg promoter with splice donor to induce overexpression of nearby genes and splice acceptors with poly adenylation signals in both orientations to inactivate genes. We have constructed transposon concatemers in the EBV vector ranging from 1-8 copies (13-35kb). Hygromycin resistance and dsRED genes have also been included in all vectors for the purpose of drug selection and identification of vector presence, respectfully. In conjunction with these vectors, we have created a multitude of constitutive and tetracycline regulatable bidirectional lentiviral vectors that express hyperactive mutants of either SB or PB, namely SB 100 or PB7, along with other useful genes such luciferase, eGFP, and Puromycin. We have been able to demonstrate that our vectors are maintained over time under hygomycin selection and that high copy numbers per cell are achievable, as measured by dsRED intensity. Additionally, PCR based excision assays have shown that transposition is occurring from the episomal concatemer and the canonical SB footprints are observed in vectors recovered from transfected cells expressing SB. Currently, we are validating this system by performing a metastasis screen using non-metastatic NIH 3T3 cells in athymic mice and an AxaC drug resistance screen in human AML cell lines. Materials & Methods Vector construction

pPBT20ncX^ACAG was used as a template to amplify the PB-SB-OncX

transposon; Nhel, Ascl, Afel and NgoMIV, Sbfi, Swal restriction enzyme sites were engineered into the forward and reverse primer, respectively. This PCR product was then TOPO-cloned into pCR-Blunt II-TOPO vector (Invitrogen) and sequence verified. PB-SB-OncX was then cloned into pENTR-PGK-dsRED within the attL-attR gateway system (Invitrogen) sites just upstream of PGK using Nhel-NgoMIV sites to NgoMPv^"- Avrll of pENTR-PGK-dsRED to produce pENTR- 1 OncX-PGK-dsRED. A second copy of PB-SB-OncX was then cloned upstream of the first PB-SB-OncX via the NgoMIV- Afel to NgoMIV-Swal of pENTR-lOncX-PGK-dsRED, again within the attL-attR sites, to create pENTR-20ncX-PGK-dsRED. One copy of PB-SB-OncX was then cloned into pCEP4 (Invitrogen) using PvuII->MscI to a single PvuII site in the MCS of pCEP4 to create pCEP4-10ncX. A second copy of PB-SB-OncX was then cloned into pCEP4- lOncX using NheI->NgoMiV to NheI->NgoMTV sites of pCEP4-10ncX to make pCEP4-20ncX. The original pCEP4 CMV promoter upsteam of the MCS was then removed by Bglll digest and self-ligation to reduce the over-all size of pCEP4-20ncX. The Gateway system DEST cassette was then PCR amplified using the pDEST40 vector (Invitrogen) as a template and cloned between the 2 copies of the PB-SB-OncX transposons in pCEP4-20ncX using Nhel- Ascl sites of both insert and vector to make pCEP4-DEST-20ncX. We then performed L-R Clonase reactions between pCEP4- DEST-20ncX & pENTR-PGK-dsRED to make _PCEP4-20ncX-dsRED, or pCEP4- DEST-20ncX & pENTR-PGK-dsRED- 1 OncX to make pCEP4-30ncX-dsRED, or pCEP4-DEST-20ncX & pENTR-PGK-dsRED-20ncX to make pCEP4-40ncX-dsRED. Another DEST cassette was then blunt cloned into pCEP4-40ncX in place of the PGK- dsRED-pA cassette, which was removed by AfeI->PvuII sites to create pCEP4-DEST- 40ncX. The L-R Clonase reaction was then performed between pCEP4-DEST-40ncX & pENTR-PGK-dsRED-20ncX to create pCEP4-60ncX. This procedure of replacing the PGK-dsRED cassette with the DEST cassette followed by L-R Clonase reaction was repeated to form pCEP4-80ncX. In order to produce the pCEP4-50ncX-PB7-ERt2 vector, CMV-PB7-ERt2-pA was first cloned into pENTR- 1 OncX-PGK-dsRED using the NruI-NgoMIV sites in pcDNA-PB7-ERt2 to the Swal-NgoMIV sites in the pENTR- lOncX-PGK-dsRED plasmid and cloned upstream of lOncX to create pENTR-CMV- PB7-ERt2-10ncX-PGK-dsRED. The L-R Clonase reaction was then performed between pENTR-CMV-PB7-ERt2-10ncX-PGK-dsRED & pCEP4-DEST-40ncX to create pCEP4-50ncX-PB7-ERt2. For construction of both the _PTol2-50ncX-PB7-ERt2 and pTol2-60ncX we began by cloning the Hygro-DEST-40ncX fragment from pCEP4- DEST-40ncX using Nrul-Pcil to the pTol2 vector cut with NcoI-EcoRV within the ITRs to produce pTol2-DEST-40ncX. The L-R Clonase reaction was then performed between pENTR-CMV-PB7-ERt2-10ncX-PG -dsRED & _PTol2-DEST-40ncX or pENTR- 20ncX-PGK-dsRED & _PTol2-DEST-40ncX to create _PCEP4-50ncX-PB7-ERt2 or pTol2-60ncX, respectively.

Cell culture

Human HEK 293T cells and mouse NIH-3T3 cells were cultured in DMEM containing 10% FBS & l%Pen/Strep. Cells were passaged every 3 days and not allowed to reach confluency.

Plasmid transfection

Plasmids were transfected by lipofection using Lipofectamine 2000 (Invitrogen), following the manufacturer's protocol.

Linker-Mediated PCR

Transposon-chromosome junctions were recovered by ligation-mediated PCR, as described by Collier et al. (2005).

Engineering cells to stably express the 'Transposon Mutagenesis System '

250,000 NIH-3T3 cells were transduced with Lentiviruses expressing SB 100- IRES-GFP at an MOI of ~1 overnight in full media containing 6 ug/mL polybrene. These cells were then expanded and Facs sorted to obtain a pure GFP⁺ population. This GFP⁺ population was then expanded to 80-90% confluency in T- 175 flasks and 40 μg of pCEP4-60ncX vector was transfected using Lipofectamine 2000. These cells were then Facs sorted for GFP ; dsRED population. These cells were then maintained under hygromycin selection at a concentration of 250 μ^ιηΐ and analyzed further.

Vector recovery from mammalian cells

Vectors were recovered from mammalian cells using the Qiagen mini prep kit, as described previously (Qiagen News, Issue No. 2/95). pCEP4-OncX SB excision PCR

PCR primers were designed to bind the PB 3' and 5' ITR and thus flank the SB- OncX portion of the PB-SB-OncX transposon, primer sequences are For: 5'- tgttatttcatgttctacttacgtgat-3' and Rev: 5'-aacaaaacttttatcgaattcctg-3'. PCR was performed using Thermo Taq 2x ReddyMix (Thermo) using the following PCR program: initial denaturing step of 95°C for 2 min; 35 cycles of denatoing at 95°C 25 s, annealing at 55°C for 35 s and extension at 72°C for 1.5 min; followed by a final extension at 72°C for 7 min.

Results

Large transposon concatemers can be created in vitro

To create a highly versatile unbiased transposon mutagenesis system a vector harboring multiple copies of a mutagenic transposon had to be created; however, initial attempts at inserting multiple copies of a mutagenic transposon into a vector to produce a concatamer of transposons failed. To this end, we utilized a combination of traditional cloning and the Gateway cloning system (Invitrogen) to produce transposon concatemer containing vectors. The PB-SB-OncX mutagenic transposon was chosen for its versatility of being able to use either Sleeping Beauty or PiggyBac transposase (Figure 1 A). Additionally, in an attempt to further increase the possible number of mutagenic transposons per cell harboring these transposon concatemer vectors, the concatemers were engineered into the Epstein Barr Virus (EBV) based episomal vector, namely pCEP4 (Invitrogen) (Figure IB).

pCEP4 contains the OriP DNA sequence and EBNA-1 gene, which allows for

EBNA-1 protein binding to OriP and subsequent recruitment of endogenous DNA replication machinery to replicate during normal cell cycle along with endogenous chromosomes. Not only are these vectors self-replicating but also maintained at high copy number per cell (up to 20 copies per cell) and stable over time under drug selection, hygromycin in the case of pCEP4. After using traditional cloning to create pCEP4- DEST-20ncX and _PENTR-PGK-dsRED-20ncX, the Gateway system L-R Clonase reaction was used to produce pCEP4-60ncX. Traditional cloning was again utilized to make pCEP4-DEST-60ncX and the L-R Clonase reaction performed to make the pCEP4-80ncX vector (Figure 2). These cycles could likely be further performed to create vectors up to lOOkb in size; the reported upper limit for EBV based vectors.

Additionally, we wanted to construct an all-in-one vector that has an inducible transposase integrated into the pCEP4 vector along with the mutagenic transposons. This system has the advantage of needing only one transfection into the cell line of interest and thus less manipulation of the cells. This could be useful for experiments where there is little time for manipulation of cells before they need to be replanted into recipient animals or culture system. One example of this is the use of primary cells that can be removed from an animal, minimally manipulated and put back into the same animal, a syngeneic animal, or an immunocompromised animal. Some cells that are used in this manner would be CD34⁺ HSCs or breast epithelial cells, both of which could be amenable to transposon mutagenesis for gene identification. To this end, we have engineered an all-in-one vector that has 5 copies of the PB-SB-OncX mutagenic transposon in addition to a tamoxifen inducible PiggyBac7 transposase, termed pCEP4- 50ncX-PB7-ERt2 (Figure 2d). The transposase is tamoxifen inducible by virtue of fusing a mutated estrogen receptor to the PB7 Transposase. As with the two-component system, the pCEP4-50ncX-PB7-ERt2 vector has dsRED and the Hygromycin resistance gene.

Transposons are mobilized from pCEP4-OncX vectors by Sleeping Beauty

To test the functionality of the mutagenesis system pCEP4-OncX vector were co- transfected into HEK 293 T human cells along with a plasmid expressing SB100-IRES- eGFP. After 24 hrs cells were expressing eGFP and dsRED indicating that they had been successfully transfected with both vectors. These cells were then passaged and expanded; mobilization of mutagenic transposons from the pCEP4-OncX vector by SB 100 transposase was assumed to be occurring during this time. To assess this assumption we recovered pCEP4-OncX vectors from the co-transfected HEK-293T cells after 1 week in culture using a modified mini-perp protocol (see Materials and Methods) and performed a pCEP4-OncX SB PCR excision assay using the recovered vectors as a template. As shown in figure 3 transposition from pCEP4-OncX vectors was occurring and all controls demonstrated expected results.

Cells can be engineered to stably express the 'Transposon Mutagenesis System '

To assess whether the transposon mutagenesis system can be maintained and active stably over time we engineered NEH-3T3 cells using lentiviruses that express SB 100- IRES-eGFP and the pCEP-60ncX vector. Cells were first transduced with a SB 100-

IRES-eGFP lentiviral vector and sorted for eGFP+ cells. These cells were then expanded and transfect with pCEP4-60ncX and FACS sorted for a eGFP⁺; dsRED⁺ population and thereafter maintained under hygromycin selection (figure 4). These cells express eGFP uniformly and dsRED at variable intensities for up to 2 months tested.

SB mediated transposition produces numerous and diverse insertions

Knowing that transposition is occurring from the pCEP4-OncX vectors by SB 100 it was pertinent to assess the extent to which this was occurring. To this end, we extracted genomic DNA from cells both engineered to express the mutagenesis system stably and also from cells transiently transfected with SB 100 expressing plasmid and the pCEP4- OncX vector and performed Ligation mediated-PCR (LM-PCR) to identify transposon insertion sites in these two cell populations. It was found that there were numerous and diverse transposon insertions with both transient and stable expression of the system. Even transient expression produced 'smears' comparable to those that have been observed previously by our lab that induced osteosarcomagenesis in mouse somatic cells undergoing SB mediated mutagenesis for 8-12 months (Figure 5).

Mice Injected with Mutagenized NIH-3T3 Cells Can Develop Lung Nodules

In order to show proof of principle that our in vitro mutagenesis system is functional we undertook a NIH-3T3 metastasis model. It has been previously reported thatNIH-3T3 cells over-expressing different forms of WT of constitutively active Ras can form lung nodules after tail vein injection into nude mice (Bradley et al., 1986). To this end, we injected 500,000 of the aforementioned NIH-3T3 cells, expressing SB 100- IRES-eGFP and the pCEP-60ncX vector, into nude mice by tail vein injection. We also injected control cell lines expressing the pCEP-60ncX or SBlOO-IRES-eGFP alone as negative controls and cells expressing a constitutively active K-Ras V12G as a positive control. The positive control cells induced nodule formation in the lungs of injected nude mice after only 1 month, in accordance with previous studies (Bradley et al., 1986). Up to 1 year after injection none of the pCEP-60ncX or SBlOO-IRES-eGFP alone cells induced lung nodules. Two animals did develop lung nodules after 5.6 and 10 months, respectively. The nodules were similar in appearance to the positive control. We are currently validating that these nodules are truly derived from the injected NIH-3T3 cells and plan to clone out transposon insertion sites to identify the causative genes.

Conclusions and Future Directions

We have successfully created and functionally validated a highly versatile, unbiased transposon mutagenesis system for use in cultured cells. Large transposon concatemers have been engineered in self-replicating, stably maintained EBV based vectors. Transposons are mobilized from the pCEP4-OncX vectors by SB transposase and identified insertion sites are numerous and diverse.

Currently, we are testing the system using PiggyBac transposase in place of SB. Experiments using transient co-transfection of PB plasmid and various pCEP4-OncX vectors into HEK 293 T cells are underway. Additionally, we have undertaken a screen to identify metastasis genes using non-metastatic NIH-3T3 cells by tail vein injection of engineered cells expressing the transposon mutagenesis system stably into nude mice. Two animals injected with the engineered NIH-3T3 cells have developed lung nodules. These nodules are currently being analyzed for transposon insertion sites. This screen was chosen because it has been shown that simple over-expression of the oncogene Ras in NIH-3T3 cells in sufficient to allow metastasis in athymic nude mice. We are also undertaking an AraC drug resistance gene identification screen in human AML cell lines to show that the system can be used to identify genes involved in such an important phenotype as drug resistance. Lastly, we have also created an integratable transposon mutagenesis system using the Tol2 transposon system for use in screens where a cell type is not amenable to EBV based episomes or where an episome is not desired. We hope to validate this system for mutagenesis in the near future.

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The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence

submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety.

Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

What is claimed is:

1. A method for altering a phenotype of a cell, comprising:

incubating a cell comprising at least 2 transposons and a polynucleotide encoding a transposase, wherein the transposase causes transposition of the transposons to genomic DNA of the cell, wherein the incubating is under conditions suitable for growth and results in mutagenized cells comprising transposons that have transposed to genomic DNA;

observing a phenotype in the mutagenized cells, wherein the phenotype is not present in control cells that do not contain the at least 2 transposons and the polynucleotide encoding a transposase.

2. The method of claim 1 wherein the cell is a tumorigenic cell, wherein the observing comprises:

exposing the mutagenized cells to a compound at a concentration that kills the control cells; and

identifying a mutagenized cell that is resistant to the compound.

3. The method of claim 2 wherein the compound is an alkylating agent, an

antimetabolite, an anthracycline, a plant alkaloid, or a topoisomerase inhibitor.

4. The method of claim 1 wherein the cell is a non-tumorigenic cell, wherein the observing comprises:

administering the mutagenized cells to an animal; and

identifying a tumor in the animal, wherein the tumor is derived from an administered mutagenized cell having a tumorigenic phenotype.

5. The method of claim 4 wherein the animal is an immunoincompetent animal.

6. The method of claim 1 wherein the cell in the incubating step is a non-tumorigenic cell, wherein the phenoype is immortalization, tumorigenesis, drug resistance, or transformation.

7. The method of claim 1 wherein the cell is a human cell.

8. The method of claim 1 wherein the transposon comprises cis-acting elements that bind a transposase, wherein the transposase is a SB transposase.

9. The method of claim 1 wherein the transposon comprises cis-acting elements that bind a transposase, wherein the transposase is a PB transposase.

10. The method of claim 1 wherein the transposon comprises a first pair of cis-acting elements that bind a SB transposase, and a second pair of cis-acting elements that bind a PB transposase.

11. The method of claim 1 wherein the transposase is a Sleeping Beauty transposase or a PiggyBac transposase.

12. The method of claim 1 wherein the method further comprises introducing into the cell a first vector comprising at least 2 transposons and a second vector comprising a

polynucleotide encoding a transposase.

13. The method of claim 12 wherein the first vector replicates in the cell.

14. The method of claim 12 wherein the first vector is integrated into genomic DNA of the cell.

15. The method of claim 12 wherein the second vector replicates in the cell.

16. The method of claim 12 wherein the second vector is integrated into genomic DNA of the cell.

17. The method of claim 1 wherein the method further comprises introducing into the cell a vector comprising at least 2 transposons and a polynucleotide encoding a transposase.

18. The method of claim 17 wherein the vector replicates in the cell.

19. The method of claim 17 wherein the vector is integrated into genomic DNA of the cell.

20. A transposon mutagenesis system to introduce a polynucleotide into genomic DNA of an in vitro cell comprising:

a first vector that comprises at least two transposons; and

a second vector that comprises a polynucleotide encoding a transposase, wherein the transposase causes transposition of the transposons from the first vector to genomic DNA of the cell.

21. A transposon mutagenesis system to introduce a polynucleotide into genomic DNA of an in vitro cell comprising:

a vector that comprises at least two transposons and a polynucleotide encoding a transposase, wherein the transposase causes transposition of the transposons from the first vector to genomic DNA of the cell.

22. A kit comprising packaging materials, a first vector that comprises at least two transposons, and a second vector that comprises a polynucleotide encoding a transposase, wherein the transposase causes transposition of the transposons from the first vector to genomic DNA of the cell.

23. A vector comprising a coding region encoding an Epstein-Barr virus nuclear antigen 1, an Epstein-Barr virus OriP origin of replication, and at least 4 transposons.

24. A cell comprising the vector of claim 23.

25. An isolated cell produced by the method of claim 1.