WO2002002782A1

WO2002002782A1 - Activation of gene expression in cloned eukaryotic genomic dna and methods of use thereof

Info

Publication number: WO2002002782A1
Application number: PCT/US2001/020256
Authority: WO
Inventors: Xiangdong Yang; Shiaoching Gong; Nathaniel Heintz
Original assignee: The Rockefeller University
Priority date: 2000-06-29
Filing date: 2001-06-26
Publication date: 2002-01-10
Also published as: AU2001270162A1

Abstract

The present invention provides methods of modulating the expression of eukaryotic genes in eukaryotic cells. The methods employ homologous recombination to insert heterologous promoters and transcriptional control sequences into independent origin based cloning vectors (IOBCVs) that comprise eukaryotic genes. The resulting modified IOBCVs can then be inserted into eukaryotic cells to express the eukaryotic genes. Included in the invention are methods of using the vectors to modulate the expression of the eukaryotic genes in cells and in animals that comprise the cells. Also included in the invention are methods of preparing IOBCV expression contigs and genomic DNA expression libraries.

Description

ACTIVATION OF GENE EXPRESSION IN CLONED EUKARYOTIC GENOMIC DNA AND METHODS OF USE THEREOF

GOVERNMENTAL SUPPORT

The research leading to the present invention was supported, at least in part, by a grant from the National Science Foundation Grant No. MCB-9316625, by the National Institutes of Health MSTP grant GM07739 and NINDS PHS 30532. Accordingly, the Government may have certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to methods of modulating the expression of eukaryotic proteins in eukaryotic cells. The methods employ homologous recombination to insert heterologous promoters into independent origin based cloning vectors (IOBCVs). The IOBCVs are then inserted into eukaryotic cells. Included in the invention are methods of using these vectors and eukaryotic cells to modulate the expression of eukaryotic genes in the cells, and in animals that comprise the cells.

BACKGROUND OF THE INVENTION

With the advances made in recombinant DNA technology over the past twenty-five years, it has become possible to produce and study recombinant proteins both in vitro and in vivo. Direct screening of complement DNA (cDNA) libraries has been, at least until very recently, the most prevalent method for determining the sequences of these proteins, and the expression of isolated cDNAs has been the predominant method for generating the corresponding recombinant proteins. Furthermore, cDNAs have also been the major form of nucleic acid used for either expressing or misexpressing genes in eukaryotic cells. One particular advantage of using cDNAs is that they are much easier to manipulate than the corresponding genomic DNAs, since cDNAs are significantly smaller than their corresponding genomic DNAs. To express a selected protein in a eukaryotic cell, a cDNA can be sub-cloned into an expression vector containing a eukaryotic promoter at the 5' end and a polyadenyation signal at its 3' end. When the vector contains a strong promoter, the gene can be expressed at high levels following the transfection of the vector into an appropriate eukaryotic cell. Thus, cDNA based expression systems have become standard methodology for producing proteins in isolated cells, as well as in transgenic animals.

However, now that complete nucleotide sequences of the genomes of individual species are capable of being determined in a relatively short period of time, with a number of important genomes having already been determined, e.g., the Drosophila genome and the human genome, or are in the process of being determined, the use of cDNA based expression systems are beginning to become the bottleneck in genetic manipulation, since obtaining a full-length cDNA is now relatively time consuming. Indeed, full length cDNAs are not necessarily available for genes that have been newly identified (i) through genomic sequencing, (ii) by homology with genes of other species, and/or (iii) through predictions by one or more gene annotation programs. In addition, cDNA libraries are inherently biased towards nucleic acids that are prevalent in the tissue sample being studied. Thus, full-length cDNAs derived from genes which are expressed solely in tissues that are difficult to obtain, and/or are expressed only under relatively rare circumstances, are difficult to isolate. This is particularly problematic since such genes often play a unique role during a specific cellular challenge, and thus may be important in a specific diseased state.

Furthermore, cDNAs generally lack introns, and endogenous introns can play an important role in the regulation of gene expression. For example, gene splicing is known to be important in mRNA nuclear export. In addition, alternative gene splicing allows multiple forms of a given gene to be expressed in the same cell, whereas a given cDNA is only one single alternatively spliced variant.

In contrast with cDNA, cloned genomic DNA has not been generally used to modulate gene expression in eukaryotic cells for a variety of reasons. First, as indicated above, the portion of genomic DNA encoding a particular protein is almost always much larger than the corresponding cDNA. For example, the average gene size of human chromosome 21 is 39kb [Hattori et ah, Nature 405:311-319 (2000)] and of human chromosome 22 is 19.2 kb [Dunham et al., Nature, 402:490-495 (1999)], whereas a typical eukaryotic cDNA is less than 3kb. Moreover, cloned eukaryotic genomic DNA generally contains the gene's own promoter and regulatory elements, which often confer the naturally restricted expression of the gene, i.e., only in particular cell types, and at a limited expression level. Therefore, it is difficult to overproduce a protein using genomic DNA in an heterologous cell type which normally does not express the gene, or expresses it at a low level. Furthermore, cloned genomic DNA may lack certain exons, endogenous regulatory elements or polyadenylation signals. Finally, large genomic DNA clones are relatively difficult to modify using conventional recombinant DNA technology. Unlike a cDNA which can be easily sub-cloned into various expression vectors, no facile methods are currently available to express a cloned genomic DNA at a high expression level, in vivo.

Several vector systems have been developed for large segments of mammalian DNA, including cosmids, and bacteriophage PI [Sternberg et al, Proc. Natl. Acad. Sci. U.S.A., 87: 103-107 (1990)]. Such vectors can be grouped together as being individual types of independent origin based cloning vectors (IOBCVs). One such IOBCV is a yeast artificial chromosome, or YAC. YACs have certain advantages over alternative large capacity cloning vectors [Burke et al, Science, 236:806-812 (1987)]. The maximum DNA insert size is 35-30 kb for cosmids, and 100 kb for bacteriophage PI, whereas the maximal DNA insert for a YAC, is greater than 1Mb. YACs have the additional advantage in that they are amenable to homologous recombination based modifications, such as insertions, deletions and point mutations in the yeast host. However, there are several critical limitations to the YAC system including difficulties in manipulating YAC DNA, chimerism and clonal instability [Green et al., Genomics, 11:658 (1991); Kouprina et al, Genomics 21:7 (1994); and Larionov et al, Nature Genet. 6:84 (1994)]. An alternative to YACs are E. coli based cloning systems based on the E. coli fertility factor that have been developed to construct large genomic DNA insert libraries. These include bacterial artificial chromosomes (BACs) and P-l derived artificial chromosomes (PACs) [Mejia et al, Genome Res. 7:179-186 (1997); Shizuya et al, Proc. Natl. Acad. Sci. 89:8794-8797 (1992); loannou et al, Nat. Genet., 6:84-89 (1994); and Hosoda et al, Nucleic Acids Res. 18:3863 (1990)]. BACs are based on the E. coli fertility plasmid (F factor); and PACs are based on the bacteriophage PI. Bacterial artificial chromosomes (BACs) and P-l derived artificial chromosomes (PACs) are large genomic clones that propagate in E. Coli as a circular plasmid [Shizya et al, PNAS 89, 8794-8797 (1992); loannou et al, Nature Genetics, 6:84-89 (1994)]. BACs and PACs have a cloning capacity of up to 700kb [Zimmer and Verrinder Gibbins, Genomics, 42:217-226 (1997)], with most libraries having an average insert size of 150kb. This means that a single BAC can hold most mammalian genes. These vectors propagate at a very low copy number (1-2 per cell) enabling genomic inserts of up to 300 kb in size to be stably maintained in recombination deficient hosts (most clones in human genomic libraries fall within the 100-200kb size range). The host cell is required to be recombination deficient to ensure that non-specific and potentially deleterious recombination events are kept to a very minimum. As a result, libraries of PACs and BACs are relatively free of the high proportion of chimeric or rearranged clones typical in YAC libraries, [Monaco et al, Trends Biotechnol 12:280-286 (1994); and Boyseu et al, Genome Research, 7:330-338 (1997)].

In addition, isolating and sequencing DNA from PACs or BACs involves simpler procedures than for YACs, and PACs and BACs have a higher cloning efficiency than YACs [Shizuya et al, Proc. Natl. Acad. Sci. 89:8794-8797 (1992); loannou et al, Nat. Genet., 6:84-89 (1994); and Hosoda et al, Nucleic Acids Res. 18:3863 (1990)]. Such advantages have made BACs and PACs important tools for physical mapping in many genomes [Woo et al, Nucleic Acids Res., 22:4922 (1994); Kim et al, Proc.Natl.Acad.Sci. 93:6297-6301 (1996); Wang et al, Genomics 24:527 (1994); and Wooster et al, Nature 378:789 (1995)]. Furthermore, the PACs and BACs are circular DNA molecules that are readily isolated from the host genomic background by classical alkaline lysis [Birnboim et al, Nucleic Acids Res. 7: 1513-1523 (1979)].

Functional characterization of a gene of interest contained by a PAC or BAC clone generally entails transferring the DNA into a eukaryotic cell for either transient or long-term expression. A transfection reporter gene, e.g., a gene encoding lacZ, together with a selectable marker, e.g., neo, can be inserted into a BAC [Mejia et al., Genome Res. 7:179-186 (1997)]. Transfected cells can be detected by staining for X-Gal to verify DNA uptake, for example, and stably transformed cells are then selected for by the antibiotic G418.

Now that the complete nucleotide sequence of the genomes of individual species are either available or rapidly becoming available, the elucidation of the complete structure of all the genes within a given organism, including the regulatory elements and coding sequences will be assessable. Furthermore, many of the eukaryotic sequences are and/or will be available on complete sets of overlapping BAC and PAC clones. For example, overlapping BAC/PAC clones are available for the complete Drosophila Genome [Adams et al, Science 287:2135-2195, 2000], as well as for human chromosomes 21 and 22. Unfortunately, currently there is no method to reliably activate the expression of the genes encoded by these BAC and PAC clones in eukaryotic cells.

Therefore, there is a need to provide methodology that can more efficiently utilize the known sequences in IOBCVs, such as YAC, BAC or PAC libraries. Furthermore, there is a need to provide methodology for expressing particular genes contained in such libraries. Such methodology may be particularly useful for expressing genes that are expressed relatively rarely and/or only at specific times (such as the genes involved in circadian rhythms or those involved in body weight homeostasis); and/or are predominantly expressed in tissues that are difficult to obtain, such as the human organ of Corti. Furthermore, there is a need to provide facile methods of manipulating particular genes that can be used in situ and/or in vivo. Furthermore, there is a need to provide cells and organisms that contain the manipulated genes.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

SUMMARY OF THE INVENTION

The present invention provides novel methods for controlling the expression of genomic DNA in eukaryotic cells. This methodology permits the modulation of the expression of essentially any gene of any eukaryotic genome. Thus, the methodology of the present invention can be used to express a particular gene of interest in a eukaryotic cell in which the gene is not normally expressed and/or express the gene of interest at a time in development (and/or time of the cell cycle) in which the gene is not normally expressed. The present invention further allows the over-expression of a gene of interest in a eukaryotic cell, or alternatively, a reduction in expression of the gene.

The present invention provides a method of modifying eukaryotic cells to contain genes that are modulated by a Heterologous Control Unit (HCU). One such method comprises inserting a HCU into an Independent Origin Based Cloning Vector (IOBCV) by homologous recombination, thereby generating a modified IOBCV. The insertion of the HCU into the now modified IOBCV enables the HCU to modulate the expression of a gene of interest comprised by the IOBCV when the modified IOBCV is introduced into a eukaryotic cell and the eukaryotic cell is in an environment that allows gene expression. The modified IOBCV is next introduced into a eukaryotic cell, where the expression of the gene of interest can be modulated by the HCU when the eukaryotic cell is in an environment that allows gene expression. The modified eukaryotic cells are also part of the present invention.

Therefore, the present invention further provides methods of modulating the expression of a gene of interest in a eukaryotic cell. One such method comprises inserting a Heterologous Control Unit (HCU) into an Independent Origin Based Cloning Vector (IOBCV) by homologous recombination, thereby generating a modified IOBCV. The insertion of the HCU into the now modified IOBCV enables the HCU to modulate the expression of a gene of interest comprised by the IOBCV when the modified IOBCV is introduced into a eukaryotic cell and the eukaryotic cell is in an environment that allows gene expression. The modified IOBCV is next introduced into a eukaryotic cell, where the expression of the gene of interest is modulated by the HCU. In a particular embodiment, the introduction of the modified IOBCV into the eukaryotic cell is performed in an environment that allows gene expression. In another embodiment, the eukaryotic cell is subsequently placed into an environment that allows gene expression.

In a preferred embodiment the HCU is inserted (or placed) in front of an exon of the gene of interest. In a related embodiment, the HCU is placed operatively upstream of the entire coding region of the gene of interest. In a particular embodiment the HCU modulates the expression of the gene of interest in the eukaryotic cell by expressing a gene product encoded by the gene of interest that is not normally expressed in that eukaryotic cell.

In a particular embodiment the HCU is a heterologous eukaryotic promoter (HEP). In another embodiment the HCU is a heterologous transcriptional regulatory element (HTRE). In still another embodiment the HCU is a heterologous eukaryotic promoter comprising one or more transcriptional regulatory elements (HEP/TRE). In yet another embodiment the HCU is a heterologous eukaryotic promoter with one or more transcriptional regulatory elements that further encodes a 5' fusion polypeptide/peptide (HTF). In still another embodiment the HCU is a multi -promoter insert (MPI). In a particular embodiment the HCU comprises a eukaryotic translation initiation sequence (e.g., a Kozak sequence). In preferred embodiment the gene of interest comprises an endogenous polyadenylation sequence. In one particular method the IOBCV integrates into the genome of the eukaryotic cell. The IOBCV used in the methods of the present invention can be a YAC. In another embodiment the IOBCV can be a BBPAC. In a particular embodiment of this type, the BBPAC is a PAC. In a preferred embodiment of this type, the BBPAC is a BAC.

In a particular embodiment the HCU modulates the expression of the gene of interest by increasing its expression. In one such embodiment the gene of interest encodes a viral antigen, and the IOBCV is used as a DNA vaccine. In an alternative embodiment, the HCU modulates the expression of the gene of interest by decreasing its expression.

The present invention also provides a method of inserting an HCU into an IOBCV that comprises introducing a shuttle vector into a host cell containing the IOBCV under conditions in which the shuttle vector can replicate and transform the host cell. One such shuttle vector comprises (i) a conditional origin of replication, (ii) a region of homology with the gene of interest, and (iii) an HCU which are situated in the shuttle vector such that the HCU is inserted operatively upstream of the coding region of the gene of interest comprised by the IOBCV following the recombination event between the region of homology of the shuttle vector and the gene of interest of the IOBCV. In a preferred embodiment of this type, after the insertion of HCU into the IOBCV, the host cell is grown under conditions in which the shuttle vector cannot replicate and is thereby diluted out. In one such embodiment the conditional origin of replication is a temperature sensitive origin of replication. In another such embodiment, the conditional origin of replication is the R6Kγ origin of replication. In a preferred embodiment of this particular type, the host cell comprising the IOBCV expresses the Pir protein.

The present invention further provides IOBCVs that comprise an HCU operatively upstream of the coding region of a gene of interest such that the HCU can modulate the expression of the gene of interest when the IOBCV is introduced into a eukaryotic cell. The HCUs are preferably placed operatively upstream of the coding region of the gene of interest in the IOBCV by homologous recombination. In a particular embodiment, the HCU has been inserted operatively upstream of the coding region of the gene of interest of the IOBCV by the transient expression of a recombination protein (e.g., RecA) in a recombination deficient host cell (e.g., a recA- cell). In a particular embodiment the HCU is a heterologous eukaryotic promoter (HEP). In another embodiment the HCU is a heterologous transcriptional regulatory element (HTRE). In still another embodiment the HCU is a heterologous eukaryotic promoter comprising one or more transcriptional regulatory elements (HEP/TRE). In yet another embodiment the HCU is a heterologous eukaryotic promoter with one or more transcriptional regulatory elements that further encodes a 5' fusion polypeptide/peptide (HTF). In still another embodiment the HCU is a multi -promoter insert (MPI). In a particular embodiment the HCU comprises a eukaryotic translation initiation sequence (e.g., a Kozak sequence). In a preferred embodiment the gene of interest comprises an endogenous polyadenylation sequence.

The present invention provides the eukaryotic cells that comprise a gene of interest that has been modulated by the methods of the present invention, including those comprising the IOBCVs modified by the methods of the present invention, and further provides the modified IOBCVs of the present invention.

The present invention also provides methods of expressing an antisense RNA of a gene of interest in a eukaryotic cell. One such method comprises inserting a HEP in the antisense orientation near the 3' end of the coding region of a gene of interest contained in an IOBCV by homologous recombination. The inserted HEP facilitates the expression of the corresponding antisense RNA after the IOBCV is introduced into a eukaryotic cell. The IOBCV is then introduced into the eukaryotic cell, and the antisense RNA for the gene of interest is expressed.

The present invention further provides methods of producing a non-human transgenic animal that comprises an HCU which modulates the expression of and/or can modulate the expression of a gene of interest. One such method comprises inserting an HCU operatively upstream of a gene of interest contained by an IOBCV by homologous recombination, thereby enabling the HCU to modulate the expression of the gene of interest when the IOBCV is introduced into a eukaryotic cell (and the eukaryotic cell is in an environment that allows gene expression). The resulting IOBCV is placed into the eukaryotic cell and the resulting eukaryotic cell is then placed into a recipient animal which allows the eukaryotic cell to develop into the non-human transgenic animal. The non-human transgenic animal thereby comprises a gene of interest that either is and/or can be modulated by the HCU. In one embodiment of this type, the eukaryotic cell is a fertilized animal zygote. In another embodiment, the eukaryotic cell is an embryonic stem cell. In a particular embodiment of this type, a modified embryonic stem cell is generated by introducing into the embryonic stem cell an IOBCV, in which an HCU has been inserted through homologous recombination to be operatively upstream of a gene of interest. In a preferred embodiment, the embryonic stem cell is a mouse embryonic stem cell. In another preferred embodiment, the IOBCV is a BBPAC in which an HCU has been inserted operatively upstream of a gene of interest through homologous recombination in a RecA" bacterial host cell that has been transiently induced to support homologous recombination. The embryonic stem cells and fertilized animal zygotes modified by the present invention are also part of the present invention.

The present invention further provides methods of producing a non-human transgenic animal that expresses an antisense RNA of a gene of interest. One such method comprises inserting a HEP in the antisense orientation near the 3' end of the coding region of a gene of interest contained by an IOBCV by homologous recombination. Again, the HEP facilitates the expression of an antisense RNA to the gene of interest when the IOBCV is introduced into a eukaryotic cell (and the eukaryotic cell is in an environment that allows gene expression). The IOBCV is next introduced into the eukaryotic cell, which is then, in turn, placed into a recipient animal. The eukaryotic cell can then develop into a non-human transgenic animal that expresses the antisense RNA of the gene of interest.

In one embodiment the IOBCV is placed into the eukaryotic cell by electroporation. In another embodiment the IOBCV is placed into the eukaryotic cell by lipofection. In still another embodiment the IOBCV is placed into the eukaryotic cell by microinjection. In yet another embodiment the IOBCV is placed into the eukaryotic cell by calcium mediated transfection. In still another embodiment the IOBCV is inserted into a viral vector that can be used to infect a animal host.

A non-human transgenic animal of the present invention can be used for a number of purposes including as a animal model for a disease that is related to and/or believed to be related to the expression of the gene of interest. In a preferred embodiment of this type, the animal model is for a human disease. The present invention also includes a series of such non-human transgenic animals that have modulated expression of individual genes of interest that are involved in a particular cellular, immunological and/or metabolic pathway, for example. Such a series of non-human transgenic animals allows the selective modulation of the expression of the individual genes of interest of the particular pathway. Alternatively, the present invention provides a method of making a single non-human transgenic animal that comprises either multiple IOBCVs in which an HCU has been inserted through homologous recombination to be operatively upstream of multiple genes of interest and/or an IOBCV in which one or more HCUs have been inserted through homologous recombination to be operatively upstream of multiple genes of interest.

The present invention further provides the non-human transgenic animals generated by the methods of the present invention. In a particular embodiment, a non-human transgenic animal is generated that contains more than one gene of interest under the influence of one or more HCUs.

The present invention also provides methods of using the IOBCVs modified by the methods of the present invention for use in in vivo and/or ex vivo gene therapy. In a particular embodiment of this type, the IOBCV has been modified to express an antisense RNA. Thus, the modified IOBCVs of the present invention can be introduced into an animal cell for ex vivo gene therapy, or into an animal host for in vivo gene therapy. Preferably the animal cell is a human cell, and the animal host is a human host. In another aspect of the present invention the IOBCVs modified by the methods of the present invention can be placed into isolated plant cells and/or plants. In one such embodiment, the modified IOBCV is used to make a plant more resistant to insects by facilitating the expression of an insecticide by the host plant. In another embodiment, the modified IOBCV is used to make a plant more nutritious by facilitating the expression of a protein rich (or modified to be rich) in an amino acid that is relatively deficient in the proteins naturally expressed by the native plant.

Methods of constructing a genomic DNA based expression library for a particular eukaryotic species are also part of the present invention. One such method comprises subcloning gene fragments of a genomic DNA for a particular eukaryotic species into IOBCVs and determining the sequence of the fragments. Exons contained by the gene fragments that encode gene products are identified and nucleotide sequences adjacent to the exons are identified. HCUs are then inserted into the IOBCVs by homologous recombination using the nucleotide sequences adjacent to the exons. The HCUs modulate the expression of the gene fragments when the IOBCVs are introduced into a eukaryotic cell (and the eukaryotic cell is in an environment that allows gene expression). In a particular embodiment, genomic DNA based expression libraries are constructed for multiple eukaryotic species.

The present invention further provides methods of over-expressing a gene of interest in a eukaryotic cell. One such method comprises inserting a HCU by homologous recombination into an IOBCV operatively upstream of the coding region of a gene of interest contained by the IOBCV thereby enabling the HCU to over-express the gene of interest when the IOBCV is introduced into a eukaryotic cell. The IOBCV then can be introduced into the eukaryotic cell, and the eukaryotic cell can be cultured in an appropriate cell culture medium under conditions that provide for over-expression of the gene of interest in the eukaryotic cell. In a particular embodiment, the gene product of the gene of interest is then purified. The purified gene product is also part of the present invention. Accordingly, it is a principal object of the present invention to provide a method for expressing eukaryotic (including viral) genes in a eukaryotic cell.

It is a further object of the present invention to provide a method of constructing a genomic DNA expression library.

It is a further object of the present invention to provide a method of performing gene therapy employing the modified independent origin based cloning vectors (IOBCVs) of the present invention.

It is a further object of the present invention to provide a method of transcribing a eukaryotic gene from a heterologous genomic gene in a eukaryotic cell without the aid of a cDNA.

It is a further object of the present invention to provide a method of modulating the expression of a gene naturally encoded by a eukaryotic cell.

It is a further object of the present invention to provide a method of expressing a gene naturally encoded by a eukaryotic embryo, at a time in the embryonic development that the gene is not normally expressed.

It is a further object of the present invention to provide a method of preventing the expression of a gene that is naturally encoded by a eukaryotic embryo, at a time in the embryonic development that the gene is normally expressed.

It is a further object of the present invention to provide a method of expressing a gene naturally encoded by a eukaryotic cell in a cell type where the gene is not naturally expressed. It is a further object of the present invention to provide a method of preventing the expression of a gene naturally encoded by a eukaryotic cell in a cell type where the gene is naturally expressed.

It is a further object of the present invention to provide a method of over-expressing a gene naturally encoded by a eukaryotic cell.

It is a further object of the present invention to provide a purified gene product from a eukaryotic cell (in vitro, in situ and or in vivo) that has been over-expressed by a method of the present invention.

It is a further object of the present invention to provide a transgenic animal that comprises a modified independent origin based cloning vectors (IOBCV) of the present invention.

It is a further object of the present invention to provide the modified IOBCVs, including modified BBPACs of the present invention.

These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1B show schematic depictions of insertion modules that can be used to place promoters, and/or regulatory elements, and/or eukaryotic translation initiation signals e.g., a Kozak sequence, and/or other heterologous nucleotide sequences into an IOBCV. A schematic depiction of an IOBCV is shown in Figure 1 A comprising two separate genomic nucleic acids in opposite orientations. Each genomic nucleic acid contains an endogenous promoter (EP) and multiple exons (rectangles). The shaded exons contain the coding sequence for the gene product. As shown, heterologous nucleotide sequences can be inserted into the IOBCV to modulate the expression of a gene of interest. HEP/TRE, TRE, HTRE and HTF, are as defined below; HP is used interchangeably with HEP.

Figure 2 is a schematic depiction of the pLD55.RecA.TGCA shuttle vector. The pLD55.RecA shuttle vector comprises a R6Kγ origin, nucleic acids that provide the shuttle vector antibiotic resistance to tetracycline (Tet) and ampicillin [AmpR(Bla)] respectively and encodes the recA protein (RecA). Ascl, Pacl, Mlul, and Pmel are restriction enzyme sites used to add the genomic homology region for the gene of interest. The R6Kγ origin of replication can only replicate in bacteria that express the pir protein, such as E. coli strain 21118 [Metcalf et al, Plasmid 35:1-13 (1996)]. The genomic homology region, e.g., here about 500 basepairs, for the targeting can be amplified by the polymeration chain reaction (PCR) subcloned into the polycloning sites containing Ascl, Pacl, Mlul, and Pmel.

Figure 3 outlines a protocol for producing and purifying biologically active proteins from eukaryotic cells comprising a modified IOBCV from the present invention using Targeted Genomic Clone Activation (TGCA).

Figure 4A is a schematic depiction of the Bacterial Artificial Chromosome, RP4BAC -662-B22, which comprises 116 kilobases (kb) containing exon 3, exon 4 and the polyadenylation signal of the human beta NGF gene at the end of the BAC insert. Exon 4 encodes the mature beta NGF polypeptide. Figure 4B shows a schematic depiction of a pLD.55recA.TGCA vector comprising a CMV promoter and enhancer, and a human beta NGF homology arm. This particular shuttle vector can be used to place the human beta NGF under the control of the CMV promoter/enhancer.

Figure 5A is a schematic depiction of the PAC, RPCI PAC 24B12, which comprises 167 kb containing exons 1-3 and the polyadenylation signal of the human GDNF gene. Exons 2 and 3 encode the mature GDNF polypeptide. Figure 5B shows a schematic depiction of a pLD.55recA.TGCA vector comprising a CMV promoter and enhancer, and a human GDNF homology arm. This particular shuttle vector can be used to place the human GDNF under the control of the CMV promoter/enhancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel procedure for inserting a heterologous eukaryotic promoter (HEP) and/or one or more heterologous transcriptional regulatory elements (HTRE) into an independent origin based cloning vector (IOBCV). The methodology disclosed herein, allows the modulation of the expression of essentially all of the genes contained by any selected eukaryotic genome. The present invention thus provides a facile method for modulating gene expression in a eukaryotic cell, including a human cell inside a human subject, e.g., for gene therapy.

One aspect of the present invention exploits the stability of BBPACs to ensure that the eukaryotic gene is an endogenous gene, not an artifactural chimeric gene. In addition, BBPACs are large enough to contain the necessary elements for DNA replication and nuclear retention in eukaryotic cells. Thus BBPACs are maintained as stable episomal plasmids when introduced into eukaryotic cells, allowing selective gene expression to be performed in practically any eukaryotic cell. BBPACs can be readily modified using homologous recombination to introduce specific modifications into a gene of interest [U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No. 09/102,490, Filed June 22, 1998; and U.S. Serial No.09/356,987, Filed July 20, 1999; the contents of which are all hereby incorporated by reference in their entireties]. In addition, BBPACs can be manipulated to express the genes they encode through gene-trapping methodology [U.S. Serial No.09/007,206, Filed January 14, 1998; and U.S. Serial No.09/102,488, Filed June 22, 1998; the contents of which are all hereby incorporated by reference in their entireties].

The present invention further provides a method, that enables the modulation of the expression of a gene encoded by eukaryotic DNA contained within a cell and/or organism. The method termed Targeted Genomic Clone Activation (TGC Activation or TGCA), uses homologous recombination to insert heterologous eukaryotic promoters and/or enhancers into cloned eukaryotic genomic DNA. The heterologous eukaryotic promoter can then modulate the expression level of an endogenous gene in a eukaryotic cell. In a particular embodiment, the method employs homologous recombination to insert a heterologous eukaryotic promoter in the antisense orientation with regard to a gene contained by a genomic clone.

Thus, the TGC Activation system can be readily applied to a variety of applications including to generate a genome-based eukaryotic expression library; to modulate the expression of proteins in eukaryotic cells; to perform functional assays in cells; to perform assays using cell extracts or supernatants derived from cells transfected with TGCA clones; to generate transgenic animals that have a gene of choice under the control of a heterologous promoter/regulatory system; to produce cells that express genes under the control of a heterologous promoter for cell based gene therapy; and to express genes in a genomic DNA-based vaccine to increase the vaccine's efficacy. Indeed, the present invention permits the critical time and place of gene expression to be controlled in experimental animal disease models, or in animal subjects (including human patients) in need of such controlled expression.

The methodology of the present invention can also be directly applied to IOBCV contigs. For example, a genomic DNA clone based expression library can be constructed for any given genome through the application of the disclosed methodology to a BBPAC contig, such as a complete set of overlapping BACs.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994)].

A "nucleic acid molecule" refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogues thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, mter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 ' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal sequence is usually required.

As used herein, the terms "protein", "polypeptide" and "peptide" are interchangeable in that they all denote polymers of amino acids. Generally, the term peptide is used to denote a polymer of amino acids that comprises 2 to 25 amino acid residues, whereas a protein denotes a polymer of amino acids that comprises 25 or more amino acid residues.

As used herein the terms "fusion protein" and "fusion peptide" are used interchangeably and encompass "chimeric proteins and/or chimeric peptides" and fusion "intein proteins/peptides". A fusion protein of the present invention comprises at least a portion of the protein or peptide encoded by a gene of interest of the present invention joined via a peptide bond to at least a portion of another protein or peptide in a chimeric/ fusion protein. For example, fusion proteins can comprise a marker protein or peptide, or a protein or peptide that aids in the isolation and/or purification of the protein or peptide encoded by a gene of interest of the present invention.

As used herein the term "eukaryotic " is used broadly, and in contrast to the term "prokaryotic". The terms "eukaryotic genes" and "eukaryotic transcriptional and translational control sequences" (such as a "eukaryotic promoter"), are also used broadly, and in contrast to "prokaryotic genes" and "prokaryotic transcriptional and translational control sequences". Thus the terms "eukaryotic genes" and "eukaryotic transcriptional and translational control sequences" as used herein include genes and transcriptional and translational control sequences from any eukaryotic organism, including yeast, and furthermore, can include viral genes and viral transcriptional and translational control sequences, but do not include prokaryotic and/or bacteriophage genes or prokaryotic and/or bacteriophage transcriptional and translational control sequences.

A molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids. An antigenic portion of a molecule can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier molecule for immunization. A molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier.

As used herein the terms "approximately" and "about" are used to signify that a value is within twenty percent of the indicated value i.e., a nucleotide sequence containing "about" 500 basepairs can contain between 400 and 600 basepairs.

As used herein a "small organic molecule" is an organic compound [or organic compound complexed with an inorganic compound (e.g., metal)] that has a molecular weight of less than 3 Kilodaltons.

A "heterologous nucleotide sequence" as used herein is a nucleotide sequence that can be covalently combined with a gene of interest of the present invention (e.g., by homologous recombination) to modify the gene of interest. Such nucleotide sequences can encode chimeric and/or fusion proteins. The heterologous nucleotide sequence can also encode peptides and/or proteins which contain regulatory and/or structural properties. In another such embodiment a heterologous nucleotide can encode a protein or peptide that can function as a means of detecting a protein or peptide encoded by a gene of interest (contained by a BAC, for example). In still another such embodiment a heterologous nucleotide can function as a means of detecting a nucleotide sequence of the present invention. A heterologous nucleotide sequence can also comprise non-coding sequences including restriction sites, transcriptional regulatory elements, promoters and the like.

A "translocation signal sequence" as used herein refers to a signal sequence that is included at the beginning of the coding sequence for a protein to be expressed on the surface of a cell. This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide. Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms. A "vector" is a replicon, such as a plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.

As used herein the term "Targeted Genomic Clone Activation" is used interchangeably with "TGC Activation" and "TGCA" and is a method of using homologous recombination to insert heterologous eukaryotic promoters and/or enhancers into cloned eukaryotic genomic DNA in IOBCVs.

As used herein an "IOBCV" is an independent origin based cloning vector. An IOBCV is a cloning vector such as a YAC, BAC or PAC that can, though is not required to, replicate independently when inserted into a cell under the appropriate conditions. An IOBCV generally comprises a nucleic acid insert which either is, or contains one or more eukaryotic genes or portions thereof. The present invention includes methodology for inserting a heterologous eukaryotic promoter (HEP) operatively upstream of a eukaryotic gene in an IOBCV. The HEP modulates (i.e., attenuates and/or controls) the transcription of the eukaryotic gene, when the IOBCV is placed into a eukaryotic cell, and the eukaryotic cell is in an environment that allows gene expression.

As used herein, a "Bacterial or Bacteriophage-Derived Artificial Chromosome" or "BBPAC" denotes a vector that is derived from a bacterium or bacteriophage such as a Bacterial Artificial Chromosome (BAC) which is an E. coli F element based cloning system, a Pi-Derived Artificial Chromosome (PAC) or a bacteriophage-based genomic vector. In one embodiment, the BBPAC encodes up to 700 kilobases of genomic sequences. In a preferred embodiment, the BBPAC encodes between 120 to 180 kilobases of genomic sequences. In one particular embodiment the BBPAC encodes 130 kilobases of genomic sequence. Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, a polyadenylation signal sequence is a particular type of a control sequence.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to, at the minimum, include the fewest number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

As used herein, a "heterologous eukaryotic promoter" or "HEP" is a eukaryotic promoter that can be placed into an IOBCV, such as a BBPAC, to modulate (i.e., attenuate and/or control) the expression of a gene of interest comprised by the IOBCV. An IOBCV can be constructed to contain a specific promoter, but preferably the HEP is inserted into the IOBCV via homologous recombination using a portion of the DNA sequence of the gene of interest which allows the insertion of the HEP at a site at which it can functionally control the expression of the gene of interest.

Although, it is preferable to replace the intrinsic promoter of the gene of interest with a HEP that is not naturally associated with the gene of interest, e.g., not part of the gene, as used herein, a HEP of the present invention can also be the natural promoter of a gene of interest that either replaces or supplements the intrinsic promoter (e.g., to replace a defective promoter with a functional promoter).

As used herein "HEP/TRE" is a Heterologous Eukaryotic Promoter (HEP) with one or more Transcriptional Regulatory Elements (TRE). As used herein the abbreviation "HEP/TRE/FP" is used interchangeably with "HTF" and is a heterologous eukaryotic promoter with one or more transcriptional regulatory elements that further encodes a 5' Fusion Polypeptide/Peptide (FP) that is inserted in frame with a coding exon of the gene of interest to form a fusion protein/peptide.

As used herein "HTRE" is an abbreviation for one or more Heterologous

Transcriptional Regulatory Elements that are inserted adjacent to the endogenous promoter of a gene in an IOBCV to modulate the expression of the gene. Preferably the HTRE is placed so that it is 5' to the promoter, which is 5' (i.e., operatively upstream) to the gene of interest.

As used herein "MPI" is used interchangeably with "Multi-Promoter Insert" and is an HCU comprising more than one promoter. Preferably all but one of these promoters is used to drive the expression of the gene of interest. A MPI of the present invention can facilitate over-production of one or more proteins, one of which is encoded by the gene of interest of the IOBCV.

A heterologous eukaryotic promoter (HEP) of the present invention is operatively upstream of an exon of a eukaryotic gene (and conversely the exon is therefore operatively downstream of the heterologous eukaryotic promoter) if the expression of the exon can be modulated (i.e., attenuated and/or controlled) by the HEP when the promoter and exon are present in a eukaryotic cell, e.g., when the transcription of the exon can be initiated in a eukaryotic cell by the binding of RNA polymerase to the heterologous eukaryotic promoter. The HEP thereby facilitates the transcription of the exon in a eukaryotic cell. Depending on the position of the HEP relative to the exons of the eukaryotic gene, the transcription of more than one exon of the eukaryotic gene can be facilitated. In a preferred embodiment, the HEP is operatively upstream from all of the coding regions of the gene, thereby facilitating the transcription of the entire protein encoded by the gene. Preferably the eukaryotic cell is a mammalian cell, or a plant cell. As used herein, a "Heterologous Control Unit" or "HCU" is a heterologous nucleotide sequence that minimally contains a heterologous transcriptional and/or translational control sequence, such as a promoter, and/or an enhancer, and/or a terminator, and the like, which can be inserted into an IOBCV so as to modulate (i.e., attenuate and/or control) the expression of a gene or portion thereof (i.e., a gene of interest) contained by the IOBCV. Examples include, a heterologous eukaryotic promoter (HEP), a heterologous transcriptional regulatory element (HTRE), a HEP/TRE, a HTF, and a MPI.

A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when these sequences effect the RNA polymerase-catalyzed transcription of the coding sequence into mRNA and/or the subsequent translation of the resulting mRNA. The mRNA may then be spliced and translated into the protein encoded by the coding sequence.

As used herein a "gene of interest" is a gene or portion thereof contained by the genomic DNA of an independent origin based cloning vector (IOBCV) that has been selected to have its expression modulated (e.g., turned on, increased, decreased, or shut off, and or made responsive to particular factors). A gene of interest can be placed into an independent origin based cloning vector for this purpose, or preferably is already contained by the independent origin based cloning vector.

As used herein a "marker" is an indicator, whose presence or absence can be used to distinguish the presence or absence of a particular nucleic acid and preferably the corresponding presence or absence of a larger DNA which contains and/or is linked to the specific nucleic acid. In a preferred embodiment the marker is a protein or a gene or portion thereof encoding the protein, and thus can be more specifically termed a "marker protein" or a "marker gene". The term "marker" (and thus marker protein or marker gene) is meant to be used extremely broadly and includes fluorescent proteins such as green fluorescent protein [U.S. Patent No. 5,625,048, Issued April 29, 1997, WO 97/26333, and WO 99/64592 the contents of which are all hereby incorporated by reference in their entireties], enzymes such as luciferase, and further includes drug resistant proteins, whose presence or absence may not solely be regarded as a means to detect cells that contain the drug resistance protein; and/or the gene or portion thereof that encode such proteins. However, drug resistance proteins and/or their corresponding genes can allow the preferential growth of cells that contain the drug resistant gene (or alternatively allow the counter-selection of cells that do not contain the drug resistant gene) and therefore bestow a type of selectable distinction which is meant to fall within the present definition of a marker.

The term "a gene which encodes a marker protein" is used herein interchangeably with the term "marker protein gene" and denotes a nucleic acid which encodes a marker protein.

A "cassette" refers to a segment of DNA that can be inserted into a vector at specific restriction sites. The segment of DNA can encode a particular polypeptide and/or contain a HCU. The cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation, when required. In a particular embodiment of the present invention a "recombination cassette" includes two homology fragments interrupted by a HEP which is to be inserted into a gene of interest contained by an IOBCV.

"Homologous recombination" refers to the insertion of a modified or foreign DNA sequence contained by a first vector into another DNA sequence contained by a second vector, or a chromosome of a cell. The first vector targets a specific site for homologous recombination. For specific homologous recombination, the first vector will contain sufficiently long regions of homology to sequences of the second vector or chromosome to allow complementary binding and incorporation of DNA from the first vector into the DNA of the second vector, or the chromosome. Longer regions of homology, and greater degrees of sequence similarity may increase the efficiency of homologous recombination.

A "recombination protein" as used herein is a protein involved in homologous recombination that can be used either alone or in conjunction with other proteins to allow homologous recombination to proceed in a cell that is otherwise recombination deficient. Examples of recombination proteins include RecA-like proteins, the rec E and rec T proteins which are encoded by the Rec E gene [Clark et al, J.Bacteriol 175:7673-82, (1993); Hall et al, J. Bacteriol 175:277-87, (1993); Kusano et al. 138:17-25, (1994); also reviewed by Clark and Sandier, Crit Rev Microgiol, 20:125- 142, 1994)], the Lambda beta protein [Berger and Cohen, J. Bacteriol. 171:3523- 3529, (1989)] and the Arabidopsis thaliana DRT100 gene product [Pang et al., Proc. Natl. Acad. Sci. 89:8073-8077, (1992)].

A "RecA-like protein" is defined herein to have the meaning generally accepted in the art except as used herein the recA protein itself is included as being a specific RecA- like protein. RecA-like proteins are proteins involved in homologous recombination and are homologs to recA [Clark et al., Critical Reviews in Microbiology 20:125-142 (1994)]. The recA protein is the central enzyme in prokaryotic homologous recombination. It catalyzes pairing and strand exchange between homologous DNA molecules, and functions in both DNA repair and genetic recombination [McKee et al, Chromosoma 7:479-488 (1996)]. A number of RecA-like proteins have been found in eukaryotic organisms and yeast [Reiss et al, Proc.NatlAcad.Sci. 93:3094- 3098 (1996)]. Two RecA-like proteins in yeast are Rad51 and Dmcl [McKee et al. (1996) supra]. Rad51 is a highly conserved RecA-like protein in eukaryotes [Peakman et al. , Proc.NatlAcad.Sci. 93: 10222-10227 (1996)] and the A. vinelandii recA gene which is functionally analogous to the E. coli recA gene [Venkatesh et al, Mol Gen Genet 224(3):482-486 (1990)].

As used herein a recombinant deficient host cell is "RecA^"" when the host cell is unable to express a RecA-like protein, including recA itself, which can support homologous recombination. In the simplest case, the gene encoding the RecA-like protein has been deleted in a RecA^" host cell. Alternatively, the RecA-host cell contains a mutation in the recA gene that impairs its function. An "IOBCV contig" contains IOBCV s comprising overlapping genomic fragments. One such IOBCV contig is a "BBPAC contig" which contains BBPACs comprising overlapping genomic fragments, e.g., a "BAC contig" which contains BACs comprising overlapping genomic fragments.

Genomic DNA.

Any eukaryotic cell potentially can serve as the source for the genomic DNA to be used in the present invention. The genomic DNA can contain regulatory and intron DNA regions in addition to coding regions. Clones derived from cDNA will not contain intron sequences. Sources envisioned by the present invention include all types of eukaryotic cells in the animal and plant kingdom. Particular sources include but are not limited to mammals including humans, chimpanzees, gorillas, orangutans, whales, porpoises, cattle, horses, goats, sheep, dogs, cats, mice, rats, and rabbits; avians including chickens, pigeons, turkeys, parrots, parakeets, and canaries; amphibians such as frogs; fish such as salmon, swordfish, trout, catfish, and zebrafish; invertebrates such as insects including beetles, ants, flies, mosquitoes, and wasps such as bees; and plants including trees, grasses, and agricultural crops including corn, wheat, soybeans, peas, carrots, potatoes, rice and tobacco.

Large genomic DNA contained in the BACs, PACs, PI and YACs or other IOBCVs having a genomic insert that is about 20kb or larger are preferable. Clones having even larger inserts, e.g., greater than about lOOkb are even more preferable due to their higher probability of containing a given eukaryotic gene within a single insert (e.g., the average size of a human gene is about 30kb in humans). Such genomic cloning systems also have a low copy number, and therefore, can be more readily modified. Figure 1 exemplifies insertion modules that can be used to place the regulatory elements into genomic clones.

The IOBCVs that can be employed in the methods of the present invention can be obtained from a number of sources. For example, YAC libraries comprising genomic DNA can be constructed by known procedures [Burke et al, Science, 236:806-812 (1987); U.S. Patent No:4,889,806, Issued 12/26/89; Green et al, Science 250:94-98 (1990] but alternatively, are now readily available. In addition, E. coli-based artificial chromosomes for human libraries have been described [Shizuya et αl, Proc. Natl. Acad. Sci. 89:8794-8797 (1992); loannou et αl., In Current Protocols in Human Genetics (ed., Dracopoli et al.) 5.15.1-5.15.24 John Wiley & Sons, New York (1996); Kim et al, Genomics 34:213-218 (1996)]. Libraries of PACs and BACs also have been constructed [reviewed in Monaco et al, Trends BiotechoL, 12:280-286 (1994)], that are readily isolated from the host genomic background for example by classical alkaline lysis plasmid preparation protocols [Birnboim et al, Nucleic Acids Res. 7:1513-1523 (1979)], or alternatively, with the use of a nucleobond kit, a boiling Prep, or by cesium gradient [Sambrook et al, (1989)]. BAC, PAC, and PI libraries are also available for a variety of species (e.g. Research Genetics, Inc., Genome Research, Inc., Texas A&M has a BAC center to make a BAC library for livestock and important crops). BACs also can be used as a component of mammalian artificial chromosomes. The use of a mouse genomic BAC library from Research Genetics is exemplified in U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No. 09/102,490, Filed June 22, 1998; and U.S. Serial No.09/356,987, Filed July 20, 1999; the contents of which are all hereby incorporated by reference in their entireties.

In addition, the BBPACs can be constructed from vectors containing the HCUs of the present invention [See Shizuya et al, Proc. Natl. Acad. Sci. 18:8794-8797 (1992); and Kim et al, Genomics 34:213-218 (1996), the contents of which are all hereby incorporated by reference in their entireties]. The HCUs can be inserted into the vectors by known methodology including homologous recombination e.g., by means described in U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No. 09/102,490, Filed June 22, 1998; and U.S. Serial No.09/356,987, Filed July 20, 1999; the contents of which are all hereby incorporated by reference in their entireties.

BBPAC contigs are available from various Genome projects, including the Human Genome Project for example. Bacterial cells that can be used to manipulate the BBPACs include any bacterial cell that can support the BBPAC and the shuttle vector, preferably the original BBPAC host cell.

Maxiprep DNA from BBPACs with HCU insertions can be prepared by a number of methods including by cesium gradient, or with commercially available columns (e.g., Nucleobond, etc.) as described in U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No. 09/102,490, Filed June 22, 1998; and U.S. Serial No.09/356,987, Filed July 20, 1999; the contents of which are all hereby incorporated by reference in their entireties. If a pool of modified BBPACs are to be used, the maxiprep DNA can be prepared from the culture containing the pool of bacteria.

Heterologous Eukaryotic Promoters and Enhancers Heterologous eukaryotic promoters can be used with or without other Transcriptional Regulatory Elements (TREs). The HEP/TREs of the present invention can be any eukaryotic promoter and/or enhancer (including viral promoters and/or enhancers). In certain situations it is desirable for the promoter to stimulate strong and ubiquitous expression in most eukaryotic cell types. In this case, it is preferable that the eukaryotic promoter is a strong promoter such as the simian virus 40 (SV40) promoter, metallothionein-1 promoter, the cytomegalo virus (CMV) promoter, the actin promoter, the elongation factor I promoter, the cauliflower mosaic virus promoter, the nopaline synthase promoter [Broido et al, Nucl. Acids Res. 17:7891- 7903 (1989)] or the herpes thymidine kinase promoter [Wagner et al, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981)].

Additionally, the promoter can be a tissue specific promoter such as the elastase I gene control region which is active in pancreatic acinar cells [Swift et al, Cell 38:639-646 (1984); Ornitz et al, Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, Hepatology 7:425-515 (1987)]; the insulin gene control region which is active in pancreatic beta cells [Hanahan, Nature 315:115-122 (1985)]; the immunoglobulin gene control region which is active in lymphoid cells [Grosschedl et al, Cell 38:647-658 (1984); Adames et al, Nature 318:533-538 (1985); Alexander et al, Mol. Cell. Biol. 7: 1436-1444 (1987)]; mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells [Leder et al, Cell 45:485-495 (1986)]; albumin gene control region which is active in liver [Pinkert et al, Genes and Devel. 1:268-276 (1987)]; alpha-fetoprotein gene control region which is active in liver [Krumlauf et al, Mol Cell. Biol. 5:1639-1648 (1985); Hammer et al, Science 235:53-58 (1987)]; alpha 1-antitrypsin gene control region which is active in the liver [Kelsey et al, Genes and Devel. 1: 161-171 (1987)];, beta-globin gene control region which is active in myeloid cells [Mogram et al, Nature 315:338-340 (1985); Kollias et al, Cell 46:89-94 (1986)]; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain [Readhead et al, Cell 48:703-712 (1987)]; myosin light chain-2 gene control region which is active in skeletal muscle [Sani, Nature 314:283-286 (1985)]; gonadotropic releasing hormone gene control region which is active in the hypothalamus [Mason et al, Science 234:1372-1378 (1986)]; the nestin promoter, the GFAP promoter, and the keratin 14 promoter.

Alternatively, the HEP can be an inducible promoter, such as the metallofhionein promoter, which is induced by exposure to heavy metals, a glucocorticoid inducible promoter, an estrogen inducible promoter, a tetracycline regulated promoter, a Gal4-regulated promoter, and the interferon regulated promoter.

In a particular embodiment the promoter can be a bi-directional promoter which would allow two adjacent genes in an IOBCV having opposite orientations to be expressed simultaneously as exemplified in U.S. Serial No.09/007,206, Filed January 14, 1998; and U.S. Serial No.09/102,488, Filed June 22, 1998; the contents of which are all hereby incorporated by reference in their entireties.

The HEPs of the present invention can also include a retroviral LTR. The regulatory elements include but are not limited to the CMV enhancer, the IgG heavy chain enhancer; eukaryotic locus control regions and insulator element. In addition, in order to translate proteins from genes lacking an immediate translation initiation site (i.e., from second coding exons), a eukaryotic translation initiation signal (e.g., a Kozak sequence) may also be supplied at the 3'end of the HEP/TRE. The transcription regulatory elements include but are not limited to eukaryotic enhancers, eukaryotic transcription factor binding sites, locus control elements, insulator elements, and matrix attachment sites.

The HEP/TREs of the present invention can further comprise a heterologous nucleotide sequence encoding a peptide or polypeptide that acts as a 5' Fusion Polypeptide (FP). Thus the HEP/TRE/FP or HTF has a HEP and TRE as defined above, and further comprises a polypeptide coding sequence. Preferably a eukaryotic translation initiation signal (e.g., a Kozak sequence) is contained in between the HEP/TRE and the heterologous nucleotide sequence encoding the FP of the HTF. The HTF, when fused in frame with an exon of a gene encoded by the cloned genomic DNA (i.e., the gene of interest), can enable the expression of a fusion protein comprising an exogenously supplied N-terminal polypeptide fused to the polypeptide encoded by the gene of interest. The N-terminal fusion polypeptide can be, for example, an epitope tag for purification and/or detection [e.g., a FLAG tag, a MYC tag, or a hemagglutinin tag (HA tag)]; a marker protein, and/or enzyme such as B-galactosidase, green fluorescent protein (GFP), alkaline phosphatase, or luciferase; a purification marker such as the IgG heavy chain, glutathione-S-transferase (GST), or protein A. Alternatively, the N-terminal fusion polypeptide can be a subcellular targeting signal such as a signal peptide, a mitochondria targeting signal, or a transmembrane signal.

In a particular embodiment, one or more heterologous transcriptional regulatory elements (HTRE) can be inserted adjacent to an endogenous promoter (see EP in Figure 1) in a cloned genomic DNA to enhance the transcription level of a given gene or portion thereof, without insertion of a heterologous promoter. In addition, the HTRE can include a eukaryotic translation initiation signal (e.g., a Kozak sequence) and or additional heterologous nucleotide sequences (e.g., encoding a fusion protein). Antibiotic resistance genes or portions thereof, such as the neo gene can also be included with the HCUs of the present invention. Introduction of HCUs into Genomic DNA Many large insert genomic clones, such as BACs, PACs and PI, are propagated in a recombination deficient E. coli host strains. Indeed, when the genomic DNA is contained in a BAC, the introduction of the HCU is preferably performed in a recombination deficient E. coli host strain and more preferably includes the transient expression of a recombination protein such as the E. coli recombination enzyme RecA to introduce the HCU into the cloned genomic DNA (See Figure 3). Such methodology has been described previously to facilitate homologous recombination in an IOBCV [U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No. 09/102,490, Filed June 22, 1998; U.S. Serial No.09/356,987, Filed July 20, 1999; U.S. Serial No.09/007,206, Filed January 14, 1998; and U.S. Serial No.09/102,488, Filed June 22, 1998; the contents of which are all hereby incorporated by reference in their entireties].

Thus, the introduction of the HCU is preferably performed with a shuttle vector having a conditional origin of replication. For example, the HCU can be subcloned into a shuttle vector that has an origin of replication that is temperature-dependent as exemplified in U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No.

09/102,490, Filed June 22, 1998; U.S. Serial No.09/356,987, Filed July 20, 1999; U.S.

Serial No.09/007,206, Filed January 14, 1998; and U.S. Serial No.09/102,488, Filed June 22, 1998 the contents of which are all hereby incorporated by reference in their entireties.

Alternatively, the replication of the shuttle vector can be dependent upon the expression of a specific protein. For example, the pLD55.recA shuttle vector contains a R6Kγ DNA origin of replication [oriR (R6Kγ)], which requires the presence of the pir protein to replicate. The R6Kγ origin of replication has been successfully used to integrate plasmids into an homologous region of the Ε.coli Genome of pir- E.coli strains [Miller and Mekalanos, J. Bacteriol 170:2575-2583 (1988); Metcalf et al, Plasmid 35:1-13 (1996); Kalogeraki and Winans, Gene 188:69-75, (1997)]. Thus, in one particular embodiment, the HCU is subcloned into the pLD55.recA shuttle vector (Figure 2). Since the pLD55.recA plasmid only replicates in the presence of the pir protein, the cloning can be performed in the 21118 E coli strain which constitutively expresses the pir protein.

A genomic DNA fragment (preferably about lOObp or longer, and more preferably about 450bp or longer) can be subcloned from the large cloned genomic DNA 3' to the HCU in the pLD5.recA shuttle vector (Figure 3). The genomic DNA is chosen so that the HCU will be placed adjacent to or fused to the exon of a gene to be modulated (e.g., over-expressed) in eukaryotic cells following the homologous recombination event. The genomic DNA sequence can be determined by direct sequencing of a large insert genomic clone through a subcloning step, or by a now commonplace genomic database search.

Alternatively, when an Expressed Sequence Tag (EST) is known, the full length coding sequence can be obtained by using the EST to design an appropriate nucleotide sequence to perform homologous recombination as disclosed herein. Thus, an HCU can be then be placed into an IOBCV that encodes the full length coding sequence that comprises the EST, thereby enabling the expression of the full length coding sequence when the IOBCV is introduced into an appropriate eukaryotic cell. This procedure also allows the retrieval of the full length mRNA and the corresponding cDNA. The eukaryotic cells transferred with modified IOBCV can produce partial or preferably full length polypeptides encoded by the gene from which the EST sequence was obtained.

In a preferred embodiment, the genomic insert can by amplified by high fidelity PCR with primers carrying appropriate restriction sites at their ends to facilitate the sub-cloning of the PCR products into the shuttle vector. Plasmid DNA of the resulting targeting plasmid can be prepared using a standard alkaline lysis method [Sambrook et al, (1989), supra], for example. Electrocompetent cells containing the large insert genomic clones can also be prepared according to standard protocols. Thus, the present invention provides a method for selectively inserting an HCU of the present invention operatively upstream of the coding sequence of a selected gene (i.e., a gene of interest) by homologous recombination. The gene of interest is preferably contained by an IOBCV. The IOBCV is either in an appropriate host cell or introduced into the appropriate host cell. In a particular embodiment, neither the IOBCV alone, nor the host cell, either alone or in combination can independently support homologous recombination. In one such embodiment, the IOBCV is a BAC, and the host cell is a RecA deficient E. coli cell.

In a particular embodiment, the HCU of the present invention can be inserted into a recombination cassette that selectively integrates into a particular nucleotide sequence operatively upstream of the coding sequence of the gene of interest when the recombination deficient cell is transiently induced to support homologous recombination. Thus, the present invention allows the integration of an HCU operatively upstream of a gene of interest of the IOBCV, thereby permitting the HCU to modulate (i.e., attenuate and/or control) the expression of the gene of interest when the resulting modified IOBCV is placed into a eukaryotic cell (and the eukaryotic cell is in an environment that allows gene expression).

In a particular embodiment, the recombination deficient host cell cannot independently support homologous recombination because the host cell is RecA^", and inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of a recombination protein (e.g., RecA) that supports recombination in the host cell. Such induction may be performed by expressing such a recombination protein contained by the recombination deficient host that is under the control of an inducible promoter, for example. In another embodiment inducing the transient expression of the recombination protein is performed with a conditional replication shuttle vector that encodes the recombination protein. In one embodiment of this type, the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) that replicates at a permissive temperature, but does not replicate at a non-permissive temperature. Thus the TSSV can encode a recombination protein that is expressed in the host cell and supports the homologous recombination between a specific nucleic acid contained by the recombination cassette (which contains the HCU) and the particular nucleotide sequence of the gene of interest contained by the IOBCV, permitting the insertion of the HCU upstream of the coding region of the gene of interest. Inducing the transient expression of the recombination protein then consists of transforming the host cell with the TSSV at a permissive temperature, and growing the host cell at a non- permissive temperature. The TSSV encoding the recombination protein is diluted out when the host cell is grown at the non-permissive temperature.

A positive BAC that contains a desired gene of interest can generally be obtained in a few days. To insert a gene of interest into a selected locus in the BAC, the region of insertion can be mapped for restriction enzyme sites. Whereas subcloning is necessary for detailed mapping, it is generally unnecessary since rough mapping is usually sufficient. As is readily apparent, other independent origin based cloning vector genomic libraries can be screened and the isolated independent origin based cloning vectors manipulated in an analogous fashion.

The conditional replication shuttle vectors of the present invention can be constructed so as to contain a recombination cassette that can selectively integrate into the nucleotide sequence of the gene of interest encoded by the IOBCV. Such conditional replication shuttle vectors can be constructed by inserting, for example, a PCR amplified recombination protein gene into an appropriate conditional replication shuttle vector which can also contain a specific drug resistant gene and/or can be subsequently modified to contain one. In a preferred embodiment of this type, the drug resistant gene can also be counter-selected against, such as with tetracycline and fusaric acid. Alternatively, in addition to the drug resistant gene the conditional shuttle vector can also contain a counter-selection gene such as a gene that confers sensitivity to galactose, for example.

In U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No. 09/102,490, Filed June 22, 1998; and U.S. Serial No.09/356,987, Filed July 20, 1999 (the contents of which are all hereby incorporated by reference in their entireties), the E. coli K12 recA gene (1.3kb) was inserted into the BamHl site of a pMBO96 vector. In this case the vector already carried a gene that bestows tetracycline resistance, and in addition contains a pSClOl temperature sensitive origin of replication, which allows the plasmid to replicate at 30 degrees but not at 43 degrees.

The recombination protein of a conditional replication shuttle vector can be controlled by either an inducible promoter or a constitutive promoter. In one particular embodiment the transient expression of the recombination protein is achieved by the transient induction of the inducible promoter in a host cell. In another embodiment, the constitutive promoter is the endogenous E. coli recA promoter.

The conditional replication shuttle vector preferably also contains at least one unique cloning site. In certain cases a building vector is used to construct the recombination cassette as described previously [U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No. 09/102,490, Filed June 22, 1998; and U.S. Serial No.09/356,987, Filed July 20, 1999, the contents of which are all hereby incorporated by reference in their entireties]. When a building vector is used for the construction of the recombination cassette, one unique site is reserved for transferring the recombination cassette containing the HCU from the building vector to the conditional replication shuttle vector. For example, a polylinker can be inserted between two specific restriction sites to create additional restriction sites that allow cloning of the recombination cassette into the conditional replication shuttle vector. In any case, the conditional replication shuttle vector created can contain a recombination cassette comprising the HCU that is situated such that after the recombination event, the HCU is operatively upstream of the gene of interest comprised by the IOBCV, (see Figure 3, below).

According to one method of the present invention the conditional replication shuttle vector is transformed into a RecA^' host cell containing the IOBCV which comprises the gene of interest. The IOBCV can also contain a gene which bestows resistance to a host cell against a corresponding toxic agent/drug such as an antibiotic or in a specific embodiment, chloramphenicol. The cells are grown under the conditions in which the conditional replication shuttle vector can replicate (e.g., when the conditional replication shuttle vector is a TSSV which replicates at 30° but not at 43°, the host cell is grown at 30 °C) and the transformants can be selected via the specific drug resistant gene (or first drug resistant gene) carried by conditional replication shuttle vector, and the second drug resistant gene carried by the independent origin based cloning vector. Since the conditional replication shuttle vector also carries the recombination protein gene, homologous recombination can occur between the conditional replication shuttle vector and the independent origin based cloning vector to form co-integrates through the sequence homology of the recombination cassette. The co-integrates then can be selected by growing the cells on plates containing the first and second drugs at non-permissive conditions (e.g. for the TSSV above, at 43 °C) so that the non-integrated, free conditional replication shuttle vectors are lost. This results in the selection for host cells carrying the integrated conditional replication shuttle vectors, (which can co-integrate either into the IOBCV, or into the host chromosome). Correct independent origin based cloning vector co-integrates can be identified by PCR or more preferably with Southern blot analyses.

When desired, the co-integrates can then be re-streaked onto plates containing the second drug, (i.e., the drug which the gene initially carried by the IOBCV protects against) and grown under non-permissive conditions overnight. A fraction of the co- integrates undergo a second recombination event (defined as resolution), through the sequence homology of the gene of interest and the recombination cassette. The shuttle vector can be constructed so that the resolved IOBCV automatically loses both the first drug resistant gene (i.e. in the example above, the specific drug resistant gene contained by the conditional replication shuttle vector) and the recombination protein gene due to the linkage arrangement of the recombination protein gene, the drug resistant gene, the HCU and the homologous nucleic acid on the conditional replication shuttle vector. In addition, the excised conditional replication shuttle vector cannot replicate under the non-permissive conditions and is therefore diluted out. The resolved independent origin based cloning vectors can be further selected for by growing the host cells (e.g., at 37°C) on plates containing the second drug and an agent that counterselects against cells containing the gene resistant to the first drug (e.g., a gene conferring tetracycline resistance may be counter-selected against with fusaric acid). The resolved IOBCV will be either the original independent origin based cloning vector or the precisely modified independent origin based cloning vector. One method to identify the correctly resolved BAC is to choose 5-10 colonies and prepare a miniprep DNA. The DNA can then be analyzed using Southern blots to detect the correct targeting events. Alternatively, the desired clones can be identified by colony hybridization using a labeled probe for the specific nucleic acid contained by the recombination cassette. Such probes are well known in the art, and include labeled nucleotides probes that hybridize to the nucleic acid sequence. Alternatively, a marker nucleic acid can be included in the recombination cassette and constructed so as to remain with the specific nucleic acid upon integration into the independent origin based cloning vector.

A modified IOBCV of the present invention can be purified by gel filtration, e.g. a column filled with SEPHAROSE CL-4B. The column can be pre-equilibrated in an appropriate buffer, as described in U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No. 09/102,490, Filed June 22, 1998; and U.S. Serial No.09/356,987, Filed July 20, 1999, the contents of which are all hereby incorporated by reference in their entireties. The purified DNA can be directly visualized with ultraviolet light after ethidium bromide staining, for example. Columns such as the SEPHAROSE CL-4B column also can efficiently separate degraded DNA from the pure linear DNA.

The present invention further provides a one step gene targeting protocol to form a co-integrate (see, Figure 3). Although final resolution is not necessary, optionally the HCU can be placed in front of an exon in the cloned genomic DNA, and the targeting vector sequence can be removed from the cloned genomic DNA using a second recombination event. For example, if a second homology sequence is placed into the initial targeting vector, a second homology recombination step can be carried out to selectively eliminate the vector plasmid while leaving the HCU in the cloned genomic DNA [U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No. 09/102,490, Filed June 22, 1998; and U.S. Serial No.09/356,987, Filed July 20, 1999; the contents of which are all hereby incorporated by reference in their entireties]. The vector can also be removed using a site specific recombination step (e.g., using the ere recombinase and one or more lox sites as described in U.S. 4,959,317 Issued September 25, 1990)

Liquid BAC modification: The present invention further provides a method of preparing modified BBPACs that can be used in high throughput procedures. One such method is based on a series of plasmids constructed for allele replacement into the bacterial chromosome [Metcalf et al, Plasmid 35: 1-13 (1996), the contents of which are hereby incorporated by reference in its entirety]. In one particular embodiment, the vector is pLD55, which contains the R6Kγ origin of replication and tet R and amp R selectable markers. The recA gene can be inserted into pLD55.recA, which now carries the recA gene for restoration of homologous recombination in the BAC strain as disclosed above. Preferably a more robust tet R gene is used since the allele present in the original pLD55 vector was not optimal for the fusaric acid negative selection that is used in the resolution step of the BAC modification procedure, yielding pLD55.recA.tet. This vector carries both the recA gene and selectable markers used in the BAC modification protocol disclosed herein, and in a particular embodiment merely substitutes the original temperature-sensitive plasmid origin described above with the conditional R6Kγ origin [Metcalf et al, Plasmid 35:1- 13 (1996)].

The R6Kγ origin is completely dependent on the pir protein, which is not generally encoded by BAC strains. Therefore, all cloning into the shuttle vector is carried out in apir+ bacterial strain [Metcalf et αl., Plasmid 35:1-13 (1996)] in which the shuttle vector can be propagated effectively. When this shuttle vector is electroporated into the BAC strains, it absolutely cannot replicate. Thus, the only way the BAC strain can contain both the chloramphenicol resistance and the tetracycline resistance markers is if the shuttle vector integrates into the BAC episome forming the cointegrant that is sought. One such protocol can include: 1. Preparation of competent cells from the BAC.

2. Electroporation of the shuttle vector into the BAC strain; (preferably two separate vials).

3. Selection in liquid culture in high ampicillin (e.g., lOOug/ml). 4. Dilute 1:1000, selection again in high ampicillin.

5. Preparation of BAC DNAs.

6. Assaying cointegrant by PCR.

Preferably two separate vials are electroporated for each BAC strain.

The Ori R6Kγ conditional replication shuttle vector takes advantage of the same selection system as disclosed herein for the temperature sensitive conditional replication shuttle vector but is preferable for liquid media high throughput procedures. Preferably a streamlined version of the vector containing only the tetR, oriR, and RecA genes is used. The Ori R6Kγ conditional replication shuttle vector can also carry a marker cassette containing a myc tag in all three reading frames followed by a stop codon, and/or an IRES/EGFP/polyA gene for creation of a fusion transcript expressing enhanced green fluorescent protein from an internal ribosome entry site (IRES). In a particular embodiment LoxP sites can surround the vector sequences. One protocol for BAC modification using this Ori R6Kγ conditional replication shuttle vector is as follows: 1. Prepare competent cells from four independent BAC isolates.

2. Electroporate with pLD55 3'trap vector.

3. Select for growth in liquid culture containing chloramphenicol and tetracycline.

4. Dilute culture, repeat selection. 5. Miniprep DNA, cleave with Notl (introduced by the pLD55 3'trap cointegration); select clone with gene located in the center of the BAC.

6. Prepare competent cells and transform 2 vials with pWM91/cre.

7. Plate on fusaric acid plates to select against BACs that still carry the tetR marker. 8. Prepare DNA and PCR assay for appropriate modification. 9. Midiprep modified BAC clone and prepare for transgenesis.

Using such a procedure, modification and characterization of multiple BACs can be performed in a relatively short period of time. Furthermore, this protocol can be fully automated by changing the fusaric acid selection from plates to liquid culture if the project is to be scaled up.

Introduction of the HCU into Genomic DNA in Recombination Competent Bacteria: In another particular embodiment, the HCU can be inserted into an IOBCV (e.g., a BBPAC) in recombination competent bacteria by homologous recombination using standard homologous recombination protocols. For example, the BBPAC DNA can be electroporated into recombination competent bacteria. The HCU and a homology sequence can be placed into a temperature sensitive vector or a R6kr containing vector as described above except, the vector no longer needs to encode a recombination protein. The targeting vector is th'en transformed into the recombination competent bacteria and the resulting homologous recombinant is selected.

Introduction of an HCU into Genomic DNA in a Single Cell Eukaryotic Organism such as Yeast: The Yeast Artificial Chromosome (YAC) also contains large genomic inserts and can be readily modified through homologous recombination. Thus, the HCUs of the present invention also can be inserted into YACs [see Burke et al., Science, 236:806-812 (1987); and U.S. Patent No:4,889,806, Issued 12/26/89 the contents of both of which are hereby incorporated by reference in their entireties] and used as described above.

Introducing Modified Genomic Clones into Eukaryotic Cells A large quantity of genomic DNA (greater than about 50 ug) can be obtained using standard protocols for the IOBCVs, e.g., BACs/PACs and YACs, such as Qiagen tip-500 columns. The plasmid DNA can then be introduced into eukaryotic cells by of any of a number of standard methods including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter [see, e.g., Wu et al, 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al, Canadian Patent Application No. 2,012,311, filed March 15, 1990]. In a particular embodiment BBPACs modified by methods of the present invention are introduced by a psoralen-inactivated adenovirus as carrier as described by [Baker et al, NAR 25: 1950-1956 (1997)]. Essentially any cell line can be used since BBPACs replicate in mammalian cells and the heterologous promoter can be selected to be active in the host eukaryotic cell of choice. For YACs in particular, spheroplast fusion can be employed.

Upon transfection, the large genomic DNA can either exist as episomal DNA or as an integrated genomic DNA. The former may or may not replicate in the host eukaryotic cells. There is evidence that episomes of large genomic DNA clones can exist independently longer in eukaryotic cells than episomes of smaller genomic DNA clones, most likely due to their ability to replicate. If the genomic DNA is integrated into the host genome, the integrated DNA can be stably maintained in the eukaryotic cells in subsequent generations. A number of eukaryotic cells can be used including immortalized cell lines, primary cells, secondary cells, stem cells, and fertilized zygotes derived from any eukaryotic organism. The transfected eukaryotic cells then can be cultured according to standard procedures and/or used to make transgenic animals.

Monitoring the Expression of the Activated Eukaryotic Genes

There are a variety of methods for monitoring the expression of a gene of interest of an IOBCV in a eukaryotic cell. For example, a cell extract can be prepared and a Northern blot can be performed, [Sambrook et al, (1989), supra] using a cDNA or genomic DNA fragment corresponding to an exon or portion thereof of the gene. A Western blot also can be used, provided that an antibody to the gene product of the gene of interest is available. Alternatively, the gene product can be tagged with an epitope that reacts with a known antibody. Yet another method is by direct detection for the expression of a fusion protein of the gene product, e.g., with green fluorescence protein or β-galactosidase. In addition, the gene product itself may have a particular activity that can be assayed, e.g., if it is an enzyme, or has a specific binding partner.

Producing Proteins from Transfected Eukaryotic Cells Preparation of the modified IOBCV comprising an HCU that is operatively upstream of a gene of interest is performed as described above. The IOBCV is then transfected into an appropriate eukaryotic cell, as indicated above. Next the eukaryotic cell is grown under conditions in which the gene product encoded by the gene of interest (in this case a protein) is expressed. The protein then can be purified by standard protein purification procedures.

Figure 3 outlines one protocol for expressing a protein by the methodology of the present invention. As exemplified, a BAC or PAC clone containing the entire coding region and the polyadenylation signal for the gene of interest is identified by directly screening the BAC or PAC library and/or through a DNA database search. A HEP/TRE is inserted through homologous recombination in front of a 5' non-coding exon or first coding exon as described above. Alternatively, a 5'fusin peptide or protein, such as an IgG heavy chain, a FLAG epitope tag, a polyhistidine tag, a glutathione-S-transferase (GST) fusion protein, or a maltose-binding (MBP) protein fusion protein could be fused in-frame with the 5'coding region of the gene, to facilitate the purification of a protein of interest. For example, GST binds glutathione conjugated to a solid support matrix, MBP binds to a maltose matrix, and polyhistidine chelates to a Ni-chelation support matrix. The fusion protein can be eluted from the specific matrix with appropriate buffers, or by treating with a protease specific for a cleavage site preferably engineered between the gene product from the gene of interest and the fusion partner (e.g., GST, MBP, or poly-His) as described above. Thus, a 5' fusion peptide or protein may be designed so that it can be readily cleaved by a protease to facilitate the purification and/or after the protein is purified.

Alternatively, a chimeric protein may be generated that comprises both the green fluorescent protein, for example, and the gene product encoded by the gene of interest, In this case, the chimeric protein can be visually detected by fluorescence. In this particular embodiment, the chimeric protein can also be used to determine the intracellular localization of the gene product encoded by the gene of interest in a eukaryotic cell.

The proteins synthesized by the methods of the present invention also can be isolated from the eukaryotic cells by more classical protein purification procedures. Initial steps may include salting in or salting out, such as in ammonium sulfate fractionations; solvent exclusion fractionations, e.g., an ethanol precipitation; detergent extractions to free membrane bound proteins using such detergents as TRITON X-100, TWEEN-20 etc.; or high salt extractions. Solubilization of proteins may also be achieved using aprotic solvents such as dimethyl sulfoxide and hexamethylphosphoramide. In addition, high speed ultracentrifugation may be used either alone or in conjunction with other extraction techniques.

Generally good secondary isolation or purification steps include solid phase absorption using calcium phosphate gel or hydroxyapatite; or solid phase binding. Solid phase binding may be performed through ionic bonding, with either an anion exchanger, such as diethylaminoethyl (DEAE), or diethyl [2-hydroxypropyl] aminoethyl (QAE) SEPHADEX or cellulose; or with a cation exchanger such as carboxymethyl (CM) or sulfopropyl (SP) SEPHADEX or cellulose. Alternative means of solid phase binding includes the exploitation of hydrophobic interactions e.g., the using of a solid support such as phenylSepharose and a high salt buffer; affinity-binding (using a binding partner of the proteins), immuno-binding, using e.g., an antibody to the protein bound to an activated support; as well as other solid phase supports including those that contain specific dyes or lectins etc. A further solid phase support technique that is often used at the end of the purification procedure relies on size exclusion, such as SEPHADEX and SEPHAROSE gels, or pressurized or centrifugal membrane techniques, using size exclusion membrane filters.

Solid phase support separations are generally performed batch-wise with low-speed centrifugations or by column chromatography. High performance liquid chromatography (HPLC), including such related techniques as FPLC, is presently the most common means of performing liquid chromatography. Size exclusion techniques may also be accomplished with the aid of low speed centrifugation.

In addition size permeation techniques such as gel electrophoretic techniques may be employed. These techniques are generally performed in tubes, slabs or by capillary electrophoresis.

Almost all steps involving protein purification employ a buffered solution. Unless otherwise specified, generally 25-100 mM concentrations of buffer salts are used. Low concentration buffers generally imply 5-25 mM concentrations. High concentration buffers generally imply concentrations of the buffering agent of between 0.1-2.0 M concentrations. Typical buffers can be purchased from most biochemical catalogues and include the classical buffers such as Tris, pyrophosphate, monophosphate and diphosphate and the Good buffers [Good et al, Biochemistry, 5:467 (1966); Good and Izawa,, Meth. EnzymoL, 24B:53 (1972); and Fergunson and Good, Anal. Biochem., 104:300 (1980)] such as Mes, Hepes, Mops, tricine and Ches.

Materials to perform all of these techniques are available from a variety of commercial sources such as Sigma Chemical Company in St. Louis, Missouri.

Microinjection of Modified Cloned Genomic DNA into Transgenic Animals IOBCVs (e.g., BBPACs) modified through methodology described above can be microinjected into a fertilized animal zygote to generate a non-human transgenic animal mammal [see, U.S. Serial No. 08/880,966, Filed June 23, 1997; U.S. Serial No. 09/102,490, Filed June 22, 1998; and U.S. Serial No.09/356,987, Filed July 20, 1999; the contents of which are all hereby incorporated by reference in their entireties]. The non-human transgenic animals generated are also part of the present invention.

Such non-human transgenic animals can be obtained through gene therapy techniques or by microinjection of the IOBCV of the present invention, for example, into an embryonic stem cell or an animal zygote as indicated above. Microinjection of BACs has been shown to be successful in a number of animals including rats, rabbits, pigs, goats, sheep, and cows [in Transgenic Animals Generation and Use e<£,L.M. Houdebine, Harwood Academic Publishers, The Netherlands (1997)]. Alternatively, a yeast artificial chromosome (YAC) can be used. In a preferred embodiment the transgenic animal is a mouse and the embryonic stem cell is a mouse embryonic stem cell.

The modulation of the expression of a gene of interest is then effected in the resulting transgenic mammal. Such modulation of expression, for example, can be used to probe the function of the gene in vivo. In addition, the methodology of the present invention can be used to augment production of certain commercially or pharmaceutically important proteins from these transgenic animals.

Thus, the present invention further provides methods of preparing transgenic mice with the modified IOBCVs (e.g., BACs) of the present invention. In a particular embodiment multiple founders for each modified BAC are generated so that the expression patterns can be analyzed at different developmental stages. The founder(s) chosen for breeding are preferably males to maximize the yield of Fl progeny.

Eukaryotic Expression Libraries One important aspect of the present invention includes a method of generating a non-biased genomic-DNA based expression library for any given eukaryotic organism. As indicated above, current cDNA based expression libraries are highly biased towards the gene expression pattern in the cell type from which the libraries were constructed. However, through the use of a complete set of overlapping BACs and PACs, a BBPAC contig for example, a genomic DNA clone based expression library can be constructed for any given genome. This library can be based on BAC/PAC clones of known sequences and gene distributions, and can be further defined with the aid of a DNA data base search for homology with known cDNAs, ESTs, and/or through the use of gene annotation programs. Therefore, the DNA sequences of a given IOBCV library can be obtained by any of a number of methods including by direct sequencing, shot gun sequencing, and/or a DNA database search (with or without a gene annotation program). The location and structure of one or more genes contained by a given BAC/PAC for example, can thus be identified. The eukaryotic genes contained by the BBPAC then can be identified and/or modified. Preferably genes that are lacking the 3'UTR are excluded. For each gene to be modified, preferably about 500kb of an homology arm is located that corresponds to the sequence of the first identified exon (and/or portion of the intron following the exon, where appropriate). This fragment can be amplified by PCR and subcloned into a pLD55.RecA vector, for example, containing a HEP/TRE. The homology is designed and cloned in such way to facilitate the insertion of HEP/TRE by homologous recombination in front of the first identified exon. If necessary, a eukaryotic translation initiation signal (e.g., a Kozak sequence) can be supplied to support the translation of the gene of interest. BBPAC targeting can then be performed (using a pLD55.RecA based targeting system for example). Preferably, it is performed in liquid medium for high-throughput preparations. The modified BBPAC clone can be characterized by PCR or Southern blots to confirm the targeting event. Modified clones may be stored in liquid nitrogen as a glycerol stock until use.

This process can be re-iterated for every gene contained by a IOBCV, (excluding ones that are missing poly-adenylation signals). This procedure can be further repeated for every gene identified in a given genome using IOBCV libraries. In a preferred embodiment, additional IOBCV clones are identified with standard screening techniques that use cDNA or genomic DNA probes.

Once an IOBCV is prepared that can express a gene of interest, it can be used to transfect eukaryotic cells. These eukaryotic cells can be used in a variety of methods including in functional studies, cDNA retrieval, for protein production and or high throughput drug screening. Furthermore, the method of generating genomic based expression libraries as disclosed herein is extremely important in the post-genomic era. Such libraries represent an unbiased and complete set of genes for any given eukaryotic genome. Such libraries have significant advantages over the current cDNA based expression system. Moreover, the sequencing of additional eukaryotic genomes, including plants and lower vertebrates provides the opportunity to generate corresponding additional genomic based expression libraries using the methodology of the present invention. Furthermore, the present invention provides a method of converting the gene sequences determined in the various genome projects, including those in silico, into biologically active reagents ready for further studies including for drug assays, diagnostics, and/or in particular instances in the treatment of diseases. For example, the expression libraries of the present invention are extremely useful in providing tens of thousands of biologically active and naturally occurring polypeptides for high throughput drug screening.

DNA Vaccines The present methodology also can be used to generate specific DNA vaccines. DNA vaccines [International Patent Publication WO 95/20660 and International Patent Publication WO 93/19183, the disclosures of which are hereby incorporated by reference in their entireties] that encode a viral protein to elicit a protective immune response have been demonstrated in numerous experimental systems [Conry et al, Cancer Res., 54:1164-1168 (1994); Cox et al, Virol, 67:5664-5667 (1993); Davis et al, Hum. Mole. Genet., 2:1847-1851 (1993); Sedegah et al, Proc. Natl. Acad. Sci., 91:9866-9870 (1994); Montgomery et al, DNA Cell Bio., 12:777-783 (1993); Ulmer et al, Science, 259:1745-1749 (1993); Wang et al, Proc. Natl. Acad. Sci., 90:4156- 4160 (1993); Xiang et al, Virology, 199:132-140 (1994); Kodihalli et al, J. Virol. 71(5):3391-3396 (1997); and Kodihalli et al, J. Virol 73(3):2094-2098 (1999)]. Genomes for many human pathogens, such as Herpes virus, have been cloned into BACs (Brune et al, TIG 16:254-259, (2000)]. BACs containing a replication-competent, packaging-defective HSV1 virus have also been shown to induce host immunity [Suter et al, Proc. Natl. Acad. Sci. USA 96:12697-12702 (1999)].

The DNA vaccines of the present invention can be introduced into the desired host by methods known in the art, e.g., scarification, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter [see, e.g., Wu et al, J. Biol. Chem. 267:963-967 (1992); Wu and Wu, J. Biol. Chem. 263:14621-14624(1988); and Hartmut et al, Canadian Patent Application No. 2,012,311, filed March 15, 1990]. Thus, a mammal can be inoculated by a parenteral route by intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular methods, by a gene gun or via other acceptable vaccine administration routes.

Furthermore, DNA-based vaccines have been shown to be more effective when the antigen is overproduced. Currently, this is achieved by using an expression vector and a small viral genomic DNA or cDNA molecule [Hasan et al., J. Immuno Methods, 229:1-22, (1999)]. However, the present invention can be applied to overexpress individual viral genes (in the context of a relatively complete viral genome in a BBPAC) to enhance host immunity by using the methodology taught herein and using the resulting BAC, for example as the DNA vaccine. This may be particularly useful to optimize the efficacy of the so-called BAC- VAC [Suter et al, Proc. Natl. Acad. Sci. USA 96:12697 -12702 (1999)].

Gene Therapy The modified IOBCVs of the present invention, i.e., IOBCVs comprising an HCU operatively upstream of a gene of interest prepared by the methods of the present invention, also can be introduced in a viral vector. Such vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt et al, Molec. Cell. Neurosci., 2:320-330 (1991)], an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. [J. Clin. Invest., 90:626-630 (1992)], and a defective adeno-associated virus vector [Samulski et al, J. Virol, 61:3096-3101 (1987); Samulski et al, J. Virol, 63:3822-3828 (1989)] including a defective adeno-associated virus vector with a tissue specific promoter, [see e.g., U.S. Patent No:6,040,172, Issued March 21, 2000].

For in vitro administration, an appropriate immunosuppressive treatment can be employed in conjunction with the viral vector, e.g., adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors [see, e.g., Wilson, Nature Medicine, (1995)]. In addition, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.

In another embodiment the IOBCV can be introduced in a retroviral vector, e.g., as described in Anderson et al, U.S. Patent No. 5,399,346; Mann et al, Cell, 33:153 (1983); Temin et al, U.S. Patent No. 4,650,764; Temin et al, U.S. Patent No. 4,980,289; Markowitz et al, J. Virol, 62:1120 (1988); Temin et al, U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995, by Dougherty et al; and Kuo et al, Blood, 82:845 (1993).

Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.

Alternatively, the vector can be introduced by lipofection. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner, et. al, Proc. Natl. Acad. Sci. U.S.A., 84:7413-7417 (1987); see Mackey, et al, Proc. Natl. Acad. Sci. U.S.A., 85:8027-8031 (1988)]. The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes [Feigner and Ringold, Science, 337:387-388 (1989)]. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting [see Mackey, et. al., 1988, supra]. Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

It is also possible to introduce the modified IOBCV as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., scarification, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter [see, e.g., Wu et al., J. Biol. Chem., 267:963-967 (1992); Wu and Wu, J. Biol. Chem., 263:14621- 14624 (1988); Hartmut et al, Canadian Patent Application No. 2,012,311, filed March 15, 1990].

Antisense Gene Inactivation The methodology of the present invention also can be applied to effect antisense gene inactivation. The methodology of the present invention can be performed to insert a HEP/TRE in the antisense orientation near the 3' end of an exon in a gene encoded in the cloned genomic DNA. When transfected into eukaryotic cells, this modified cloned genomic DNA will transcribe an antisense RNA, which can interfere with the function of the endogenous gene through antisense inhibition or through dS RNA mediated gene silencing.

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule [See Weintraub, Sci. Amer. 262:40-46 (1990); Marcus-Sekura, Nucl. Acid Res, 15: 5749-5763 (1987); Marcus-Sekura Anal.Biochem., 172:289-295 (1988); Brysch et al, Cell Mol. Neurobiol, 14:557-568 (1994)]. Preferably, the antisense molecule employed is complementary to a substantial portion of the mRNA. In the cell, the antisense molecule hybridizes to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of greater than about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, though larger molecules that are essentially complementary to the entire mRNA are more likely to be effective. Antisense methods have been used to inhibit the expression of many genes in vitro [Marcus-Sekura, Anal.Biochem., 172:289-295 (1988); Hambor et al, Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014 (1988)] and in situ [Arima et al, Antisense Nucl. Acid Drug Dev. 8:319-327 (1998); Hou et al. Antisense Nucl. Acid Drug Dev. 8:295-308 (1998); U.S. Patent No. 5,726,020, Issued March 10, 1998; and U.S. Patent No. 5,731,294, Issued March 24, 1998, all of which are incorporated by reference in their entireties].

The present invention may be better understood by reference to the following non- limiting Examples, which are provided as being exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

EXAMPLE 1 Targeted Genomic Clone Activation of Human Nerve Growth Factor

(NGF) Beta Gene in a Bacterial Artificial Chromosome

Introduction NGF beta is a target derived neurotrophic factor responsible for the survival and maintenance of sympathetic and sensory neurons [Levi-Montalcini et al, TIN, 19:514-520 (1996)]. Moreover, NGF beta also exerts a modulatory role on sensory, nociceptive nerves during adulthood and appears to be responsible for the hypalgesia during tissue inflammation [Levi-Montalcini et al, TIN, 19:514-520 (1996)]. In a recent Phase II clinical trial, recombinant human NGF beta protein was found to be safe and was shown to have preliminary efficacy in patients with symptomatic diabetic polyneuropathy [Apfel et al, Neurology 51:695-702 (1998)]. Functional recombinant NGF beta is very easily assayed in culture by its ability to promote neuronal differentiation of PC12 cells [Greene and Tischler, Proc. Natl. Acad. Sci. USA 73:2424-8 (1976); reviewed by Levi et al. , Molecular Neurobiology 2:201-226 (1988)].

In humans, NGF beta is synthesized as a prepro-NGF peptide which is subsequently processed into mature beta NGF [Ulrich et al, Nature 303:821-826 (1983)]. In mouse, the protein is encoded by at least four exons, spans 40kb genomic DNA, and the last exon encodes the mature beta NGF peptide.

Results Identification of a Human Bacterial Artificial Chromosome Containing the beta-NGF Gene: A database search of Genbank using the human beta-NGF cDNA sequence identified one full length human genomic clone, RP4-662-B22, which contains exon 3, exon 4 and the polyadenylation signal of the human beta NGF gene at the end of the BAC insert (Fig.4A). Since exon 4 encodes the mature beta NGF polypeptide, this BAC was selected for performing Targeted Genomic Clone Activation.

The CMV promoter and CMV enhancer are first subcloned into the pLD55.RecA vector (Fig.4B) as described above to create the pLD.55recA.TGCA vector. Then about 500bp of genomic DNA corresponding to the human nerve growth factor (hNGF) exon3, and the immediate intron sequence 3' to the exon are amplified by PCR and subcloned downstream to the CMV promoter to generate the pLD55.RecA.TGCA.NGF vector (or pLD55.NGF vector). The PLD55.NGF vector is used to modify the RP4-662B22 BAC as described above. Correct insertion of the CMV promoter in front of exon 3 of the beta NGF gene in the BAC is confirmed by Southern blot analysis.

In excess of 50 ug of modified BAC DNA is prepared using Qiagen tip-500 columns. 5-10 ug of modified RP4-662-B22 BAC DNA is transfected into COS cells using 20 ug of lipofectamine (BRL-Life Technologies) as described by Kim et al [Genome Res. 8:804-12 (1998)]. The assay for the NGF in the supernatant on PC12 neurite extension is performed according to a standard protocol [Greene and Tischler, Proc. Natl Acad. Sci. USA 73:2424-8 (1976); and Bruce and Heinrich, Neurobio Aging 10:89-94 (1989)].

EXAMPLE 2

Targeted Genomic Clone Activation of Glia Derived Neurotrophic Factor from a PI -derived Artificial Chromosome (PAC).

Introduction GDNF was initially identified as a glioma cell line derived neurotrophic factor for midbrain dopaminergic neurons [Lin et al, Science 260:1130-1132 (1993)]. GDNF also acts as a survival factor in vitro for a variety of neurons, including motor neurons from spinal cord [Henderson et al, Science 266:1062-1064 (1994)], and cerebellar Purkinje cells [Mount et al, Proc. Natl. Acad. Sci. USA 92:9092-9096 (1995)]. Moreover, GDNF has been shown in preclinical study to improve clinical symptoms in rodent and non-human primate models of Parkinson's Disease [Reviewed by Grondin and Gash, J. Neurol. 245, sup 35-42 (1998)].

Human GDNF is encoded by three exons covering about 28kb of genomic DNA [Grimm et al, Hum. Mol. Gen. 7:1873-1886 (1998)]. A human PAC (RPCI PAC 24B12) has been identified to contain the entire coding region of the GDNF gene (Fig. 5A ) [Grimm et al, Hum. Mol. Gen. 7:1873-1886 (1998)]. RPCI PAC 24B12 can be obtained from Oakland Children's Hospital.

Results The CMV promoter and CMV enhancer are first subcloned into the pLD55.RecA vector, as described in Example 1, to create the pLD.55recA.CMV vector. Then about 500bp of genomic DNA corresponding to the GDNF 5' promoter is inserted after the TATAA box. The GDNF exonl sequence then is amplified by PCR and subcloned downstream to the CMV promoter to create the pLD55.recA.TGCA.GDNF vector (or ρLD55.GDNF vector). The PLD55.GDNF vector is used to modify the RPCI PAC 24B12, as described above. Correct insertion of the CMV promoter 5' of exon 1 of the GDNF gene in the PAC is confirmed by Southern blot analysis. An excess of 50 ug of the modified BAC DNA is prepared using Qiagen tip-500 columns. 5-10 ug of the modified RPCI PAC 24B 12 DNA is transfected into COS cells using 20 ug of lipofectamine (BRL-Life Technologies) as described in Kim et al. [Genome Res. 8: 804-12 (1998)].

An assay for the GDNF in the supernatant to determine whether the embryonic Purkinje cell survival is enhanced and/or differentiation occurs is performed as described Mount et al. [Proc. Natl. Acad. Sci. USA 92:9092-9096 (1995)].

The present invention is not to be limited in scope by the specific embodiments describe herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all base sizes, and all molecular weight or molecular mass values, given for nucleic acids are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A method of modifying a eukaryotic cell comprising:

(a) inserting a Heterologous Control Unit (HCU) into an Independent Origin Based Cloning Vector (IOBCV) by homologous recombination, thereby generating a modified IOBCV; wherein said inserting enables the HCU to modulate the expression of a gene of interest comprised by the IOBCV when the modified IOBCV is introduced into a eukaryotic cell and the eukaryotic cell is in an environment that allows gene expression; and

(b) introducing the modified IOBCV into the eukaryotic cell; wherein the expression of the gene of interest is modulated by the HCU in the eukaryotic cell when the eukaryotic cell is in an environment that allows gene expression.

2. The method of Claim 1 wherein said inserting comprises the placement of the HCU in front of an exon of the gene of interest.

3. The method of Claim 1 wherein the HCU is selected from the group consisting of a heterologous eukaryotic promoter (HEP), a heterologous transcriptional regulatory element (HTRE), a heterologous eukaryotic promoter comprising one or more transcriptional regulatory elements (HEP/TRE), a heterologous eukaryotic promoter with one or more transcriptional regulatory elements that further encodes a 5' fusion polypeptide/peptide (HTF), and a multi -promoter insert (MPI).

4. The method of Claim 3 wherein the HCU comprises a eukaryotic translation initiation sequence.

5. The method of Claim 1 wherein the gene of interest comprises an endogenous polyadenylation sequence.

6. The method of Claim 1 wherein the IOBCV integrates into the genome of the eukaryotic cell.

7. The method of Claim 1 wherein the IOBCV is a YAC.

8. The method of Claim 1 wherein the IOBCV is a BBPAC.

9. A method of modulating the expression of the gene of interest in the eukaryotic cell of Claim 1 comprising placing the eukaryotic cell into an environment that allows gene expression.

10. The method of Claim 9 wherein the HCU modulates the expression of the gene of interest in the eukaryotic cell by expressing a gene product encoded by the gene of interest that is not normally expressed in that eukaryotic cell.

11. The method of Claim 10 wherein the gene of interest encodes a viral antigen, and the IOBCV is used as a DNA vaccine.

12. The method of Claim 9 wherein the HCU modulates the expression of the gene of interest by increasing its expression.

13. The method of Claim 1 wherein the HCU modulates the expression of the gene of interest by decreasing its expression.

14. The method of Claim 1 wherein inserting the HCU into the IOBCV comprises introducing a shuttle vector into a host cell containing the IOBCV under conditions in which the shuttle vector can replicate and transform the host cell; wherein the shuttle vector comprises a conditional origin of replication, the HCU, and a region of homology with the gene of interest; wherein the region of homology and the HCU are situated in the shuttle vector such that the HCU is inserted operatively upstream of the coding region of the gene of interest comprised by the IOBCV after the region of homology and the gene of interest undergo a recombination event; and wherein following the insertion of HCU into the IOBCV, the host cell is grown under conditions in which the shuttle vector cannot replicate.

15. The method of Claim 14 wherein the conditional origin of replication is a temperature sensitive origin of replication.

16. The method of Claim 14 wherein the conditional origin of replication is the R6Kγ origin of replication; and wherein the host cell expresses the Pir protein.

17. An IOBCV that comprises a Heterologous Control Unit (HCU) operatively upstream of the coding region of a gene of interest; wherein said HCU has been placed operatively upstream of the coding region of the gene of interest by homologous recombination; and wherein said HCU can modulate the expression of the gene of interest when the IOBCV is introduced into a eukaryotic cell and the eukaryotic cell is in an environment that allows gene expression.

18. A eukaryotic cell that comprises a gene of interest that has been modulated by the method of Claim 1.

19. A method of producing a non-human transgenic animal that has a gene of interest modulated by a Heterologous Control Unit (HCU) comprising:

(a) inserting an HCU into an Independent Origin Based Cloning Vector (IOBCV) by homologous recombination, thereby generating a modified IOBCV; wherein said inserting enables the HCU to modulate the expression of a gene of interest comprised by the IOBCV when the modified IOBCV is introduced into a eukaryotic cell;

(b) introducing the modified IOBCV into the eukaryotic cell; wherein the expression of the gene of interest is modulated by the HCU in the eukaryotic cell; and

(c) placing the eukaryotic cell into a recipient animal, wherein the eukaryotic cell develops into the non-human transgenic animal; wherein the eukaryotic cell is selected from the group consisting of a fertilized animal zygote and an embryonic stem cell; and wherein the gene of interest is modulated by the HCU in the non-human transgenic animal.

20. The method of Claim 19 wherein the embryonic stem cell is a mouse embryonic stem cell

21. The method of Claim 19 wherein the IOBCV is a BBPAC in which an HCU has been inserted through homologous recombination in a RecA' bacterial host cell that has been transiently induced to support homologous recombination.

22. The method of Claim 19 wherein said introducing the modified IOBCV into the eukaryotic cell is selected from the group consisting of electroporation, lipofection, microinjection, calcium mediated transfection, and viral infection.

23. A method of modifying an embryonic stem cell comprising introducing an IOBCV into a non-human embryonic stem cell therein modifying the embryonic stem cell; wherein the IOBCV comprises a gene of interest in which a HCU has been inserted operatively upstream of the coding region of the gene of interest by homologous recombination.

24. The non-human embryonic stem cell of Claim 23.

25. A method of constructing a genomic DNA based expression library for a particular eukaryotic species comprising:

(a) subcloning gene fragments of a genomic DNA for a particular eukaryotic species into an Independent Origin Based Cloning Vector (IOBCV);

(b) determining the sequence of the fragments; wherein exons contained by the gene fragments that encode gene products are identified; and wherein nucleotide sequences adjacent to the exons are also identified; and

(c) inserting Heterologous Control Units (HCUs) into the IOBCVs by homologous recombination using the nucleotide sequences adjacent to the exons, thereby generating modified IOBCVs; wherein said inserting enables the HCUs to modulate the expression of the gene fragments when the modified IOBCVs are introduced into a eukaryotic cell and the eukaryotic cell is in an environment that allows gene expression.

26. A method of over-expressing a gene of interest in a eukaryotic cell comprising:

(a) inserting a Heterologous Control Unit (HCU) by homologous recombination into an Independent Origin Based Cloning Vector (IOBCV) operatively upstream of the coding region of a gene of interest contained by the IOBCV; wherein said inserting enables the HCU to over-express the gene of interest when the IOBCV is introduced into a eukaryotic cell and the eukaryotic cell is in an environment that allows gene expression;

(b) introducing the IOBCV into the eukaryotic cell; and (c) culturing the eukaryotic cell in an appropriate cell culture medium under conditions that provide for over-expression of the gene of interest in the eukaryotic cell.

27. The method of Claim 26 further comprising the step of purifying the gene product of the gene of interest.

28. The purified form of the gene product of Claim 27.

29. A method of expressing an antisense RNA of a gene of interest in a eukaryotic cell comprising:

(a) inserting a Heterologous Eukaryotic Promoter (HEP) in the antisense orientation near the 3' end of the coding region of a gene of interest contained by an Independent Origin Based Cloning Vector (IOBCV) by homologous recombination; wherein said inserting enables the HEP to facilitate the expression of an antisense RNA of the gene of interest when the IOBCV is introduced into a eukaryotic cell and the eukaryotic cell is in an environment that allows gene expression; and

(b) introducing the IOBCV into the eukaryotic cell; wherein the expression of the antisense RNA of the gene of interest is expressed by the eukaryotic cell.

30. A method of producing a non-human transgenic animal that expresses an antisense RNA of a gene of interest comprising: a) inserting a Heterologous Eukaryotic Promoter (HEP) in the antisense orientation near the 3' end of the coding region of a gene of interest contained by an Independent Origin Based Cloning Vector (IOBCV) by homologous recombination; wherein said inserting enables the HEP to facilitate the expression of an antisense RNA to the gene of interest when the IOBCV is introduced into a eukaryotic cell and the eukaryotic cell is in an environment that allows gene expression;

(b) introducing the IOBCV into the eukaryotic cell; wherein the antisense RNA of the gene of interest is expressed by the eukaryotic cell; and

(c) placing the eukaryotic cell into a recipient animal, wherein the eukaryotic cell develops into the non-human transgenic animal; wherein the eukaryotic cell is selected from the group consisting of a fertilized animal zygote and an embryonic stem cell; and wherein the non-human transgenic animal expresses the antisense RNA of the gene of interest.

31. The non-human transgenic animal of Claim 30.

32. The non-human transgenic animal of Claim 19.