WO2012051615A1 - Novel vectors for production of glycosylated interferon - Google Patents

Abstract

Novel glycosylated interferons such as interferon-α 2a, interferon-α 2b, or interferon-β 1a (IFN-α 2a, IFN-α 2b, or IFN-β la) are provided, wherein these interferons have glycosylation sites that are not present in wild type interferon. Novel compositions for the production of these glycosylated interferons are provided. The compositions comprise components of vectors, such as a vector backbone, a promoter, and a gene of interest that encodes a glycosylated interferon such as IFN-α 2a, IFN-α 2b, or IFN-β 1a, and the vectors comprising these components. In certain embodiments, these vectors are transposon-based vectors. Also provided are methods of making these compositions and methods of using these compositions for the production glycosylated interferons, such as IFN-α 2a, IFN-α 2b, or IFN-β 1a, containing two glycosylation sites that are not present in wild type interferon.

Description

NOVEL VECTORS FOR PRODUCTION OF GLYCOSYLATED INTERFERON

PRIOR RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 61/393,868, filed October 15, 2010, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods for the production of interferon (IFN) which is glycosylated. In particular, the disclosure relates to transposon based vectors and their use in methods for the efficient expression of a glycosylated interferon.

BACKGROUND OF THE INVENTION

Interferons are a family of proteins, produced by cells of the immune system, that provide protection against viruses, bacteria, tumors, and other foreign substances that may invade the body. There are three classes of interferons, and each class has different, but overlapping effects. Interferons attack a foreign substance, by slowing, blocking, or changing its growth or function.

Interferon alpha (IFN-a) proteins are closely related in structure, containing 165 or 166 amino acids, including four conserved cysteine residues which form two disulfide bridges. The IFN-a proteins include twelve different protein types (e.g., 1 , 2, etc.) which are encoded by about fourteen genes, and each of the protein types is further broken down into different subtypes (e.g., a, b, etc.). To date, interferon alpha 2 (IFN-a2) has been used predominantly as a therapeutic. Pegylated and/or non-pegylated forms of interferon alpha 2a (IFN-a 2a (also sometimes referred to as INF-a 2a)) and interferon alpha 2b (IFN-a 2b (also sometimes referred to as INF-a 2b)) have received FDA approval for the treatment of hairy cell leukemia, malignant melanoma, follicular lymphoma, condylomata acuminate, AIDS-related Kaposi sarcoma, and chronic hepatitis B and C. IFN-a 2a, IFN-a 2b, and IFN-a 2c differ only by one or two amino acids from one another. Human leukocyte subtype IFN-aLe has been used in several European countries for adjuvant treatment of patients with stage lib to stage III cutaneous melanoma after two initial cycles of dacarbazine (DTIC). In addition, IFN-β proteins have been used as therapeutics. For example, IFN- la and IFN- lb have been used to treat and control multiple sclerosis, by slowing progression and activity in relapsing-remitting multiple sclerosis and by reducing attacks in secondary progressive multiple sclerosis.

The manufacture of therapeutic interferons such as IFN-a 2a, IFN-a 2b, IFN- la, and IFN- lb is an expensive process. Companies using recombinant techniques to manufacture these proteins are working at capacity and usually have a long waiting list to access their fermentation facilities. What is needed, therefore, are new, efficient, and economical approaches to make interferons, such as IFN-a 2a, IFN-a 2b, IFN-β la, and IFN-β lb, in vitro or in vivo.

SUMMARY

The present invention addresses these needs by providing novel isolated interferon proteins comprising glycosylation sites that are not present in wild type interferon. The present invention also provides isolated nucleic acids encoding an interferon comprising glycosylation sites that are not present in wild type interferon. In certain embodiments, the isolated interferon proteins comprises two or more glycosylation sites that are not present in wild type interferon. In certain embodiments, the interferon comprises four additional glycosylation sites that are not present in wild type interferon. The isolated interferon protein in some aspects may be IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb. In certain embodiments, the isolated interferon is selected from the group consisting of SEQ ID NOs:44 and 46-53. In some embodiments, the isolated interferon is a human interferon. In certain embodiments, the interferon is encoded by a sequence selected from the group consisting of the isolated nucleic acid sequence at base pairs 7927-8424 of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43; the isolated nucleic acid sequence at base pairs 7927-8427 of SEQ ID NO:39; and the isolated nucleic acid sequence at base pairs 7266-7763 of SEQ ID NO:36.

The present invention addresses these needs by providing novel compositions which can be used to transfect cells for production of a glycosylated interferon such as IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb. These compositions also can be used for the production of transgenic animals that can transmit the gene encoding an interferon to their offspring. The present disclosure provides compositions and methods for efficient production of glycosylated interferons such as IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb, particularly human interferons such as hIFN-α 2a, hIFN-a 2b, hlFN- ia, or hlFN-pib. These methods enable production of large quantities of interferons such as IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb. In some embodiments, when the present compositions are used for in vitro expression, the interferon such as IFN-a 2a, IFN-a 2b, IFN- ia, or IFN- lb is produced at a level of between about 25 g protein/month and about 4 kg protein/month.

The present invention provides compositions comprising vectors and components of vectors that facilitate efficient production of glycosylated interferons such as IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb, particularly human interferons such as hIFN-α 2a, hIFN-α 2b, hlFN- la, or hlFN- ib. The present invention provides compositions that include components of vectors such as a vector backbone (SEQ ID NOs: l-15 ), a novel promoter (SEQ ID NOs: 14-16), and a gene of interest that encodes for a glycosylated inteferon such as IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb. The present vectors further comprise an insulator element located between the transposon insertion sequences and the multicloning site on the vector. In one embodiment, the insulator element is selected from the group consisting of an HS4 element, a lysozyme replicator element, a combination of a lysozyme replicator element and an HS4 element, a matrix attachment region element, a ubiquitin chromatin operating element (UCOE), or a combination thereof. The expression vectors comprising these components are shown as SEQ ID NOs:33-43. In one embodiment these vectors are transposon-based vectors. The present invention also provides methods of making these compositions and methods of using these compositions for the production of an interferon such as glycosylated IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb. In one embodiment, the interferon is human (h) glycosylated IFN-a 2a, hIFN-α 2b, hlFN- ia, or hlFN- lb.

In N-linked glycosylation, an oligosaccharide is linked through the amide nitrogen of asparagine (Asn). Formation of the N-linked oligosaccharides begins in the endoplasmic reticulum (ER) lumen and continues in the Golgi apparatus. A specific sequence, Asn-X-Thr (or Ser) in which X can be any amino acid except proline or aspartic acid, is required. There are occasions where this sequence is not glycosylated if the protein conformation makes the sequence unavailable to the glycotransferase. However, in the case of 0-, N-linked IFN a2b, it has been demonstrated that the molecule has both O- and N-linked glycosylation.

It is to be understood that different cells may be transfected with one of the presently disclosed compositions, provided the cells contain protein synthetic biochemical pathways for the expression of the gene of interest. For example, both prokaryotic cells and eukaryotic cells may be transfected with one of the disclosed compositions. In certain embodiments, animal or plant cells are transfected. Animal cells include, for example, mammalian cells and avian cells. Animal cells that may be transfected include, but are not limited to, Chinese hamster ovary (CHO) cells, CHO-K1 cells, chicken embryonic fibroblasts, HeLa cells, Vera cells, FAO (liver cells), human 3T3 cells, A20 cells, EL4 cells, HepG2 cells, J744A cells, Jurkat cells, P388D1 cells, RC-4B/c cells, SK-N-SH cells, Sp2/mIL-6 cells, SW480 cells, 3T6 Swiss cells, human ARPT-19 (human pigmented retinal epithelial) cells, PerC 6 cells, embryonic duck cells, LMH cells, LMH2a cells, tubular gland cells, or hybridomas.

In one embodiment, avian cells are transfected with one of the disclosed compositions. In a specific embodiment, avian hepatocytes, hepatocyte-related cells, or tubular gland cells are transfected. In certain embodiments, chicken cells are transfected with one of the disclosed compositions. In one embodiment, chicken tubular gland cells, chicken embryonic fibroblasts, chicken LMH2A cells, or chicken LMH cells are transfected with one of the disclosed compositions. Chicken LMH and LMH2A cells are chicken hepatoma cell lines; LMH2A cells have been transformed to express estrogen receptors on their cell surface.

In other embodiments, mammalian cells are transfected with one of the disclosed compositions. In one embodiment, Chinese hamster ovary (CHO) cells, ARPT-19 cells, HeLa cells, Vera cells, FAO (liver cells), human 3T3 cells, or hybridomas are transfected for IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb production. In a specific embodiment, CHO-K1 cells or ARPT-19 cells are transfected with one of the disclosed compositions.

These vectors also may be used in vivo to transfect germline cells in animals such as birds which can be bred and which then pass an IFN transgene through several generations. These vectors also may be used for the production of an IFN in vivo, for example, for deposition in an egg. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the structure of three different hybrid promoters. Figure 1A is a schematic of the Version 1 CMV/Oval promoter 1 (ChOvp/CMVenh/CMVp; SEQ ID NO: 16). Figure IB is a schematic of the Version 2 CMV/Oval promoter (SEQ ID NO: 17; ChSDRE/ CM Venh/ChNRE/ CM Vp) . Figure 1C is a schematic of the Version 4 CMV.Ovalp vs.4 Hybrid Promoter (SEQ ID NO: 18; ChSDRE/CMVenh/CMVp).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides isolated interferon proteins comprising glycosylation sites that are not present in wild type interferon. The present invention also provides isolated nucleic acids encoding an interferon comprising glycosylation sites that are not present in wild type interferon. In certain embodiments, the isolated interferon proteins comprises two or more glycosylation sites that are not present in wild type interferon. In certain embodiments, the interferon comprises four additional glycosylation sites that are not present in wild type interferon. The isolated interferon in some aspects may be IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb. In certain embodiments, the isolated interferon is selected from the group consisting of SEQ ID NOs:44 and 46-53. In some embodiments, the isolated interferon is a human interferon.

The present invention provides novel vectors and vector components for use in transfecting cells for production of glycosylated interferons such as hIFN-α 2a, hIFN-α 2b, hlFN- ia, or hlFN- ib in vitro or in vivo. The present invention also provides methods to make these vector components, methods to make the vectors themselves, and methods for using these vectors to transfect cells such that the transfected cells produce the interferon. The interferon may be any interferon such as IFN-a 2a, IFN-a 2b, IFN-pla, hlFN-plb, hIFN-α Le, hIFN-γ, or others known to one of skill in the art. In some embodiments, the interferon is a human interferon such as hIFN-α 2a, hIFN-α 2b, hlFN- la, or hlFN- lb.

Any cell with protein synthetic capacity may be used for this purpose. Animal cells are the preferred cells, particularly mammalian cells and avian cells. Animal cells that may be transfected include, but are not limited to, Chinese hamster ovary (CHO) cells, CHO-K1 cells, chicken embryonic fibroblasts, HeLa cells, Vera cells, FAO (liver cells), human 3T3 cells, A20 cells, EL4 cells, HepG2 cells, J744A cells, Jurkat cells, P388D1 cells, RC-4B/c cells, SK-N-SH cells, Sp2/mIL-6 cells, SW480 cells, 3T6 Swiss cells, human ARPT-19 (human pigmented retinal epithelial) cells, PerC 6 cells, embryonic duck cells, LMH cells, LMH2a cells, tubular gland cells, or hybridomas. Avian cells include, but are not limited to, LMH, LMH2a cells, embryonic duck cells, chicken embryonic fibroblasts, and tubular gland cells.

As used herein, the terms "interferon," "IFN," "interferon a 2," "IFN-a 2a," "IFN-a 2b," "IFN-β la," and "IFN- lb" refer to an interferon protein that is encoded by a gene that is either a naturally occurring or a codon-optimized gene. As used herein, the term "codon-optimized" means that the DNA sequence has been changed such that where several different codons code for the same amino acid residue, the sequence selected for the gene is the one that is most often utilized by the cell in which the gene is being expressed. For example, in some embodiments, the interferon gene is expressed in LMH or LMH2A cells and includes codon sequences that are preferred in that cell type. In one embodiment, the interferon gene is an hIFN-α 2a gene, an hIFN-a 2b gene, an hlFN- ia gene, or an hlFN- ib gene. In other embodiments, the interferon is an interferon other than IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb, the sequence of which may be found by one of skill in the art in sequence databases such as GenBank.

In one embodiment, the vectors of the present invention contain a gene encoding an interferon such as IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb for the production of such protein by transfected cells in vitro. In other embodiments, the interferon such as IFN-a 2a, IFN-a 2b, IFN- la, or IFN- lb for the production of such protein by transfected cells in vivo.

A. Vectors & Vector Components

The following paragraphs describe the novel vector components and vectors employed in the present invention.

1. Backbone Vectors

The backbone vectors provide the vector components minus the gene of interest (GOI) that codes for the interferon. In one embodiment, transposon-based vectors are used as described further under sections l .a. through l .p. Any of these backbone vectors may be employed to make expression vectors for interferon production.

a. Transposon-Based Vector Tn-MCS #5001 (p5001) (SEQ ID NO:l) Linear sequences were amplified using plasmid DNA from pBluescriptll sk(-) (Stratagene, La Jolla, CA), pGWIZ (Gene Therapy Systems, San Diego, CA), pNK2859 (Dr. Nancy Kleckner, Department of Biochemistry and Molecular Biology, Harvard University), and synthetic linear DNA constructed from specifically designed DNA Oligonucleotides (Integrated DNA Technologies, Coralville, IA). PCR was set up using the above referenced DNA as template, electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. DNA bands corresponding to the expected size were excised from the gel and purified from the agarose using Zymo Research's Clean Gel Recovery Kit (Orange, CA). The resulting products were cloned into the Invitrogen's PCR Blunt II Topo plasmid (Carlsbad, CA) according to the manufacturer's protocol.

After sequence verification, subsequent clones were selected and digested from the PCR Blunt II Topo Vector (Invitrogen Life Technologies, Carlsbad, CA) with corresponding enzymes (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. The linear pieces were ligated together using Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. Ligated products were transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed bacterial cells were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C then spread to LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in a minimum of 250 ml of LB/amp broth. Plasmid DNA was harvested using Qiagen's Maxi-Prep Kit according to the manufacturer's protocol (Chatsworth, CA). The DNA was used as a sequencing template to verify that the pieces were ligated together accurately to form the desired vector sequence. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System. Once a clone was identified that consisted of the desired sequence, the DNA was isolated for use in cloning in specific genes of interest.

b. Preparation of Transposon-Based Vector TnX-MCS #5005 (p5005)

This vector (SEQ ID NO:2) is a modification of p5001 (SEQ ID NO:l) described above in section l .a. The MCS extension was designed to add unique restriction sites to the multiple cloning site of the pTn-MCS vector (SEQ ID NO: l), creating pTnX-MCS (SEQ ID NO:2), in order to increase the ligation efficiency of constructed cassettes into the backbone vector. The first step was to create a list of all non-cutting enzymes for the current pTn-MCS DNA sequence (SEQ ID NO: l). A linear sequence was designed using the list of enzymes and compressing the restriction site sequences together. Necessary restriction site sequences for Xhol and PspOMI (New England Biolabs, Beverly, MA) were then added to each end of this sequence for use in splicing this MCS extension into the pTn-MCS backbone (SEQ ID NO:l). The resulting sequence of 108 bases is SEQ ID NO: 16 shown in the Appendix. A subset of these bases within this 108 base pair sequence corresponds to bases 4917-5012 in SEQ ID NO:4 (discussed below).

For construction, the sequence was split at the Narl restriction site and divided into two sections. Both 5' forward and 3' reverse oligonucleotides (Integrated DNA Technologies, San Diego, CA) were synthesized for each of the two sections. The 5' and 3' oligonucleotides for each section were annealed together, and the resulting synthetic DNA sections were digested with Narl then subsequently ligated together to form the 108 bp MCS extension (SEQ ID NO: 16). PCR was set up on the ligation, electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. DNA bands corresponding to the expected size were excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA). The resulting product was cloned into the PCR Blunt II Topo Vector (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's protocol.

After sequence verification of the MCS extension sequence (SEQ ID NO: 16), a clone was selected and digested from the PCR Blunt II Topo Vector (Invitrogen Life Technologies, Carlsbad, CA) with Xhol and PspoMI (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. The pTn-MCS vector (SEQ ID NO:l) also was digested with Xhol and PspOMI (New England Biolabs, Beverly, MA) according to the manufacturer's protocol, purified as described above, and the two pieces were ligated together using Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according the manufacturer's protocol. Transformed bacterial cells were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C then spread onto LB agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). All plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on an ultraviolet transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in a minimum of 250 mis of LB/amp broth. Plasmid DNA was harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). The DNA was then used as a sequencing template to verify that the changes made in the vector were the desired changes and that no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System. Once a clone was identified that contained the multiple cloning site extension, the DNA was isolated and used for cloning specific genes of interest.

c. Preparation of Transposon-Based Vector TnHS4FBV #5006 (p5006)

This vector (SEQ ID NO:3) is a modification of p5005 (SEQ ID NO:2) described above in section l .b. The modification includes insertion of the HS4 Peta globin insulator element on both the 5' and 3' ends of the multiple cloning site. The 1241 bp HS4 element was isolated from chicken genomic DNA and amplified through polymerase chain reaction (PCR) using conditions known to one skilled in the art. The PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. DNA bands corresponding to the expected size of the HS4 Peta globin insulator element were excised from the agarose gel and purified using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).

Purified HS4 DNA was digested with restriction enzymes Notl, Xhol, PspOMI, and Mlul (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. The digested DNA was then purified using a Zymo DNA Clean and Concentrator kit (Orange, CA). To insert the 5' HS4 element into the MCS of the p5005 vector (SEQ ID NO:2), HS4 DNA and vector p5005 (SEQ ID NO:2) were digested with Notl and Xhol restriction enzymes, purified as described above, and ligated using Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. To insert the 3' HS4 element into the MCS of the p5005 vector (SEQ ID NO:2), HS4 and vector p5005 DNA (SEQ ID NO:2) were digested with PspOMI and Mlul, purified, and ligated as described above. Ligated product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed bacterial cells were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C then spread onto LB agar plates supplemented with 100 μg/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al., 1989), electrophoresed on a 1% agarose gel, and visualized on an ultraviolet transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 mis of LB/amp broth and plasmid DNA was harvested using a Qiagen Maxi-Prep Kit according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). The DNA was then used as sequencing template to verify that any changes made in the vector were the desired changes and that no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System. Once a clone was identified that contained both HS4 elements, the DNA was isolated and used for cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli bacteria containing the plasmid of interest were grown in 500 ml of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed.

d. Preparation of Transposon-Based Vector pTnlO HS4FBV #5012

This vector (SEQ ID NO:4) is a modification of p5006 (SEQ ID NO:3) described above under section I .e. The modification includes a base pair substitution in the transposase gene at base pair 1998 of p5006 (SEQ ID NO:3). The corrected transposase gene was amplified by PCR from template DNA, using PCR conditions known to one skilled in the art. PCR product of the corrected transposase was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. DNA bands corresponding to the expected size were excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).

Purified transposase DNA was digested with restriction enzymes Nrul and Stul (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction digests using a Zymo DNA Clean and Concentrator kit (Zymo Research). To insert the corrected transposase sequence into the MCS of the p5006 vector (SEQ ID NO:3), the transposase DNA and the p5006 vector (SEQ ID NO:3) were digested with Nrul and Stul, purified as described above, and ligated using a Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed cells were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C before spreading onto LB agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). All plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on an ultraviolet transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth. The plasmid DNA was harvested using a Qiagen Maxi-Prep Kit according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). The DNA was then used as a sequencing template to verify that the changes made in the vector were desired changes and that no further changes or mutations occurred. All sequencing was performed using a Beckman Coulter CEQ 8000 Genetic Analysis System. Once a clone was identified that contained the corrected transposase sequence, the DNA was isolated and used for cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli bacteria containing the plasmid of interest was grown in 500 mL of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed.

e. Preparation of Transposon-Based Vector pTn-10 MARFBV #5018

This vector (SEQ ID NO:5) is a modification of p5012 (SEQ ID NO:4) described above under section l .d. The modification includes insertion of the chicken 5' Matrix Attachment Region (MAR) on both the 5' and 3' ends of the multiple cloning site. To accomplish this, the 1.7 kb MAR element was isolated from chicken genomic DNA and amplified by PCR. PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. DNA bands corresponding to the expected size were excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).

Purified MAR DNA was digested with restriction enzymes Notl, Xhol, PspOMI, and Mlul (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from agarose using a Zymo DNA Clean and Concentrator kit (Zymo Research, Orange CA). To insert the 5' MAR element into the MCS of p5012, the purified MAR DNA and p5012 were digested with Not I and Xho I, purified as described above, and ligated using Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. To insert the 3' MAR element into the MCS of p5012, the purified MAR DNA and p5012 were digested with PspOMI and Mlul, purified, and ligated as described above. Ligated product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed cells were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C and then spread onto LB agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). All plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on an ultraviolet transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in a minimum of 250 ml of LB/amp broth, and plasmid DNA was harvested using a Qiagen Maxi-Prep Kit according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). Column purified DNA was used as a sequencing template to verify that the changes made in the vector were the desired changes and that no further changes or mutations occurred. All sequencing was performed using a Beckman Coulter CEQ 8000 Genetic Analysis Systyem. Once a clone was identified that contained both MAR elements, the DNA was isolated and used for cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli bacteria containing the plasmid of interest were grown in 500 mL of LB broth (supplemented with an appropriate antibiotic) at 37°C in a shaking incubator. Plasmid DNA was isolated from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed. f. Preparation ofTransposon-Based Vector pTn- 10 PuroMAR #5021 (p5021) This vector (SEQ ID NO:6) is a modification of p5018 (SEQ ID NO:5) described above in section I .e. The modification includes insertion of the puromycin (puro) gene into the multiple cloning site adjacent to one of the MAR insulator elements. To accomplish this, the 602 bp puromycin gene was amplified by PCR from the vector pMOD Puro (Invitrogen Life Technologies, Carlsbad, CA). Amplified PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).

Purified DNA from the puromycin gene was digested with the restriction enzymes BsiWI and Mlul (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from agarose using a Zymo DNA Clean and Concentrator kit (Zymo Research). To insert the puro gene into the MCS of p5018, puro and p5018 were digested with BsiWI and Mlul, purified as described above, and ligated using Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed cells were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C then spread onto LB agar plates supplemented with 100 μg/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al., 1989), electrophoresed on a 1% agarose gel, and visualized on an ultraviolet transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in a minimum of 250 ml of LB/amp broth. The plasmid DNA was harvested using a Qiagen Maxi-Prep Kit according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). The DNA was used as a sequencing template to verify that the changes made in the vector were desired changes and that no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis Systyem. Once a clone was identified that contained the puro gene, the DNA was isolated and used for cloning in specific genes of interest. All plasmid DNA was isolated by standard procedures. Briefly, E. coli containing the plasmid of interest was grown in 500 ml of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed.

g. Preparation of Transposon-Based Vector TnLysRep #5020

The vector (SEQ ID NO: 7) included the chicken lysozyme replicator (LysRep or LR2) insulator elements to prevent gene silencing. Each LysRep element was ligated 3' to the insertion sequences (IS) of the vector. To accomplish this ligation, a 930 bp fragment of the chicken LysRep element (GenBank # NW 060235) was amplified using PCR conditions known to one skilled in the art. Amplified PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).

Purified LysRep DNA was sequentially digested with restriction enzymes Not I and Xho I (5 'end) and Mlu I and Apa I (3 'end) (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research). To insert the LysRep elements between the IS left and the MCS in pTnX-MCS (SEQ ID NO:2), the purified LysRep DNA and pTnX-MCS were digested with Not I and Xho I, purified as described above, and ligated using a Stratagene T4 Ligase Kit (Stratagene, Inc. La Jolla, CA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen' s protocol. Transformed bacteria were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread to LB media (broth or agar) plates supplemented with 100 μg/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C, and resulting colonies picked to LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on an ultraviolet transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). Column purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was done on a Beckman Coulter CEQ 8000 Genetic Analysis Systyem. Once a clone was identified that contained the 5' LysRep DNA, the vector was digested with Mlu I and Apa I as was the purified LysRep DNA. The same procedures described above were used to ligate the LysRep DNA into the backbone and verify that it was correct. Once a clone was identified that contained both LysRep elements, the DNA was isolated for use in cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli containing the plasmid were grown in 500 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed.

h. Preparation of Transposon-Based Vector TnGenMAR #5022

A vector was designed for inserting a desired coding sequence into the genome of eukaryotic cells, and is given below as SEQ ID NO:8. The vector of SEQ ID NO: 8 was constructed and its sequence verified.

This vector is a modification of p5021 (SEQ ID NO:6) described above under section l .f. The modification includes insertion of the gentamycin gene in the multiple cloning site which is adjacent to one of the MAR insulator elements. To accomplish this ligation, the 1251 bp gentamycin gene was isolated from the vector pS65T-Cl(ClonTech Laboratories, using PCR conditions known to one skilled in the art. Amplified PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).

Purified gentamycin DNA was digested with restriction enzyme BsiW I and Mlu I (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research). To insert the gentamycin gene into the MCS of the p5018 vector (SEQ ID NO:5), the purified gentamycin DNA and the p5018 vector (SEQ ID NO:5) were digested with BsiW I and Mlu I, purified as described above, and ligated using a Stratagene T4 Ligase Kit (Stratagene, Inc. La Jolla, CA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen' s protocol. Transformed bacteria were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread to LB (Luria-Bertani media (broth or agar)) plates supplemented with 100 g/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C, and resulting colonies picked to LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). Column purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was done on a Beckman Coulter CEQ 8000 Genetic Analysis System. Once a clone was identified that contained the gentamycin gene, the DNA was isolated for use in cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli containing the plasmid was grown in 500 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed.

i. Construction of the MCS Extension

The MCS extension (SEQ ID NO: 19) was designed to add unique restriction sites to the multiple cloning site of the pTn-MCS vector (SEQ ID NO: l), creating pTnX-MCS (SEQ ID NO:2), in order to increase ligation efficiency of constructed cassettes into the backbone vector. The first step was to create a list of all non-cutting enzymes for the current pTn-MCS DNA sequence (SEQ ID NO:l). A linear sequence was then designed using the list of enzymes and compressing the restriction-site sequences together. Necessary restriction site sequences for Xhol and PspOMI were then added to each end of this sequence for use in splicing this MCS extension into the pTn-MCS backbone (SEQ ID NO: l). The resulting sequence of 108 bases is SEQ ID NO: 19 shown in Appendix A.

For construction, the sequence was split at the Narl restriction site and divided into two sections. Both 5' forward and 3 'reverse oligonucleotides were synthesized for each of the two sections. The 5' and 3' oligonucleotides for each section were annealed together, and the resulting synthetic DNA sections were digested with Narl then subsequently ligated together to form the 108 bp MCS extension (SEQ ID NO: 19). PCR was set up on the ligation, and the resulting product was cloned into the PCR Blunt II Topo Vector from Invitrogen. A clone was selected, digested from topo, and ligated into the pTn-MCS backbone vector (SEQ ID NO: l) with Xhol and PspOMI. A final clone was selected after sequence verification (SEQ ID NO:2). The resulting 102 bp DNA sequence of the MCS extension matches the theoretical sequence above, from the Xhol site to the PspOMI site.

The selected pTn-MCS + extension clone above (SEQ ID NO:2) was then used to construct the kTn-10 PURO-MAR Flanked BV vector (SEQ ID NO:6). The Lysozyme Matrix Attachment Region (MAR) sequence was inserted into the backbone on both the 5 'end of the MCS extension between the Notl and Xhol restriction sites, and on the 3 'end of the MCS extension between the Mlul and PspOMI restriction sites. In addition, the PURO cassette was added to the backbone vector between the BsiWI and Mlul restriction sites. The addition of these elements resulted in a loss of available restriction sites for use in ligation of constructed cassettes. The restriction sites available for use from the multiple cloning site extension for this pTn-PURO-MAR Flanked BV (SEQ ID NO:6) are found in the 77 base pairs between Xhol and BsiWI.

j. Preparation of Low Expression CMV Tn PuroMAR Flanked Backbone #5024

(p5024)

This vector (SEQ ID NO:9) is a modification of p5018 (SEQ ID NO:5), which includes the deletion of the CMV Enhancer region of the transposase cassette. The CMV enhancer was removed from p5018 by digesting the backbone with Mscl and Afel restriction enzymes (New England Biolabs, Beverly, MA). The digested product was electrophoresed, stained with Syber Safe DNA Gel Stain (Invitrogen Life Technologies, Carlsbad, CA), and visualized on a Visi- Blue transilluminator (UVP Laboratory Products, Upland, CA). A band corresponding to the expected size of the backbone without the enhancer region was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).

Backbone DNA from above was re-circularized using an Epicentre Fast Ligase Kit (Epicentre Biotechnologies, Madison, WI) according to the manufacturer's protocol. The ligation was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed cells were incubated in 250 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C then spread onto LB agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). All plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on an ultraviolet transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in 5ml of LB/amp broth. Plasmid DNA was harvested using Fermentas' Gene Jet Plasmid Miniprep Kit according to the manufacturer's protocol (Glen Burnie, MD). The DNA was then used as a sequencing template to verify that any changes made in the vector were desired changes and that no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System. Once a clone was identified containing the replacement promoter fragment, the DNA was isolated and used for cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli bacteria containing the plasmid of interest were grown in a minimum of 500 ml of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed.

k. Preparation of Low Expression CMV Tn PuroMAR Flanked Backbone #5025

(p5025)

This vector (SEQ ID NO: 10) is a modification of p5021 (SEQ ID NO:6), which includes the deletion of the CMV Enhancer from the CMV enhanced promoter 5' to the transposase gene. The CMV enhancer was removed from p5021 by digesting the backbone with Mscl and Afel restriction enzymes (New England Biolabs, Beverly, MA). The digested product was electrophoresed, stained with Syber Safe DNA Gel Stain (Invitrogen Life Technologies, Carlsbad, CA), and visualized on a Visi-Blue transilluminator (UVP Laboratory Products, Upland, CA). A band corresponding to the expected size of the backbone without the enhancer region was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).

Backbone DNA from above was re-circularized using an Epicentre Fast Ligase Kit (Epicentre Biotechnologies, Madison, WI) according to the manufacturer's protocol. The ligation was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed cells were incubated in 250 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C then spread onto LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). All plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on an ultraviolet transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in 5 ml of LB/amp broth. Plasmid DNA was harvested using Fermentas' Gene Jet Plasmid Miniprep Kit according to the manufacturer's protocol (Glen Burnie, MD). The DNA was then used as a sequencing template to verify that any changes made in the vector were desired changes and that no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System. Once a clone was identified containing the replacement promoter fragment, the DNA was isolated and used for cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli bacteria containing the plasmid of interest were grown in a minimum of 500 ml of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 iL of PCR-grade water and stored at -20°C until needed. 1. Preparation of Low Expression SV40 promoter Tn PuroMAR Flanked Backbone #5026 (p5026)

This vector (SEQ ID NO: l 1) is a modification of p5018 (SEQ ID NO:5), which includes the replacement of the CMV Enhanced promoter of the transposase cassette, with the SV40 promoter from pS65T-Cl (Clontech, Mountainview, CA). The CMV enhanced promoter was removed from p5018 by digesting the backbone with Mscl and Afel restriction enzymes. (New England Biolabs, Beverly, MA). The digested product was electrophoresed, stained with Syber Safe DNA Gel Stain (Invitrogen Life Technologies, Carlsbad, CA), and visualized on a Visi- Blue transilluminator (UVP Laboratory Products, Upland, CA). A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA). The SV40 promoter fragment was amplified to add the 5' and 3' cut sites, Mscl and Ascl, respectively. The PCR product was then cloned into pTopo Blunt II backbone (Invitrogen Life Technologies, Carlsbad, CA). Sequence verified DNA was then digested out of the pTopo Blunt II backbone (Invitrogen Life Technologies, Carlsbad, CA), with Mscl and Afel restriction enzymes (New England Biolabs, Beverly, MA). The digested product was electrophoresed, stained with Syber Safe DNA Gel Stain (Invitrogen Life Technologies, Carlsbad, CA), and visualized on a Visi-Blue transilluminator (UVP Laboratory Products, Upland, CA). A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).

Purified digestion product was ligated into the excised backbone DNA using Epicentre's Fast Ligase Kit (Madison, WI) according to the manufacturer's protocol. The ligation product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed cells were incubated in 250 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37° C before then spread onto LB agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). All plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on an ultraviolet transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in 5 ml of LB/amp broth. The plasmid DNA was harvested using a Fermentas' Gene Jet Plasmid Miniprep Kit according to the manufacturer's protocol (Glen Burnie, MD). The DNA was then used as sequencing template to verify that any changes made in the vector were desired changes and that no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System. Once a clone was identified that contained the replacement promoter fragment, the DNA was isolated for use in cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli bacteria containing the plasmid of interest were grown in a minimum of 500 mL of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed.

m. Preparation of Low Expression SV40 promoter Tn PuroMAR Flanked Backbone #5027 (p5027)

This vector (SEQ ID NO: 12) is a modification of p5021 (SEQ ID NO:6), which includes the replacement of the CMV Enhanced promoter of the transposase cassette, with the SV40 promoter from pS65T-Cl (Clontech, Mountainview, CA). The CMV enhanced promoter was removed from p5021 by digesting the backbone with Mscl and Afel restriction enzymes (New England Biolabs, Beverly, MA). The digested product was electrophoresed, stained with Syber Safe DNA Gel Stain (Invitrogen Life Technologies, Carlsbad, CA), and visualized on a Visi- Blue transilluminator (UVP Laboratory Products, Upland, CA). A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA). The SV40 promoter fragment was amplified to add the 5' and 3' cut sites, Mscl and Ascl, respectively. The PCR product was then cloned into pTopo Blunt II backbone (Invitrogen Life Technologies, Carlsbad, CA). Sequence verified DNA was then digested out of the pTopo Blunt II backbone (Invitrogen Life Technologies, Carlsbad, CA), with Mscl and Afel restriction enzymes (New England Biolabs, Beverly, MA). The digested product was electrophoresed, stained with Syber Safe DNA Gel Stain (Invitrogen Life Technologies, Carlsbad, CA), and visualized on a Visi-Blue transilluminator (UVP Laboratory Products, Upland, CA). A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).

Purified digestion product was ligated into the excised backbone DNA using Epicentre's Fast Ligase Kit (Madison, WI) according to the manufacturer's protocol. The ligation product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed cells were incubated in 250 μΐ of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C before being spread onto LB agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). All plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on an ultraviolet transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in 5 ml of LB/amp broth. The plasmid DNA was harvested using a Fermentas' Gene Jet Plasmid Miniprep Kit according to the manufacturer's protocol (Glen Burnie, MD). The DNA was then used as sequencing template to verify that any changes made in the vector were desired changes and that no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System. Once a clone was identified that contained the replacement promoter fragment, the DNA was isolated for use in cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli bacteria containing the plasmid of interest were grown in a minimum of 500 mL of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 iL of PCR-grade water and stored at -20°C until needed.

n. Preparation ofTnX-MCS-HNRP-CBX3 Vs. 1 #5035 (p5035) (SEQ ID NO:13)

This vector is a modification of p5005 (SEQ ID NO: 2) described above under section Lb. The modification includes a C to G base pair substitution in the transposase gene at bp 1998 of p5005, encoding an aspartic acid to glutamic acid residue change in the transposase. The corrected transposase gene was isolated from template DNA using PCR conditions known to one skilled in the art. PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. DNA bands corresponding to the expected size were excised from the gel and purified using Zymo Research's Clean Gel Recovery Kit (Orange, CA). The resulting product was cloned into the PCR Blunt II Topo Vector (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's protocol.

After sequence verification, a clone was selected and digested from the PCR Blunt II Topo Vector (Invitrogen Life Technologies, Carlsbad, CA) with restriction enzymes Nru I and Stu I (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified using a Zymo Research's DNA Clean and Concentrator kit (Orange, CA). The modified pTn-MCS vector was also digested with Nru I and Stul (New England Biolabs, Beverly, MA) according to the manufacturer's protocol, purified as described above, and the two pieces were ligated together using Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen' s protocol. Transformed bacteria were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C then spread onto LB (Luria-Bertani) plates supplemented with 100 μg/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C, and the resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining.

After sequence verification, a clone was selected for insertion of the HNRP-CBX3 Vs.1 sequence. The desired HNRP-CBX3 sequence was amplified from synthesized DNA template (Integrated DNA Technologies, Coralville, IA), electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. DNA bands corresponding to the expected size were excised from the gel and purified using Zymo Research's Gel Recovery Kit (Orange, CA). The resulting product was cloned into the PCR Blunt II Topo Vector (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. After sequence verification, a clone was selected and digested from the PCR Blunt II Topo Vector (Invitrogen Life Technologies, Carlsbad, CA) with BstX I and Xho I (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified using a Zymo Research's DNA Clean and Concentrator kit (Orange, CA). The modified pTn-MCS vector was also digested with BstX I and Xho I (New England Biolabs, Beverly, MA) according to the manufacturer's protocol, purified as described above, and the two pieces were ligated together using Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen's protocol. Transformed bacteria were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C, then spread onto LB (Luria-Bertani) plates supplemented with 100 μg/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C, and the resulting colonies picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al., 1989), electrophoresed on a 1% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA was harvested using Qiagen's Maxi-Prep Kit (according to the manufacturer's protocol (Chatsworth, CA). Purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System. Once a clone was identified that contained the HNRP-CBX3 Vs.l sequence, the DNA was isolated for use in cloning specific genes of interest.

o. Preparation of Transposon-Based Vector TnX-MCS-HNRP-CBX3 Vs.2 #5036 (p5036)(SEQ ID NO: 14)

This vector is a modification of p5005 (SEQ ID NO: 2) described above under section Lb. The modification includes a C to G base pair substitution in the transposase gene at bp 1998 of p5005, encoding an aspartic acid to glutamic acid residue change in the transposase. The corrected transposase was isolated from template DNA using PCR conditions known to one skilled in the art. PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. DNA bands corresponding to the expected size were excised from the gel and purified using Zymo Research's Clean Gel Recovery Kit (Orange, CA). The resulting product was cloned into the PCR Blunt II Topo Vector (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's protocol.

After sequence verification, a clone was selected and digested from the PCR Blunt II Topo Vector (Invitrogen Life Technologies, Carlsbad, CA) with restriction enzymes Nru I and Stu I (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using Zymo Research's DNA Clean and Concentrator kit (Orange, CA). The modified pTn-MCS vector was also digested with Nru I and Stul (New England Biolabs, Beverly, MA) according to the manufacturer's protocol, purified as described above, and the two pieces were ligated together using Stratagene's T4 Ligase Kit (La Jo 11a, CA) according to the manufacturer's protocol. Ligated product was then transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed bacteria were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread onto LB (Luria-Bertani) plates supplemented with 100 g/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C, and resulting colonies picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining.

After sequence verification, a clone was selected for insertion of the HNRP-CBX3 Vs.2 sequence. The desired HNRP-CBX3 sequence was amplified from synthesized DNA template (Integrated DNA Technologies, Coralville, IA), electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. DNA bands corresponding to the expected size were excised from the gel and purified using Zymo Research's Clean Gel Recovery Kit (Orange, CA). The resulting product was cloned into Invitrogen' s PCR Blunt II Topo Vector (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. After sequence verification, a clone was selected and digested from the PCR Blunt II Topo Vector with BstX I and Xho I (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified using Zymo Research's DNA Clean and Concentrator kit (Orange, CA). The modified pTn-MCS vector was also digested with BstX I and Xho I (New England Biolabs, Beverly, MA) according to the manufacturer's protocol, purified as described above, and the two pieces were ligated together using Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen' s protocol. Transformed bacteria were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread onto LB (Luria-Bertani) plates supplemented with 100 μg/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C. The resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using Qiagen's Maxi-Prep Kit according to the manufacturer's protocol (Chatsworth, CA). Column purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System. Once a clone was identified that contained the HNRP-CBX3 Vs.2 sequence, the DNA was isolated for use in cloning in specific genes of interest.

p. Preparation ofTransposon-Based Vector kTn-10 CMV-PuroMAR #5037 (p5037)(SEQ ID NO: 15)

This vector is a modification of p5021 (SEQ ID NO:6) described above in section l .f The modification includes replacement of the SV40 promoter 5' to the puromycin (puro) gene with the CMV promoter. To accomplish this, the 90 bp CMV promoter was amplified by polymerase chain reaction (PCR) from the vector pGWIZ (Gene Therapy Systems, San Diego, CA). Amplified PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA). Purified DNA from the CMV promoter was digested with the restriction enzymes BspHI and Mlul (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from agarose using a Zymo DNA Clean and Concentrator kit (Zymo Research, Orange, CA).

To insert the CMV promoter into the backbone vector p5021 (SEQ ID NO: 6), the CMV promoter and the p5021 DNA (SEQ ID NO:6) were digested with BspHI and Mlul, purified as described above, and ligated using Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed cells were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C then spread onto LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in a minimum of 250 ml of LB/amp broth. The plasmid DNA was harvested using a Qiagen Maxi-Prep Kit according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). The DNA was used as a sequencing template to verify that changes made in the vector were desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis Systyem. Once a clone was identified that contained the puro gene, the DNA was isolated (see below) and used for cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid of interest was grown in 500 ml of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed.

2. Promoters

A second embodiment of this invention are hybrid promoters that comprise elements from the constitutive CMV promoter and the estrogen inducible ovalbumin promoter. The goal of designing these promoters was to couple the high rate of expression associated with the CMV promoter with the estrogen inducible function of the ovalbumin promoter. To accomplish this goal, three hybrid promoters, designated versions 1, 2, and 4 (SEQ ID NOs: 16-18, respectively)(Figure 1), were designed, built, and tested in cell culture using a gene other than an interferon gene.

a. Version I CMV/Oval promoter 1 = ChOvp/CMVenh/CMVp

Hybrid promoter version 1 (SEQ ID NO: 16) was constructed by ligating the chicken ovalbumin promoter regulatory elements to the 5' end of the CMV enhancer and promoter. A schematic is shown in Figure 1 A. Hybrid promoter version 1 was made by PCR amplifying nucleotides 1090 to 1929 of the ovalbumin promoter (GenBank # J00895) from the chicken genome and cloning this DNA fragment into the pTopo vector (Invitrogen, Carlsbad, CA). Likewise, nucleotides 245-918 of the CMV promoter and enhancer were removed from the pgWiz vector (ClonTech, Mountain View, CA) and cloned into the pTopo vector. By cloning each fragment into the multiple cloning site of the pTopo vector, an array of restriction enzyme sites were available on each end of the DNA fragments which greatly facilitated cloning without PCR amplification. Each fragment was sequenced to verify it was the correct DNA sequence. Once sequence verified, the pTopo clone containing the ovalbumin promoter fragment was digested with Xho I and EcoR I, and the product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA). The pTopo clone containing the CMV promoter was treated in the same manner to open up the plasmid 5 ' to the CMV promoter; these restriction enzymes also allowed directional cloning of the ovalbumin promoter fragment upstream of CMV.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli containing the plasmid were grown in 500 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed.

b. Version 2 CMV/Oval promoter = ChSDRE/CMVenh/ChNRE/CMVp

Hybrid promoter version 2 (SEQ ID NO: 17) consisted of the steroid dependent response element (SDRE) ligated 5' to the CMV enhancer (enh) and the CMV enhancer and promoter separated by the chicken ovalbumin negative response element (NRE).

A schematic is shown in Figure IB. Hybrid promoter version 2 was made by PCR amplifying the steroid dependent response element (SDRE), nucleotides 1100 to 1389, and nucleotides 1640 to 1909 of the negative response element (NRE) of the ovalbumin promoter (GenBank # J00895) from the chicken genome and cloning each DNA fragment into the pTopo vector. Likewise, nucleotides 245-843 of the CMV enhancer and nucleotides 844-915 of the CMV promoter were removed from the pgWiz vector and each cloned into the pTopo vector. By cloning each piece into the multiple cloning site of the pTopo vector, an array of restriction enzyme sites were available on each end of the DNA fragments which greatly facilitated cloning without PCR amplification.

Each fragment was sequenced to verify it was the correct DNA sequence. Once sequence verified, the pTopo clone containing the ovalbumin SDRE fragment was digested with Xho I and EcoR I to remove the SDRE, and the product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA). The pTopo clone containing the CMV enhancer was treated in the same manner to open up the plasmid 5 ' to the CMV enhancer; these restriction enzymes also allowed directional cloning of the ovalbumin SDRE fragment upstream of CMV. The ovalbumin NRE was removed from pTopo using NgoM IV and Kpn I; the same restriction enzymes were used to digest the pTopo clone containing the CMV promoter to allow directional cloning of the NRE.

The DNA fragments were purified as described above. The new pTopo vectors containing the ovalbumin SDRE/CMV enhancer and the NRE/CMV promoter were sequence verified for the correct DNA sequence. Once sequence verified, the pTopo clone containing the ovalbumin SDRE/CMV enhancer fragment was digested with Xho I and NgoM IV to remove the SDRE/CMV Enhancer, and the product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA). The pTopo clone containing the NRE/CMVpromoter was treated in the same manner to open up the plasmid 5' to the CMV enhancer. These restriction enzymes also allowed directional cloning of the ovalbumin SDRE fragment upstream of CMV. The resulting promoter hybrid was sequence verified to insure that it was correct.

All plasmid DNA was isolated by standard procedures. Briefly, E. coli containing the plasmid were grown in 500 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 μΐ, of PCR-grade water and stored at -20°C until needed.

c. Version 4 ChSDRE VCMV enhancer YCMV promoter

Hybrid promoter version 4 (SEQ ID NO: 18) consisted of the steroid dependent response element (SDRE) ligated 5 ' to the CMV enhancer (enh) and the CMV promoter.

A schematic is shown in Figure 1C. Hybrid promoter version 4 was made by PCR amplifying the steroid dependent response element (SDRE), nucleotides 441-620 of the ovalbumin promoter (GenBank # J00895) from the chicken genome and cloning each DNA fragment into the pTopo vector. Likewise, nucleotides 245-918 of the CMV enhancer and CMV promoter were removed from the pgWiz vector and each cloned into the pTopo vector. By cloning each piece into the multiple cloning site of the pTopo vector, an array of restriction enzyme sites were available on each end of the DNA fragments which greatly facilitated cloning without PCR amplification.

Each fragment was sequenced to verify it was the correct DNA sequence. Once sequence verified, the pTopo clone containing the ovalbumin SDRE fragment was digested with Xho I and EcoR I to remove the SDRE, and the product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet trans illuminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA). The pTopo clone containing the CMV enhancer/promoter was treated in the same manner to open up the plasmid 5 ' to the CMV enhancer; these restriction enzymes also allowed directional cloning of the ovalbumin SDRE fragment upstream of CMV.

The DNA fragments were purified as described above. The new pTopo vector containing the ovalbumin SDRE/CMV enhancer/promoter was sequence verified for the correct DNA sequence. Once sequence verified, the pTopo clone containing the ovalbumin SDRE/CMV enhancer/promoter fragment was digested with Xho I and NgoM IV to remove the SDRE/CMV Enhancer/promoter, and the product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA). All plasmid DNA was isolated by standard procedures. Briefly, E. coli containing the plasmid was grown in 500 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 μΐ, of PCR-grade water and stored at -20°C until needed.

3. Transposases and Insertion Sequences and Insulator Elements

In a further embodiment of the present invention, the transposase found in the transposase-based vector is an altered target site (ATS) transposase and the insertion sequences are those recognized by the ATS transposase. However, the transposase located in the transposase-based vectors is not limited to a modified ATS transposase and can be derived from any transposase. Transposases known in the prior art include those found in AC7, Tn5SEQl, Tn916, Tn951, Tnl721, Tn 2410, Tnl681, Tnl, Tn2, Tn3, Tn4, Tn5, Tn6, Tn9, TnlO, Tn30, TnlOl, Tn903, Tn501, TnlOOO (γδ), Tnl681, Tn2901, AC transposons, Mp transposons, Spm transposons, En transposons, Dotted transposons, Mu transposons, Ds transposons, dSpm transposons and I transposons. According to the present invention, these transposases and their regulatory sequences are modified for improved functioning as follows: a) the addition one or more Kozak sequences comprising any one of SEQ ID NOs:20 to 29 at the 3' end of the promoter operably-linked to the transposase; b) a change of the codons for the first several amino acids of the transposase, wherein the third base of each codon was changed to an A or a T without changing the corresponding amino acid; c) the addition of one or more stop codons to enhance the termination of transposase synthesis; and/or, d) the addition of an effective polyA sequence operably-linked to the transposase to further enhance expression of the transposase gene.

Although not wanting to be bound by the following statement, it is believed that the modifications of the first several N-terminal codons of the transposase gene increase transcription of the transposase gene, in part, by increasing strand dissociation. It is preferable that between approximately 1 and 20, more preferably 3 and 15, and most preferably between 4 and 12 of the first N-terminal codons of the transposase are modified such that the third base of each codon is changed to an A or a T without changing the encoded amino acid. In one embodiment, the first ten N-terminal codons of the transposase gene are modified in this manner. It is also preferred that the transposase contain mutations that make it less specific for preferred insertion sites and thus increases the rate of transgene insertion as discussed in U.S. Patent No. 5,719,055.

In some embodiments, the transposon-based vectors are optimized for expression in a particular host by changing the methylation patterns of the vector DNA. For example, prokaryotic methylation may be reduced by using a methylation deficient organism for production of the transposon-based vector. The transposon-based vectors may also be methylated to resemble eukaryotic DNA for expression in a eukaryotic host.

Transposases and insertion sequences from other analogous eukaryotic transposon-based vectors that can also be modified and used are, for example, the Drosophila P element derived vectors disclosed in U.S. Patent No. 6,291,243; the Drosophila mariner element described in Sherman et al. (1998); or the sleeping beauty transposon. See also Hackett et al. (1999); D. Lampe et al, 1999. Proc. Natl. Acad. Sci. USA, 96: 11428-11433; S. Fischer et al, 2001. Proc. Natl. Acad. Sci. USA, 98:6759-6764; L. Zagoraiou et al, 2001. Proc. Natl. Acad. Sci. USA, 98: 11474-11478; and D. Berg et al. (Eds.), Mobile DNA, Amer. Soc. Microbiol. (Washington, D.C., 1989). However, it should be noted that bacterial transposon-based elements are preferred, as there is less likelihood that a eukaryotic transposase in the recipient species will recognize prokaryotic insertion sequences bracketing the transgene.

Many transposases recognize different insertion sequences, and therefore, it is to be understood that a transposase-based vector will contain insertion sequences recognized by the particular transposase also found in the transposase-based vector. In a preferred embodiment of the invention, the insertion sequences have been shortened to about 70 base pairs in length as compared to those found in wild-type transposons that typically contain insertion sequences of well over 100 base pairs.

While the examples provided below incorporate a "cut and insert" TnlO based vector that is destroyed following the insertion event, the present invention also encompasses the use of a "rolling replication" type transposon-based vector. Use of a rolling replication type transposon allows multiple copies of the transposon/transgene to be made from a single transgene construct and the copies inserted. This type of transposon-based system thereby provides for insertion of multiple copies of a transgene into a single genome. A rolling replication type transposon-based vector may be preferred when the promoter operably-linked to gene of interest is endogenous to the host cell and present in a high copy number or highly expressed. However, use of a rolling replication system may require tight control to limit the insertion events to non-lethal levels. Tnl, Tn2, Tn3, Tn4, Tn5, Tn9, Tn21, Tn501, Tn551, Tn951, Tnl721, Tn2410 and Tn2603 are examples of a rolling replication type transposon, although Tn5 could be both a rolling replication and a cut and insert type transposon.

The present vectors may further comprise an insulator element located between the transposon insertion sequences and the multicloning site on the vector. In one embodiment, the insulator element is selected from the group consisting of an HS4 element, a lysozyme replicator element, a combination of a lysozyme replicator element and an HS4 element, and a matrix attachment region element, a ubiquitin chromatin operating element (UCOE) or a combination thereof.

4. Other Promoters and Enhancers

The first promoter operably-linked to the transposase gene and the second promoter operably-linked to the gene of interest can be a constitutive promoter or an inducible promoter. Constitutive promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter, herpes simplex virus 1 (HSV1) immediate early promoter, SV40 promoter, lysozyme promoter, early and late CMV promoters, early and late HSV promoters, ?-actin promoter, tubulin promoter, Rous-Sarcoma virus (RSV) promoter, and heat-shock protein (HSP) promoter. Inducible promoters include tissue-specific promoters, developmentally-regulated promoters and chemically inducible promoters. Examples of tissue-specific promoters include the glucose-6- phosphatase (G6P) promoter, vitellogenin promoter, ovalbumin promoter, ovomucoid promoter, conalbumin promoter, ovotransferrin promoter, prolactin promoter, kidney uromodulin promoter, and placental lactogen promoter. The G6P promoter sequence may be deduced from a rat G6P gene untranslated upstream region provided in GenBank accession number U57552.1. Examples of developmentally-regulated promoters include the homeobox promoters and several hormone induced promoters. Examples of chemically inducible promoters include reproductive hormone induced promoters and antibiotic inducible promoters such as the tetracycline inducible promoter and the zinc-inducible metallothionine promoter.

Other inducible promoter systems include the Lac operator repressor system inducible by IPTG (isopropyl beta-D-thiogalactoside) (Cronin, A. et al. 2001. Genes and Development, v. 15), ecdysone-based inducible systems (Hoppe, U. C. et al. 2000. Mol. Ther. 1 : 159-164); estrogen- based inducible systems (Braselmann, S. et al. 1993. Proc. Natl. Acad. Sci. 90: 1657-1661); progesterone-based inducible systems using a chimeric regulator, GLVP, which is a hybrid protein consisting of the GAL4 binding domain and the herpes simplex virus transcriptional activation domain, VP 16, and a truncated form of the human progesterone receptor that retains the ability to bind ligand and can be turned on by RU486 (Wang, et al. 1994. Proc. Natl. Acad. Sci. 91 :8180-8184); CID-based inducible systems using chemical inducers of dimerization (CIDs) to regulate gene expression, such as a system wherein rapamycin induces dimerization of the cellular proteins FKBP12 and FRAP (Belshaw, P. J. et al. 1996. J. Chem. Biol. 3:731-738; Fan, L. et al. 1999. Hum. Gene Ther. 10:2273-2285; Shariat, S.F. et al. 2001. Cancer Res. 61 :2562-2571; Spencer, D.M. 1996. Curr. Biol. 6:839-847). Chemical substances that activate the chemically inducible promoters can be administered to the animal containing the transgene of interest via any method known to those of skill in the art.

Other examples of cell-specific and constitutive promoters include but are not limited to smooth-muscle SM22 promoter, including chimeric SM22alpha/telokin promoters (Hoggatt A.M. et al, 2002. Circ Res. 91(12): 1151-9); ubiquitin C promoter (Biochim Biophys Acta, 2003. Jan. 3;1625(l):52-63); Hsf2 promoter; murine COMP (cartilage oligomeric matrix protein) promoter; early B cell-specific mb-1 promoter (Sigvardsson M., et al., 2002. Mol. Cell Biol. 22(24):8539-51); prostate specific antigen (PSA) promoter (Yoshimura I. et al, 2002, J. Urol. 168(6):2659-64); exorh promoter and pineal expression-promoting element (Asaoka Y., et al., 2002. Proc. Natl. Acad. Sci. 99(24): 15456-61); neural and liver ceramidase gene promoters (Okino N. et al, 2002. Biochem. Biophys. Res. Commun. 299(1): 160-6); PSP94 gene promoter/enhancer (Gabril M.Y. et al., 2002. Gene Ther. 9(23): 1589-99); promoter of the human FAT/CD36 gene (Kuriki C, et al, 2002. Biol. Pharm. Bull. 25(11): 1476-8); VL30 promoter (Staplin W.R. et al, 2002. Blood October 24, 2002); and, IL-10 promoter (Brenner S., et al, 2002. J. Biol. Chem. December 18, 2002). Additional promoters are shown in Table 1.

Examples of avian promoters include, but are not limited to, promoters controlling expression of egg white proteins, such as ovalbumin, ovotransferrin (conalbumin), ovomucoid, lysozyme, ovomucin, g2 ovoglobulin, g3 ovoglobulin, ovoflavoprotein, ovostatin (ovomacroglobin), cystatin, avidin, thiamine-binding protein, glutamyl aminopeptidase minor glycoprotein 1, minor glycoprotein 2; and promoters controlling expression of egg-yolk proteins, such as vitellogenin, very low-density lipoproteins, low density lipoprotein, cobalamin-binding protein, riboflavin-binding protein, biotin-binding protein (Awade, 1996. Z. Lebensm. Unters. Forsch. 202: 1-14). An advantage of using the vitellogenin promoter is that it is active during the egg-laying stage of an animal's life-cycle, which allows for the production of the protein of interest to be temporally connected to the import of the protein of interest into the egg yolk when the protein of interest is equipped with an appropriate targeting sequence. In some embodiments, the avian promoter is an oviduct-specific promoter. As used herein, the term "oviduct-specific promoter" includes, but is not limited to, ovalbumin; ovotransferrin (conalbumin); ovomucoid; 01, 02, 03, 04 or 05 avidin; ovomucin; g2 ovoglobulin; g3 ovoglobulin; ovoflavoprotein; and ovostatin (ovomacroglobin) promoters.

When germline transformation occurs via cardiovascular, intraovarian or intratesticular administration, or when hepatocytes are targeted for incorporation of components of a vector through non-germ line administration, liver-specific promoters may be operably-linked to the gene of interest to achieve liver-specific expression of the transgene. Liver-specific promoters of the present invention include, but are not limited to, the following promoters, vitellogenin promoter, G6P promoter, cholesterol-7-alpha-hydroxylase (CYP7A) promoter, phenylalanine hydroxylase (PAH) promoter, protein C gene promoter, insulin-like growth factor I (IGF-I) promoter, bilirubin UDP-glucuronosyltransferase promoter, aldolase B promoter, furin promoter, metallothionine promoter, albumin promoter, and insulin promoter.

Also included in this invention are modified promo ters/enhancers wherein elements of a single promoter are duplicated, modified, or otherwise changed. In one embodiment, steroid hormone -binding domains of the ovalbumin promoter are moved from about -3.5 kb to within approximately the first 1000 base pairs of the gene of interest. Modifying an existing promoter with promoter/enhancer elements not found naturally in the promoter, as well as building an entirely synthetic promoter, or drawing promoter/enhancer elements from various genes together on a non-natural backbone, are all encompassed by the current invention.

Accordingly, it is to be understood that the promoters contained within the transposon- based vectors of the present invention may be entire promoter sequences or fragments of promoter sequences. The constitutive and inducible promoters contained within the transposon- based vectors may also be modified by the addition of one or more Kozak sequences comprising any one of SEQ ID NOs:20-29. As indicated above, the present invention includes transposon-based vectors containing one or more enhancers. These enhancers may or may not be operably-linked to their native promoter and may be located at any distance from their operably-linked promoter. A promoter operably-linked to an enhancer and a promoter modified to eliminate repressive regulatory effects are referred to herein as an "enhanced promoter." The enhancers contained within the transposon-based vectors may be enhancers found in birds, such as an ovalbumin enhancer, but are not limited to these types of enhancers. In one embodiment, an approximately 675 base pair enhancer element of an ovalbumin promoter is cloned upstream of an ovalbumin promoter with 300 base pairs of spacer DNA separating the enhancer and promoter. In one embodiment, the enhancer used as a part of the present invention comprises base pairs 1-675 of a chicken ovalbumin enhancer from GenBank accession #S82527.1. The polynucleotide sequence of this enhancer is provided in SEQ ID NO:30.

Also included in some of the transposon-based vectors of the present invention are cap sites and fragments of cap sites. In one embodiment, approximately 50 base pairs of a 5' untranslated region wherein the capsite resides are added on the 3' end of an enhanced promoter or promoter. An exemplary 5' untranslated region is provided in SEQ ID NO:31. A putative cap-site residing in this 5' untranslated region preferably comprises the polynucleotide sequence provided in SEQ ID NO:32.

In one embodiment of the present invention, the first promoter operably-linked to the transposase gene is a constitutive promoter and the second promoter operably-linked to the gene of interest is a cell specific promoter. In the second embodiment, use of the first constitutive promoter allows for constitutive activation of the transposase gene and incorporation of the gene of interest into virtually all cell types, including the germline of the recipient animal. Although the gene of interest is incorporated into the germline generally, the gene of interest may only be expressed in a tissue-specific manner to achieve gene therapy. A transposon-based vector having a constitutive promoter operably-linked to the transposase gene can be administered by any route, and in several embodiments, the vector is administered to the cardiovascular system, directly to an ovary, to an artery leading to the ovary or to a lymphatic system or fluid proximal to the ovary. In another embodiment, the transposon-based vector having a constitutive promoter operably-linked to the transposase gene can be administered to vessels supplying the liver, muscle, brain, lung, kidney, heart or any other desired organ, tissue or cellular target. In another embodiment, the transposon-based vector having a constitutive promoter operably-linked to the transposase gene can be administered to cells for culture in vitro.

It should be noted that cell- or tissue-specific expression as described herein does not require a complete absence of expression in cells or tissues other than the preferred cell or tissue. Instead, "cell-specific" or "tissue-specific" expression refers to a majority of the expression of a particular gene of interest in the preferred cell or tissue, respectively.

When incorporation of the gene of interest into the germline is not preferred, the first promoter operably-linked to the transposase gene can be a tissue-specific or cell-specific promoter. For example, transfection of a transposon-based vector containing a transposase gene operably-linked to a liver specific promoter such as the G6P promoter or vitellogenin promoter provides for activation of the transposase gene and incorporation of the gene of interest in the cells of the liver in vivo, or in vitro, but not into the germline and other cells generally. In another example, transfection of a transposon-based vector containing a transposase gene operably-linked to an oviduct specific promoter such as the ovalbumin promoter provides for activation of the transposase gene and incorporation of the gene of interest in the cells of the oviduct in vivo or into oviduct cells in vitro, but not into the germline and other cells generally. In this embodiment, the second promoter operably-linked to the gene of interest can be a constitutive promoter or an inducible promoter. In one embodiment, both the first promoter and the second promoter are an ovalbumin promoter. In embodiments wherein tissue-specific expression or incorporation is desired, it is preferred that the transposon-based vector is administered directly to the tissue of interest, to the cardiovascular system which provides blood supply to the tissue of interest, to an artery leading to the organ or tissue of interest or to fluids surrounding the organ or tissue of interest. In one embodiment, the tissue of interest is the oviduct and administration is achieved by direct injection into the oviduct, into the cardiovascular system, or an artery leading to the oviduct. In another embodiment, the tissue of interest is the liver and administration is achieved by direct injection into the cardiovascular system, the portal vein or hepatic artery. In another embodiment, the tissue of interest is cardiac muscle tissue in the heart and administration is achieved by direct injection into the coronary arteries or left cardiac ventricle. In another embodiment, the tissue of interest is neural tissue and administration is achieved by direct injection into the cardiovascular system, the left cardiac ventricle, a cerebrovascular or spinovascular artery. In yet another embodiment, the target is a solid tumor and the administration is achieved by injection into a vessel supplying the tumor or by injection into the tumor.

Accordingly, cell specific promoters may be used to enhance transcription in selected tissues. In birds, for example, promoters that are found in cells of the fallopian tube, such as ovalbumin, conalbumin, ovomucoid and/or lysozyme, are used in the vectors to ensure transcription of the gene of interest in the epithelial cells and tubular gland cells of the fallopian tube, leading to synthesis of the desired protein encoded by the gene and deposition into the egg white. In liver cells, the G6P promoter may be employed to drive transcription of the gene of interest for protein production. Proteins made in the liver of birds may be delivered to the egg yolk. Proteins made in transfected cells in vitro may be released into cell culture medium.

In order to achieve higher or more efficient expression of the transposase gene, the promoter and other regulatory sequences operably-linked to the transposase gene may be those derived from the host. These host specific regulatory sequences can be tissue specific as described above or can be of a constitutive nature.

Table 1

Muscle

Tissue Factor Pathway Inhibitor - low level expression in endothelial vascular smooth muscle TFPI 13 and smooth muscle cells of vascular system cardiac specific MLC2v 14, 26 ventricular myosin light chain

BMP response element that directs cardiac CAR3 18 cardiac specific expression

high level, muscle spec expression skeletal C5-12 22 to drive target gene

AdmDys,

skeletal AdmCTLA4Ig 32 muscle creatine kinase promoter smooth muscle PDE5A 41 chromosome 4q26, phosphodiesterase

use intronic splicing elements to

restrict expression to smooth

smooth muscle AlphaTM 45 muscle vs skeletal

skeletal myostatin 48 fiber type-specific expression of myostatin

Endocrine/nervous

glucocorticoid GR IB-IE 4, 12 glucocorticoid receptor promoter/ all cells neuroblastoma M2-2 8, 36 M2 muscarinic receptor

amyloid beta-protein; 30 bp fragment brain Abeta 16 needed for PC 12 and glial cell expression neuron-specific; high in hippocampus, brain enolase 21 intermediate in cortex, low in cerebellum clusters acetylcholine receptors at synapses rapsyn 29 neuromuscular junction

express limited to neurons in central and peripheral nervous system and specific endocrine cells in adenohypophysis, neuropeptide precursor VGF 39 adrenal medulla, GI tract and pancreas mammalian nervous use of methylation to control tissue system BMP/RA 46 specificity in neural cells.

central and peripheral

noradrenergic neurons Phox2a/Phox2b 47 regulation of neuron differentiation brain BAI1-AP4 55 spec to cerebral cortex and hippocampus

Gastrointestinal

UDP

glucoronsyltransferase UGT1A7 11 gastric mucosa

UGT1A8 11 small intestine and colon

UGT1A10 11 small intestine and colon

Protein kinase C betall (PKCbetall);

colon cancer PKCbetall 20 express in colon cancer to selectively kill it. Cancer

tumor suppressor 4. IB 4.1B 5 2 isoforms, 1 spec to brain, 1 in kidney nestin nestin 63 second intron regulates tissue specificity cancer spec promoter hTRT/hSPAl 68 dual promoter system for cancer specificity

Blood/lymph system

Thyroid spec.— express to kill thyroid

Thyroid thyroglobulin 10 tumors

Thyroid calcitonin 10 medullary thyroid tumors

Thyroid GR 1A 12

regulation controlled by DREAM thyroid thyroglobulin 50 transcriptional repressor

arterial endothelial cells ALK1 60 activin receptor-like kinase

Nonspecific

R A polymerase II 7

gene silencing Gnasxl, Nespas 31

beta-globin beta globin 53

Cardiac M2-1 8 M2 muscarinic receptor

IL-17 induced transcription in airway

Lung hBD-2 19 epithelium

pulmonary surfactant

protein SP-C 62 Alveolar type II cells

use in ciliated epithelial cells for CF ciliated cell-specific prom FOZJ1 70 treatment

surfactant protein

expression SPA-D 73 Possible treatment in premature babies

Clara cell secretory

protein CCSP 75

Dental

extracellular matrix protein dentin teeth/bone DSPP 28 sialophosphoprotein

Adipose

endothelial PAS domain— role in adipogenesis EPAS1 33 adipocyte differentiation

Epidermal

differentiated epidermis involucrin 38

stratum granulosum and stratum desmosomal protein CDSN 58 corneum of epidermis Liver

liver spec albumin Albumin 49

serum alpha-fetoprotein AFP 56 liver spec regulation

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B. Methods of Transfecting Cells

1. Transfection of LMH or LMH2A Cells in vitro

DNA

IFN expression vector DNA {e.g., any one of SEQ ID NOs:33 to 43) is prepared in either methylating or non-methylating bacteria, and is endotoxin- free. Agarose gels showed a single plasmid of the appropriate size. DNA was resuspended in molecular biology grade, sterile water at a concentration of at least 0.5 g/ l. The concentration was verified by spectrophotometry, and the 260/280 ratio was 1.8 or greater. A stock of each DNA sample, diluted to 0.5 ^ ΐ in sterile, molecular biology grade water, was prepared in the cell culture lab, and this stock used for all transfections. When not in use, the DNA stocks were kept frozen at -30 C in small aliquots to avoid repeated freezing and thawing.

Transfection

The transfection reagent used for LMH cells or LMH2A cells was FuGENE 6 (Roche Applied Science). This reagent was used at a 1 :6 ratio ^g of DNA: μΐ of transfection reagent) for all transfections in LMH or LMH2A cells. The chart below shows the amount of DNA and FuGENE 6 used for typical cell culture formats (T25 and T75 tissue culture flasks). If it is necessary to perform transfections in other formats, the amounts of serum free medium (SFM), FuGENE 6 and DNA are scaled appropriately based on the surface area of the flask or well used. The diluent (SFM) is any serum-free cell culture media appropriate for the cells and it does not contain any antibiotics or fungicides. Table 2

Protocol

1. Cells used for transfection were split 24-48 hours prior to the experiment, so that they were actively growing and 50-80% confluent at the time of transfection.

2. FuGENE was warmed to room temperature before use. Because FuGENE is sensitive to prolonged exposure to air, the vial was kept tightly closed when not in use. The vial of FuGENE was returned to the refrigerator as soon as possible.

3. The required amount of FuGENE was pipetted into the SFM in a sterile microcentrifuge tube. The fluid was mixed gently but thoroughly, by tapping or flicking the tube, and incubated for 5 minutes at room temperature.

4. The required amount of DNA was added to the diluted FuGENE and mixed by vortexing for one second.

5. The mixture was incubated at room temperature for 1 hour.

6. During the incubation period, media on cells was replaced with fresh growth media. This media optionally contained serum, if needed, but did not contain antibiotics or fungicides unless absolutely required, as this can reduce the transfection efficiency.

7. The entire volume of the transfection complex was added to the cells. The flask was rocked to mix thoroughly.

8. The flasks were incubated at 37 C and 5% C0₂.

9. Cells were fed and samples obtained as required. After the first 24 hours, cells were optionally fed with media containing antibiotics and/or fungicides, if desired.

2. Transfection of Other Cells

The same methods described above for LMH and LMH2A cells are used for transfection of chicken tubular gland cells or other cell types such as Chinese hamster ovary (CHO) cells, CHO-K1 cells, chicken embryonic fibroblasts, HeLa cells, Vera cells, FAO (liver cells), human 3T3 cells, A20 cells, EL4 cells, HepG2 cells, J744A cells, Jurkat cells, P388D1 cells, RC-4B/c cells, SK-N-SH cells, Sp2/mIL-6 cells, SW480 cells, 3T6 Swiss cells, human ARPT-19cells, PerC 6 cells, and embryonic duck cells.

C. Purification of Interferon Alpha 2b

The purification methods are described here with respect to IFN-a 2b, but the methods are similarly applicable to other interferons (e.g., IFN-a 2a, IFN-β la, IFN-β lb).

1. Media preparation

Media containing recombinant 3xFlag-IFN-a 2b produced by transfected cells is harvested and immediately frozen. Later the medium is thawed, filtered through a 0.45 micron cellulose acetate bottle-top filter to ensure that all particulate is removed prior to being loaded on the column.

2. Affinity Purification

The medium containing recombinant 3xFlag-IFN-a 2b produced by transfected cells is subjected to affinity purification using an Anti-Flag M2 Affinity Gel (Sigma, product code A2220) loaded onto a Poly-Prep Chromatography Columns (BioRad, catalog 731-1550). A slurry of anti-flag M2 gel is applied to Poly- Prep Chromatography Column and the column is equilibrated at 1 ml/min with wash buffer (Tris Buffered Saline (TBS)) for 30 column volumes. After equilibration was complete, the prepared medium containing 3xFlag-IFN from cultured and transfected cells is applied to the column.

The media sample passes through the column, and the column is washed for 10 column volumes with TBS. Next, 8 column volumes elution buffer (100 mM Tris, 0.5 M NaCl, pH 2.85) are run through the column, followed by 4 column volumes of TBS, and the eluent is collected. The eluent is immediately adjusted to a final pH of 8.0 with the addition of 1 M Tris, pH 8.0.

The eluent is transferred to an Amicon Ultra- 15 (that was pre- washed with TBS) and centrifuged at 3,500 x g until the sample is concentrated to the desired volume.

3. Size exclusion chromatography

The concentrated eluent from the affinity purification procedure is then subjected to size exclusion chromatography as a final polishing step in the purification procedure. First, a superdex 75 10/300 GL column (GE Healthcare) is equilibrated with TBS. Multiple size exclusion runs are done in which a sample volume of 400 μΐ for each run is passed over the column. Fractions containing 3xFlag-IFN from each run are then pooled, transferred to an Amicon Ultra- 15, and concentrated to the desired final volume.

The purification procedure is evaluated at various stages using a sandwich ELISA assay (See section D.l . below). SDS-PAGE analysis with subsequent Coomassie blue staining is done to indicate both molecular weight and purity of the purified 3xFlag-IFN (See section D.2. below).

D. Interferon Alpha 2b Detection

1. Interferon Alpha 2b (IFN-a2b) Measurement with ELISA

IFN-a 2b is measured using the following sandwich ELISA protocol:

1. Dilute monoclonal anti-IFN-a 2b (Abeam, Cat. #ab9388) 1 :1000 in 2x-carbonate, pH 9.6 such that the final working dilution concentration is 2 μg/mL. This same antibody also recognizes IFN-a 2a.

2. Add 100 xL of the diluted antibody into to the appropriate wells of the ELISA plate.

3. Allow 96-well plate to coat overnight at 4°C or for 1 hour at 37°C.

4. Wash the ELISA plate five times with wash buffer (lx TBS/0.05% TWEEN).

5. Transfer 200 of blocking buffer (1.5% bovine serum albumen (BSA)/lx TBS/0.05% TWEEN) to the appropriate wells of the ELISA plate and allow 96-well plate to block overnight @ 4°C or for 45 minutes at room temperature.

6. Dilute the purified fusion 3xFlag-IFN-a 2b standard (clone #206) in negative control media (5% FCS/Waymouth, Gibco) such that the final working dilution concentration is 16 ng/mL.

7. Dilute test samples in negative control media (5%> FCS/Waymouth , Gibco).

8. Remove the blocking buffer by manually "flicking" the ELISA plate into the sink.

9. Add the diluted samples and fusion protein standards into 96-well plate and incubate the ELISA plate at room temperature for 1 hour.

10. Dilute fresh Anti FLAG M2 Alkaline Phosphatase Antibody 1 :8,000 (Sigma, Cat. # A9469) such that the final working dilution concentration is 125 ng/mL.

11. Add 100 xL of the diluted antibody into to the appropriate wells of the ELISA plate.

12. Incubate the ELISA plate at room temperature for 1 hour. 13. Dilute the p-nitrophenyl phosphate substrate solution in IX diethanolamine (DEA) substrate buffer, pH 9.8 (KPL, Cat.# 50-80-02) such that the final working dilution concentration is 1 mg/mL.

14. Wash the ELISA plate five times with wash buffer (lx TBS/0.05% TWEEN).

15. Add 100 of the diluted p-nitrophenyl phosphate substrate solution to the appropriate wells of the ELISA plate

16. Use plate reader, took the absorbance readings at 405 nm of the ELISA plate at 30, 60, 90, and 120 minute intervals.

Culture medium is applied to the ELISA either in an undiluted or slightly diluted manner. 3xFlag-IFN-a 2b is detected in this assay. The 3xFlag-IFN-a 2b levels are determined by reference to the 3xFlag-IFN-a 2b standard curve.

The purification procedure is evaluated at various stages using a sandwich ELISA assay (See section D. l . above). SDS-PAGE analysis with subsequent Coomassie blue staining or Western blotting is done to indicate both molecular weight and purity of the purified 3xFlag-IFN (See section D.2. below).

2. Detection of Interferon Alpha 2b Expression with Immunoblotting

SDS-PAGE:

Sample mixtures, including negative control media, are heated for 8 minutes at 100°C and loaded onto a 10-20% Tris-HCl gel. The samples are run at 200 V for 1 hour 10 minutes in Tris-Glycine-SDS buffer.

3x-Flag detection:

1. The finished gel is placed into the Western blot transfer buffer for 2 minutes. This equilibrated the gel in the buffer used for the transfer.

2. The gel is rehydrated for 1 minute in Western blot transfer buffer. A sheet of nitrocellulose paper is cut to the exact size of the gel to be transferred.

3. The electrophoretic transfer occurs for 50 minutes at 100 V.

4. The blot is removed from the transfer apparatus and blocked with 5.0% milk in TBS/TWEEN 20. Blocking occurs for 1 hour at 37°C.

5. The blot is washed four times for 5 minutes per wash in TBS/TWEEN 20. 6. The blot is incubated in Anti-FLAG M2 (Sigma, Cat. # A9469) conjugated with alkaline phosphatase diluted appropriately 1 :5,000 with 1% gelatin in TBS/TWEEN 20 for 1 hour at room temperature.

7. The blot is washed four times for 5 minutes per wash in TBS/TWEEN 20.

8. Antibody bound to antigen is detected by using the BCIP/NBT Liquid Substrate System (KPL). The substrate solution is applied until color was detected (5-10 minutes).

9. Color formation (enzyme reaction) is stopped by rinsing blots with distilled H₂0.

10. The blot is air-dried on a paper towel.

Interferon detection:

1. The interferon can also be detected directly with an anti-interferon antibody as follows. The finished gel is placed into the Western blot transfer buffer for 2 minutes. This equilibrated the gel in the buffer used for the transfer.

2. The gel is rehydrated for 1 minute in Western blot transfer buffer. A sheet of nitrocellulose paper is cut to the exact size of the gel to be transferred.

3. The electrophoretic transfer occurs for 50 minutes at 100 V.

4. The blot is removed from the transfer apparatus and blocked with 5.0% MILK in TBS/TWEEN 20. Blocking occurs for 1 hour at 37 °C.

5. The blot is washed four times for 5 minutes per wash in TBS/TWEEN 20.

6. The blot is incubated in monoclonal anti-IFN-a 2b (abeam, Cat # ab9388) diluted appropriately 1 :2,000 with 1% gelatin in TBS/TWEEN 20 for 1 hour at room temperature.

7. The blot is washed three times for 5 minutes per wash in TBS/TWEEN 20.

8. The blot is incubated in anti-mouse IgG (abeam, Cat # ab6729) conjugated with alkaline phosphatase diluted appropriately 1 : 10,000 with 1% gelatin in TBS/TWEEN 20 for 1 hour at room temperature.

9. The blot is washed four times for 5 minutes per wash in TBS/TWEEN 20.

10. Antibody bound to antigen is detected by using the 5-bromo,4-chloro,3-indolylphosphate (BCIP)/ nitrobluetetrazolium (NBT) Liquid Substrate System (KPL). The substrate solution is applied until color was detected (5-10 minutes).

11. Color formation (enzyme reaction) is stopped by rinsing blots with dH₂0.

12. The blot is air-dried on a paper towel. 3. Vectors for Interferon Production

The vectors of the present invention employ some of the vector components (backbone vectors and promoters) described in the previous sections and also include the multiple cloning site (MCS) comprising the gene of interest. In one embodiment, the gene of interest encodes for a glycosylated human interferon. In certain embodiments, the gene of interest encodes a human IFN-a 2a, IFN-a 2b, or IFN- la protein. The vectors SEQ ID NOs:33-43 all contain a gene of interest encoding a glycosylated interferon protein.

E. Methods of In Vivo Administration

The polynucleotide cassettes may be delivered through the vascular system to be distributed to the cells supplied by that vessel. For example, the compositions may be administered through the cardiovascular system to reach target tissues and cells receiving blood supply. In one embodiment, the compositions may be administered through any chamber of the heart, including the right ventricle, the left ventricle, the right atrium or the left atrium. Administration into the right side of the heart may target the pulmonary circulation and tissues supplied by the pulmonary artery. Administration into the left side of the heart may target the systemic circulation through the aorta and any of its branches, including but not limited to the coronary vessels, the ovarian or testicular arteries, the renal arteries, the arteries supplying the gastrointestinal and pelvic tissues, including the celiac, cranial mesenteric and caudal mesenteric vessels and their branches, the common iliac arteries and their branches to the pelvic organs, the gastrointestinal system and the lower extremity, the carotid, brachiocephalic and subclavian arteries. It is to be understood that the specific names of blood vessels change with the species under consideration and are known to one of ordinary skill in the art. Administration into the left ventricle or ascending or descending aorta supplies any of the tissues receiving blood supply from the aorta and its branches, including but not limited to the testes, ovary, oviduct, and liver. Germline cells and other cells may be transfected in this manner. For example, the compositions may be placed in the left ventricle, the aorta or directly into an artery supplying the ovary or supplying the fallopian tube to transfect cells in those tissues. In this manner, follicles could be transfected to create a germline transgenic animal. Alternatively, supplying the compositions through the artery leading to the oviduct would preferably transfect the tubular gland and epithelial cells. Such transfected cells could manufacture a desired protein or peptide for deposition in the egg white. Administration of the compositions through the left cardiac ventricle, the portal vein or hepatic artery would target uptake and transformation of hepatic cells. Administration may occur through any means, for example by injection into the left ventricle, or by administration through a cannula or needle introduced into the left atrium, left ventricle, aorta or a branch thereof.

Intravascular administration further includes administration in to any vein, including but not limited to veins in the systemic circulation and veins in the hepatic portal circulation. Intravascular administration further includes administration into the cerebrovascular system, including the carotid arteries, the vertebral arteries and branches thereof.

Intravascular administration may be coupled with methods known to influence the permeability of vascular barriers such as the blood brain barrier and the blood testes barrier, in order to enhance transfection of cells that are difficult to affect through vascular administration. Such methods are known to one of ordinary skill in the art and include use of hyperosmotic agents, mannitol, hypothermia, nitric oxide, alkylglycerols, lipopolysaccharides (Haluska et al, Clin. J. Oncol. Nursing 8(3): 263-267, 2004; Brown et al, Brain Res., 1014: 221-227, 2004; Ikeda et al, Acta Neurochir. Suppl. 86:559-563, 2004; Weyerbrock et al, J. Neurosurg. 99(4):728-737, 2003; Erdlenbruch et al, Br. J. Pharmacol. 139(4):685-694, 2003; Gaillard et al, Microvasc. Res. 65(1):24-31, 2003; Lee et al, Biol. Reprod. 70(2):267-276, 2004)).

Intravascular administration may also be coupled with methods known to influence vascular diameter, such as use of beta blockers, nitric oxide generators, prostaglandins and other reagents that increase vascular diameter and blood flow.

Administration through the urethra and into the bladder would target the transitional epithelium of the bladder. Administration through the vagina and cervix would target the lining of the uterus and the epithelial cells of the fallopian tube.

The polynucleotide cassettes may be administered in a single administration, multiple administrations, continuously, or intermittently. The polynucleotide cassettes may be administered by injection, via a catheter, an osmotic mini-pump or any other method. In some embodiments, a polynucleotide cassette is administered to an animal in multiple administrations, each administration containing the polynucleotide cassette and a different transfecting reagent.

In a preferred embodiment, the animal is an egg-laying animal, and more preferably, an avian, and the transposon-based vectors comprising the polynucleotide cassettes are administered into the vascular system, preferably into the heart. The vector may be injected into the venous system in locations such as the jugular vein and the metatarsal vein. In one embodiment, between approximately 1 and 1000 μg, 1 and 200 μg, 5 and 200 μg, or 5 and 150 μg of a transposon-based vector containing the polynucleotide cassette is administered to the vascular system, preferably into the heart. In a chicken, it is preferred that between approximately 1 and 300 μg, or 5 and 200 μg are administered to the vascular system, preferably into the heart, more preferably into the left ventricle. The total injection volume for administration into the left ventricle of a chicken may range from about 10 μΐ to about 5.0 ml, or from about 100 μΐ to about 1.5 ml, or from about 200 μΐ to about 1.0 ml, or from about 200 μΐ to about 800 μΐ. It is to be understood that the total injection volume may vary depending on the duration of the injection. Longer injection durations may accommodate higher total volumes. In a quail, it is preferred that between approximately 1 and 200 μg, or between approximately 5 and 200 μg are administered to the vascular system, preferably into the heart, more preferably into the left ventricle. The total injection volume for administration into the left ventricle of a quail may range from about 10 μΐ to about 1.0 ml, or from about 100 μΐ to about 800 μΐ, or from about 200 μΐ to about 600 μΐ. It is to be understood that the total injection volume may vary depending on the duration of the injection. Longer injection durations may accommodate higher total volumes. The microgram quantities represent the total amount of the vector with the transfection reagent.

In another embodiment, the animal is an egg-laying animal, and more preferably, an avian. In one embodiment, between approximately 1 and 150 μg, 1 and 100 μg, 1 and 50 μg, preferably between 1 and 20 μg, and more preferably between 5 and 10 μg of a transposon-based vector containing the polynucleotide cassette is administered to the oviduct of a bird. In a chicken, it is preferred that between approximately 1 and 100 μg, or 5 and 50 μg are administered. In a quail, it is preferred that between approximately 5 and 10 μg are administered. Optimal ranges depending upon the type of bird and the bird's stage of sexual maturity. Intraoviduct administration of the transposon-based vectors of the present invention result in a PCR positive signal in the oviduct tissue, whereas intravascular administration results in a PCR positive signal in the liver, ovary and other tissues. In other embodiments, the polynucleotide cassettes is administered to the cardiovascular system, for example the left cardiac ventricle, or directly into an artery that supplies the oviduct or the liver. These methods of administration may also be combined with any methods for facilitating transfection, including without limitation, electroporation, gene guns, injection of naked DNA, and use of dimethyl sulfoxide (DMSO). U.S. Patent No. 7,527,966, U.S. Publication No. 2008-0235815, U.S. Publication No. 2010/0081789 and PCT Publication No. WO 2005/062881 are hereby incorporated by reference in their entirety.

In specific embodiments, the disclosed backbone vectors are defined by the following annotations:

SEQ ID NO:l (pTnMCS (base vector, without MCS extension) Vector #5001

Bp 1 - 130 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bpl-130

Bp 133 - 1812 CMV promoter/enhancer taken from vector pGWIZ (Gene Therapy Systems) bp 229-1873

Bp 1813 - 3018 Transposase, modified from TnlO (GeneBank accession #J01829)Bp 108- 1316

Bp 3019 - 3021 Engineered stop codon

Bp 3022 - 3374 Non-coding DNA from vector pNK2859

Bp 3375 - 3417 Lambda DNA from pNK2859

Bp 3418 - 3487 70bp of IS 10 left from TnlO

Bp 3494 - 3700 Multiple cloning site from pBluescriptll sk(-), thru the Xmal site Bp 924-718 Bp 3701 - 3744 Multiple cloning site from pBluescriptll sk(-), from the Xmal site thru the

Xhol site. These base pairs are usually lost when cloning into pTnMCS. Bp

717-673

Bp 3745 - 4184 Multiple cloning site from pBluescriptll sk(-), from the Xhol site bp 672-235

Bp 4190 - 4259 70 bp of IS 10 from TnlO

Bp 4260 - 4301 Lambda DNA from pNK2859

Bp 4302 - 5167 Non-coding DNA from pNK2859

Bp 5168 - 7368 pBluescriptll sk(-) base vector (Stratagene, INC) bp 761-2961

SEQ ID NO:2 pTnX-MCS (Vector #5005) pTNMCS (base vector) with MCS extension

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) Bp 4-135

Bp 133 - 1785 CMV Promoter/Enhancer from vector pGWIZ (Gene Therapy Systems) Bp 1786 - 3018 Transposase, modified from TnlO (GeneBank accession #J01829) Bp 81-1313

Bp 3019 - 3021 Engineered stop codon

Bp 3022 - 3374 Non-coding DNA from vector pNK2859

Bp 3375 - 3416 Lambda DNA from pNK2859

Bp 3417 - 3486 70bp of IS 10 left from TnlO (GeneBank accession #J01829 Bp 1-70)

Bp 3487 - 3704 Multiple cloning site from pBluescriptll sk(-), thru Xmal

Bp 3705 - 3749 Multiple cloning site from pBluescriptll sk(-), from Xmal thru Xhol

Bp 3750 - 3845 Multiple cloning site extension from Xhol thru PspOMI

BP 3846 - 4275 Multiple cloning site from pBluescriptll sk(-), from PspOMI

Bp 4276 - 4345 70 bp of IS10 from TnlO (GeneBank accession #J01829 Bp 70-1)

Bp 4346 - 4387 Lambda DNA from pNK2859

Bp 4388 - 5254 Non-coding DNA from pNK2859

Bp 5255 - 7455 pBluescriptll sk(-) base vector (Stratagene, INC) Bp 761-2961

SEQ ID NO:3 HS4 Flanked BV (Vector #5006)

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) Bp 4-135

Bp 133 - 1785 CMV Promoter/Enhancer from vector pGWIZ (Gene Therapy Systems) Bp

229-1873, including the combination of 2 Nrul cut sites

Bp 1786 - 3018 Transposase, modified from TnlO (GeneBank accession #J01829) Bp 81-1313 Bp 3019 - 3021 Engineered stop codon

Bp 3022 - 3374 Non-coding DNA from vector pNK2859

Bp 3375 - 3416 Lambda DNA from pNK2859

Bp 3417 - 3490 70bp of IS 10 left from TnlO (GeneBank accession #J01829 Bp 1-70)

Bp 3491 - 3680 Multiple cloning site from pBluescriptll sk(-), thru Notl Bp 926-737

Bp 3681 - 4922 HS4 - Beta-globin Insulator Element from Chicken gDNA

Bp 4923 - 5018 Multiple cloning site extension Xhol thru Mlul

Bp 5019 - 6272 HS4 - Beta-globin Insulator Element from Chicken gDNA

Bp 6273 - 6342 70 bp of IS10 from TnlO (GeneBank accession #J01829 Bp 70-1)

Bp 6343 - 6389 Lambda DNA from pNK2859 6390 - 8590 pBluescriptll sk(-) base vector (Stratagene, INC) Bp 761-2961

SEQ ID NO:4 pTn-10 HS4 Flanked Backbone (Vector #5012)

Bp 1-132 Remaining of Fl (-) Ori from pBluescript II sk(-)(Statagene Bp 4-135).

Bp 133-1806 CMV Promoter / Enhancer from vector pGWIZ (Gene Therapy Systems) Bp.

229-1873.

Bp 1807-3015 Tn-10 transposase, from pNK2859 (GeneBank accession #J01829 Bp. 81- 1313).

Bp 3016-3367 Non-coding DNA, possible putative poly A, from vector pNK2859.

Bp 3368-3410 Lambda DNA from pNK2859.

Bp 3411-3480 70bp of IS 10 left from TnlO (GeneBank accession #J01829 bp. 1-70

Bp 3481 -3674 Multiple cloning site from pBluescript II sk(-), thru Notl Bp. 926-737.

Bp 3675-4916 Chicken Beta Globin HS4 Insulator Element (Genbank accession

#NW_060254.0).

Bp 4917-5012 Multiple cloning site extension Xho I thru Mlu I.

Bp 5013-6266 Chicken Beta Globin HS4 Insulator Element (Genbank accession

#NW_060254.0).

Bp 6267-6337 70bp of IS 10 left from TnlO (GeneBank accession #J01829 bp. 1-70

Bp 6338-6382 Lambda DNA from pNK2859.

Bp 6383-8584 pBluescript II sk(-) Base Vector (Stratagene, Inc. Bp. 761-2961).

SEQ ID NO:5 pTN-10 MAR Flanked BV (Vector 5018)

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - 148 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 149 - 747 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843)

Bp 748 - 822 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-918)

Bp 823 - 943 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 944 - 1769 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040- 1865) Bp 1770 - 1777 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873) Bp 1778 - 1806

Bp 1807 - 3015

Bp 3016 - 3367

Bp 3368 - 3410

Bp 3411 - 3480

Bp 3481 - 3651

Bp 3652 - 3674

Bp 3675 - 5367

Bp 5368 - 5463

Bp 5464 - 7168

Bp 7169 - 7238

Bp 7239 - 7281

Bp 7282 - 9486

SEQ ID NO:6 Vector #5021 pTN-10 PURO - MAR Flanked BV

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - - 148 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 149 - 747 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843)

Bp 748 - 822 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-918)

Bp 823 - 943 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 944 - 1769 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040-1865)

Bp 1770 - - 1777 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 1778 - 1806 TN10 DNA, 3 'end from Genbank Accession #J01829 bp79 - 107

Bp 1807 - 3015 Transposon, modified from TnlO GenBank Accession #J01829 Bp 108-1316

Bp 3016 - 3367 Putative PolyA from vector pNK2859

Bp 3368 - 3410 Lambda DNA from pNK2859

Bp 3411 - 3480 70bp of IS 10 left from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 3481 - 3651 pBluescriptll sk(-) base vector (Stratagene, INC) Bp 3652 - 3674 Multiple cloning site from pBluescriptll sk(-)thru NotI, Bp 759-737

Bp 3675 - 5367 Lysozyme Matrix Attachment Region (MAR)

Bp 5368 - 5445 Multiple Cloning Site Extension from pTn X-MCS, Xhol thru BsiWI

Bp 5446 - 5758 HSV-TK polyA from pS65TCl bp 3873-3561

BP 5759 - 6389 Puromycin resistance gene from pMOD PURO (invivoGen)

Bp 6390 - 6775 SV40 promoter from pS65TCl, bp 2232-2617

Bp 6776 - 8486 Lysozyme Matrix Attachment Region (MAR)

Bp 8487 - 8556 70 bp of IS10 from TnlO (GenBank Accession #J01829 Bp 70-1)

Bp 8557 - 8599 Lambda DNA from pNK2859

Bp 8600 - 10804 pBluescriptll sk(-) base vector (Stratagene, INC).

SEQ ID NO:7 (Vector 5020 pTN-10 PURO - LysRep2 Flanked BV)

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - - 148 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 149 - 747 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843)

Bp 748 - 822 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-918)

Bp 823 - 943 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 944 - 1769 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040-1865)

Bp 1770 - 1777 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 1778 - 1806 TN10 DNA, 3 'end from Genbank Accession #J01829 bp79 - 107

Bp 1807 - 3015 Transposon, modified from TnlO GenBank Accession #J01829 Bp 108-1316

Bp 3016 - 3367 Putative PolyA from vector pNK2859

Bp 3368 - 3410 Lambda DNA from pNK2859

Bp 3411 - 3480 70bp of IS 10 left from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 3481 - 3484 Synthetic DNA added during construction

Bp 3485 - 3651 pBluescriptll sk(-) base vector (Stratagene, F C) bp 926-760

Bp 3652 - 3674 Multiple cloning site from pBluescriptll sk(-)thru NotI, Bp 759-737 Bp 3675 - 4608 Lysozyme Rep2 from gDNA (corresponds to Genbank Accession #NW_060235)

Bp 4609 - 4686 Multiple Cloning Site Extension from pTn X-MCS, Xhol thru BsiWI

Bp 4687 - 4999 HSV-TK polyA from pS65TCl bp 3873-3561

Bp 5000 - 5028 Excess DNA from pMOD PURO (invivoGen)

BP 5029 - 5630 Puromycin resistance gene from pMOD PURO (invivoGen) bp 717-116

Bp 5631 - 6016 SV40 promoter from pS65TCl, bp 2232-2617

Bp 6017 - 6022 Mlul RE site

Bp 6023 - 6956 Lysozyme Rep2 from gDNA (corresponds to Genbank Accession

#NW_060235)

Bp 6957 - 6968 Synthetic DNA added during construction including a PspOMI RE site

Bp 6969 - 7038 70 bp of IS10 from TnlO (GenBank Accession #J01829 Bp 70-1)

Bp 7039 - 7081 Lambda DNA from pNK2859

Bp 7082 - 7085 Synthetic DNA added during construction

Bp 7086 - 9286 pBluescriptll sk(-) base vector (Stratagene, INC) bp 761-2961

SEQ ID NO:8 (Vector #5022; pTN-10 Gen - MAR Flanked BV)

Bp 1 - 5445 pTN-10 MAR Flanked BV, ID #5018

Bp 5446 - 5900 HSV-TK polyA from Taken from pIRES2-ZsGreenl , bp 4428-3974

Bp 5901 - 6695 Kanamycin/Neomycin (G418) resistance gene, taken from pIRES2-

ZsGreenl, Bp 3973-3179

Bp 6696 - 7046 SV40 early promoter/enhancer taken from pIRES2-ZsGreenl ,bp 3178-2828 Bp 7047 - 7219 Bacterial promoter for expression of KAN resistance gene, taken from pIRES2-ZsGree.il, bp 2827-2655

Bp 7220 - 11248 pTN-10 MAR Flanked BV, bp 5458-9486

SEQ ID NO:9 pTN-10 MAR Flanked BV Vector #5024

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - 154 pGWIZ base vector (Gene Therapy Systems) bp 229-244 Bp 155 - 229 CMV promoter (from vector pGWIZ, Gene Therapy Systems bp 844-918 Bp 230 - 350 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 351 - 1176 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040-1865)

Bp 1177 - 1184 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 1185 - 1213 TN10 DNA, 3 'end from Genbank Accession #J01829 bp79 - 107

Bp 1214 - 2422 Transposon, modified from TnlO GenBank Accession #J01829 bp 108-1316 Bp 2423 - 2774 Putative PolyA from vector pNK2859

Bp 2775 - 2817 Lambda DNA from pNK2859

Bp 2818 - 2887 70bp of IS 10 left from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 2888 - 3058 pBluescriptll sk(-) base vector (Stratagene, INC) Bp 3059 - 3081 Multiple cloning site from pBluescriptll sk(-)thru Notl,

Bp 3082 - 4774 Chicken 5' Lysozyme Matrix Attachment Region (MAR) from chicken gDNA corresponding to GenBank Accession #X98408

Bp 4775 - 4870 Multiple Cloning Site Extension from pTn X-MCS, Xhol thru Mlul

Bp 4871 - 6575 Chicken 3' Lysozyme Matrix Attachment Region (MAR) from chicken gDNA corresponding to GenBank Accession #X98408

Bp 6576 - 6645 70 bp of IS10 from TnlO (GenBank Accession #J01829 Bp 70-1)

Bp 6646 - 6688 Lambda DNA from pNK2859

Bp 6689 - 8893 pBluescriptll sk(-) base vector (Stratagene, INC)

SEQ ID NO: 10 Vector #5025 pTN-10 ( CMV Enh.)PURO - MAR Flanked BV

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - 154 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 155 - 229 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-918)

Bp 230 - 350 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 351 - 1176 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040- 1865) Bp 1 177 - 1184 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 1 185 - 1213 TN10 DNA, 3 'end from Genbank Accession #J01829 bp79 - 107

Bp 1214 - 2422 Transposon, modified from Tn 10 GenBank Accession # JO 1829 Bp 108-1316 Bp 2423 - 2774 Putative PolyA from vector pNK2859

Bp 2775 - 2817 Lambda DNA from pNK2859

Bp 2818 - 2887 70bp of IS 10 left from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 2888 - 3058 pBluescriptll sk(-) base vector (Stratagene, INC)

Bp 3059 - 3081 Multiple cloning site from pBluescriptll sk(-)thru NotI, Bp 759-737

Bp 3082 - 4774 Lysozyme Matrix Attachment Region (MAR) from chicken gDNA corresponding to GenBank Accession #X98408

Bp 4775 - 4852 Multiple Cloning Site Extension from pTn X-MCS, Xhol thru BsiWI

Bp 4853 - 5165 HSV-T polyA from pS65TCl bp 3873-3561

BP 5166 - 5796 Puromycin resistance gene from pMOD PURO (invivoGen)

Bp 5797 - 6182 SV40 promoter from pS65TCl, bp 2232-2617

Bp 6183 - 7893 Lysozyme Matrix Attachment Region (MAR)

Bp 7894 - 7963 70 bp of IS10 from TnlO (GenBank Accession #J01829 Bp 70-1)

Bp 7964 - 8010 Lambda DNA from pNK2859

Bp 801 1 - 10211 pBluescriptll sk(-) base vector (Stratagene, INC) bp 761-2961

SEQ ID NO: 11 Vector #5026 pTN-10 MAR Flanked BV #5026

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - 154 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 155 - 540 SV40 promoter from pS65TCl bp 2232-2617

Bp 541 - 661 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 662 - 1487 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040-1865)

Bp 1488 - 1495 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 1496 - 1524 TN10 DNA, 3 'end from Genbank Accession #J01829 bp79 - 107 Bp 1525 - 2733

Bp 2734 - 3085

Bp 3086 - 3128

Bp 3129 - 3198

Bp 3199 - 3369

Bp 3370 - 3392

Bp 3393 - 5085

corresponding to GenBank Accession #X98408

Bp 5086 - 5181

Bp 5182 - 6886

corresponding to GenBank Accession #X98408

Bp 6887 - 6956

Bp 6957 - 6999

Bp 7000 - 9204

SEQ ID NO: 12 pTN-10 SV 40 Pr.PURO - MAR Flanked BV Vector #5027

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene)bp 4-135

Bp 133 - - 154 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 155 - ^■ 540 SV40 Promoter from pS65TCl, Bp 2232-2617

Bp 541 - ^■ 661 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 662 - ^■ 1487 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040-1865)

Bp 1488 - 1495 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 1496 - 1524 TN10 DNA, 3 'end from Genbank Accession #J01829 bp79 - 107

Bp 1525 - 2733 Transposon, modified from TnlO GenBank Accession #J01829 Bp 108-1316

Bp 2734 - 3085 Putative PolyA from vector pNK2859

Bp 3086 - 3128 Lambda DNA from pNK2859

Bp 3129 - 3198 70bp of IS 10 left from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 3199 - 3369 pBluescriptll sk(-) base vector (Stratagene, INC) Bp 3370 -3392 Multiple cloning site from pBluescriptll sk(-)thru Notl, Bp 759-737

Bp 3393 -5085 Lysozyme Matrix Attachment Region (MAR) from chicken gDNA GenBank

Accession #X98408.

Bp 5086 -5163 Multiple Cloning Site Extension from pTn X-MCS, Xhol thru BsiWI

Bp 5164 -5476 HSV-TK polyA from pS65TCl bp 3873-3561

BP 5477 -6107 Puromycin resistance gene from pMOD PURO (invivoGen)

Bp 6108 -6499 SV40 promoter from pS65TCl, bp 2232-2617

Bp 6500 - 8204 Lysozyme Matrix Attachment Region (MAR)

Bp 8205 -8274 70 bp of IS10 from TnlO (GenBank Accession #J01829 Bp 70-1)

Bp 8275 -8317 Lambda DNA from pNK2859

Bp 8318 - 10522

SEQ ID NO: 13 Tn 10 X-MCS HNRP-CBX3 Vs.l BV 5035

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) Bp 4-135

Bp 133- - 1785

Bp 1786 -3018

Bp 3019 -3021

Bp 3022 -3374

Bp 3375 -3416

Bp 3417 -3486

Bp 3487 -3673

Bp 3674 -3899

Bp 3900 -3978

Bp 3979 -4833

Bp 4834 -4935

BP 4936 -5365

Bp 5366 -5435

Bp 5436 -5477

Bp 5478 -6344

Bp 6345 -8545 SEQ ID NO: 14 Tn 10 X-MCS HNRP-CBX3 Vs.2 BV 5036

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) Bp 4-135

Bp 133 - - 1785 CMV Promoter/Enhancer from vector pGWIZ (Gene Therapy Systems)

Bp 1786 - 3018

Bp 3019 - 3021

Bp 3022 - 3374

Bp 3375 - 3416

Bp 3417 - 3486

Bp 3487 - 3673

Bp 3674 - 3899

Bp 3900 - 3978

Bp 3979 - 4833

Bp 4834 - 4993

Bp 4994 - 5096

BP 5097 - 5525

Bp 5526 - 5595

Bp 5596 - 5637

Bp 5638 - 6504

Bp 6505 - 8705

SEQ ID NO:15 (CMV-PURO) in kTN-10 MAR Flanked BV 5037

Bp 1 - ^■ 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - 148 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 149 - 747 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843)

Bp 748 - 822 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-918)

Bp 823 - 943 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 944 - 1769 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040- 1865)

Bp 1770 - 1777 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873) Bp 1778 - 1806

Bp 1807 - 3015

Bp 3016 - 3367

Bp 3368 - 3410

Bp 3411 - 3480

Bp 3481 - 3484

Bp 3485 - 3651

Bp 3652 - 3674 Multiple cloning site from pBluescriptll sk(-)thru Notl, Bp 759-737

Bp 3675 - 5367

Bp 5368 - 5445

Bp 5446 - 5758 HSV-TK polyA from pS65TCl bp 3873-3561

Bp 5759 - 5787 Excess DNA from pMOD PURO (invivoGen)

BP 5788 - 6389 Puromycin resistance gene from pMOD PURO (invivoGen) bp 717-1

Bp 6390 - 6464 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 918-844)

Bp 6465 - 8163 Lysozyme Matrix Attachment Region (MAR)

Bp 8164 - 8175

Bp 8176 - 8245

Bp 8246 - 8291 Lambda DNA from pNK2859

BBpp 88229922 -- 1100449933 pBluescriptll sk(-) base vector (Stratagene, INC) bp 761-2961

In specific embodiments, the disclosed hybrid promoters are defined by the following annotations:

SEQ ID NO: 16 (CMV/Oval promoter Version 1 = ChOvp/CMVenh/CMVp)

Bp 1 - 840: Corresponds to bp 421-1260 from the chicken ovalbumin promoter,

GenBank accession number

Bp 841- 1439: CMV Enhancer bp 245-843 taken from vector pGWhiz CMV promoter and enhancer bp 844-918 taken from vector pGWhiz (includes the CAAT box at 857-861 and the TATA box at 890-896).

Bp 1440 - 1514 CMV promoter SEQ ID NO: 17 (CMV/Oval promoter Version 2 = ChSDRE/CMVenh/ChNRE/CMVp)

Bp 1 - 180: Chicken steroid dependent response element from ovalbumin promoter

Bp 181 - 779: CMV Enhancer bp 245-843 taken from vector pGWhiz

Bp 780 - 1049: Chicken ovalbumin promoter negative response element

Bp 1050-1124: CMV promoter bp 844-918 taken from vector pGWhiz (includes the

CAAT box at 857-861 and the TATA box at 890-896. Some references overlap the enhancer to different extents.)

SEQ ID NO:18 CMV.Oval promoter Version 4 Hybrid Promoter

Bp 1 - 186 Chicken Ovalbumin enhanced promoter, SDRE region (taken from

GenBank Accession #: J00895 bp 441-620); includes synthetic DNA from vector construction (EcoRI cut site at 3 ' end for ligation)

Bp 187 - 863 CMV enhancer/promoter (bp 245-899 of gWIZ blank vector, CTC, bp

900-918 of gWIZ blank)

In specific embodiments, the disclosed expression vectors are defined by the following annotations:

SEQ ID NO:33 kTN-10 Puro/Mar(CMV.Ovalp vs.l/Conss(-AA)/Mat.hIFN-oc2b(N,N- gly)/OvpyA)

Bp 1 - 5381 kTN-10 Puro/Mar FBV (bp 1-5381)

Bp 5382 - 6228 Chicken Ovalbumin Promoter (bp 1090-1929), including synthetic DNA added during vector construction (EcoPJ cut site used for ligation)on 3 ' end

Bp 6229 - 6905 CMV enhancer/promoter (bp 245-899 of gWIZ blank vector), including

Xhol cut site + bp 900-918 of CMVpromoter from gWIZ blank vector

Bp 6906 - 7866 CMV intron A' (bp 919-1873 of gWIZ; includes CMV immediate-early gene,

Exonl; CMV intron A; CMV immediate-early gene, partial Exon 2), including synthetic DNA added during vector construction (Sail cut site used for ligation) on 3 ' end

Bp 7867 - 7926 Chicken Conalbumin Signal Sequence + Kozak sequence (from GenBank

Accession # X02009) Bp 7927 - 8430 Human Interferon alpha-2b (IFN-a 2b) gene, taken from GenBank

Accession # J00207 (bp 580-1077); changed codon encoding ASP to ASN at bp 211-213 to create N-glycosylation site, changed codon encoding LEU to ASN at bp 457-459 of the human hIFN-α 2b sequence to create N- glycosylation site. Start codon omitted; including synthetic DNA added during vector construction (BamHI cut site used for ligation) on 3' end

Bp 8431 - 9346 Chicken Ovalbumin PolyA site (taken from GenBank Accession # J00895

(bp 8260-9176)

Bp 9347 - 14752 Puro/Mar Backbone (bp 5399-10804)

SEQ ID NO:34 kTN-10 Puro/Mar (CMV.Ovalp vs.l/Conss(-AA)/Mat.E.O.hIFN-oc2a(N- gly)/OvpyA

Bp 1 - 5381 kTN-10 PURO MAR BV (bp 1-5381)

Bp 5382 - 6228 Chicken Ovalbumin Promoter (bp 1090-1929), including synthetic DNA added during vector construction (EcoRI cut site used for ligation) on 3 ' end

Bp 6229 - 6905 CMV enhancer/promoter (bp 245-899 of gWIZ blank vector) with Xhol cut site + bp 900-918 of CMVpromoter from gWIZ blank vector )

Bp 6906 - 7866 CMV intron A' (bp 919-1873 of gWIZ; includes CMV immediate-early gene, Exonl; CMV intron A; CMV immediate-early gene, partial Exon 2), including synthetic DNA added during vector construction (Sail cut site used for ligation) on 3 ' end

Bp 7867 - 7926 Chicken Conalbumin Signal Sequence + Kozak sequence (from GenBank

Accession # X02009)

Bp 7927 - 8430 Human Interferon alpha-2a N-glycosylated (IFN-a 2a) gene, taken from

GenBank sequence, changed codon encoding ASP to ASN at bp 211-213 to create N-glycosylation site and expression codon optimized for optimal protein expression; Start codon omitted, including synthetic DNA added during vector construction BamHI cut site used for ligation) on 3 ' end

Bp 8431 - 9346 Chicken Ovalbumin PolyA (taken from GenBank Accession #J00895, bp

8260-9176)

Bp 9347 - 14752 kTn-10 PURO MAR BV (bp 5399-10804) SEQ ID NO:35 KTn-10 Puro/Mar (CMV.Ovalp vs.l/Conss(-AA)/Mat.E.O.hIFN-oc2a(N,N- gly)/OvpyA

Bp 1 - 5381 kTN-10 PURO MAR BV (bp 1-5381)

Bp 5382 - 6228 Chicken Ovalbumin Promoter (bp 1090-1929), including synthetic DNA

added during vector construction (EcoRI cut site used for ligation) on 3 ' end

Bp 6229 - 6905 CMV enhancer/promoter (bp 245-899 of gWIZ blank vector) with Xhol cut site + bp 900-918 of CMVpromoter from gWIZ blank vector (from D.H. Clone 10; used this site to add on the CMViA')

Bp 6906 - 7866 CMV intron A' (bp 919-1873 of gWIZ; includes CMV immediate-early gene,

Exonl; CMV intron A; CMV immediate-early gene, partial Exon 2), including synthetic DNA added during vector construction (Sail cut site used for ligation) on 3 ' end

Bp 7867 - 7926 Chicken Conalbumin Signal Sequence + Kozak sequence (from GenBank

Accession # X02009)

Bp 7927 - 8430 Human Interferon alpha-2a Ν,Ν-glycosylated (IFN-a 2a) gene, taken from

GenBank Accession # J00207 (bp 580-1077); changed codon encoding ARG to LYS at bp 67-69 to create 2a sequence, changed codon encoding ASP to ASN at bp 211-213 to create N-glycosylation site, changed codon encoding LEU to ASN at bp 457-459 to create N-glycosylation site; expression codon optimized for optimal protein expression; Start codon omitted, including synthetic DNA added during vector construction BamHI cut site used for ligation) on 3 ' end

Bp 8431 - 9346 Chicken Ovalbumin PolyA (taken from GenBank Accession # J00895, bp

8260-9176)

Bp 9347 - 14752 kTn-10 PURO MAR BV (bp 5399-10804)

SEQ ID NO:36 Puro/Mar (CMV.Ovalp vs.4/Conss(-AA)/hIFN-oc (N,N-gly)/OvpyA)

Bp 1 - 5381 kTN-10 Puro/Mar FBV (bp 1-5381) from GenBank Accession #: J00895 bp

441-620); includes synthetic DNA from vector construction (EcoRI cut site at 3 ' end for ligation) Bp 5568 - 6244 CMV enhancer/promoter (bp 245-899 of gWIZ blank vector), including Xhol cut site + bp 900-918 of CMVpromoter from gWIZ blank vector (from D.H. Clone 10; used this site to add on the CMViA')

Bp 6245 - 7205 CMV intron A' (bp 919-1873 of gWIZ; includes CMV immediate-early gene,

Exonl; CMV intron A; CMV immediate-early gene, partial Exon 2), including synthetic DNA added during vector construction (Sail cut site used for ligation) on 3 ' end

Bp 7206 - 7265 Chicken Conalbumin Signal Sequence + Kozak sequence (from GenBank

Accession # X02009)

Bp 7266 - 7769 Human Interferon alpha-2b (IFN-a 2b) gene, taken from GenBank Accession

# J00207 (bp 580-1077); changed codon encoding ASP to ASN at bp 211-213 to create N-glycosylation site, changed codon encoding LEU to ASN to create N-glycosylation site at bp 457-459 of the hIFN-α 2b sequence. Start codon omitted; including synthetic DNA added during vector construction (BamHI cut site used for ligation) on 3 ' end

Bp 7770 - 8685 Chicken Ovalbumin PolyA site (taken from GenBank Accession # J00895 (bp

8260-9176)

Bp 8686 - 14091 Puro/Mar Backbone (bp 5399-10804)

SEQ ID NO:37 5021-Puro/Mar (CMV.Ovalp vs.l/Conss(-AA)/Mat.hIFN-oc (N,N- gly)LYS/OvpyA)

Bp 1 - 5381 kTN-10 Puro/Mar FBV (bp 1-5381)

Bp 5382 - 6228 Chicken Ovalbumin enhanced promoter (taken from GenBank Accession #:

J00895 bp 421-1260) with EcoRI cut site at 3' end for ligation

Bp 6229 - 6905 CMV enhancer/promoter (bp 245-899 of gWIZ blank vector), including Xhol cut site + bp 900-918 of CMVpromoter from gWIZ blank vector

Bp 6906 - 7866 CMV intron A' (bp 919-1873 of gWIZ; includes CMV immediate-early gene,

Exonl; CMV intron A; CMV immediate-early gene, partial Exon 2), including synthetic DNA added during vector construction (Sail cut site used for ligation) on 3 ' end Bp 7867 - 7926 Chicken Conalbumin Signal Sequence+ Kozak sequence (from GenBank

Accession # X02009)

Bp 7927 - 8430 Human Interferon alpha-2b (IFN-a 2b) gene, taken from GenBank Accession #

J00207 (bp 580-1077); changed codon encoding ASP to ASN at bp 211-213 to create N-glycosylation site, changed codon encoding LEU to ASN to create N- glycosylation site at bp 457-459 of the hIFN-α sequence; changed codons encoding amino acids 156 and 157 of the mature protein from Asn/Leu (AAC TTG) to Lys/Asn (AAA AAC)(bp 466-471). Start codon omitted; including synthetic DNA added during vector construction (BamHI cut site used for ligation) on 3 ' end

Bp 8431 - 9346 Chicken Ovalbumin PolyA site (taken from GenBank Accession # J00895 (bp

8260-9176)

Bp 9347 - 14752 Puro/Mar Backbone (bp 5399-10804)

SEQ ID NO:38 5021-Puro/Mar (CMV.Ovalp vs.l/Conss(-AA)/Mat.hIFN-oc (N,N- gly)SER/OvpyA)

Bp 1 - 5381 kTN- 10 Puro/Mar flanked backbone vector (bp 1 -5381 )

Bp 5382 - 6228 Chicken Ovalbumin enhanced promoter (taken from GenBank Accession #:

J00895 bp 421-1260) with EcoRI cut site at 3' end for ligation Bp 6229 - 6905 CMV enhancer/promoter (bp 245-899 of gWIZ blank vector), including Xhol cut site + bp 900-918 of CMV promoter from gWIZ blank vector

Bp 6906 - 7866 CMV intron A' (bp 919-1873 of gWIZ; includes CMV immediate-early gene,

Exonl; CMV intron A; CMV immediate-early gene, partial Exon 2), including synthetic DNA added during vector construction (Sail cut site used for ligation) on 3 ' end

Bp 7867 - 7926 Chicken Conalbumin Signal Sequence + Kozak sequence (from GenBank

Accession # X02009)

Bp 7927 - 8430 Human Interferon alpha-2b (IFN-a 2b) gene, taken from GenBank Accession

# J00207 (bp 580-1077); changed codon encoding ASP to ASN at bp 211-213 to create N-glycosylation site, changed codon encoding LEU to ASN to create N,N-glycosylation at bp 457-459 of the hIFN-α 2b sequence; changed codon encoding amino acid 155 of mature protein from Thr(ACA) to Ser(TCA) (bp 463-465). Start codon omitted; including synthetic DNA added during vector construction (BamHI cut site used for ligation) on 3 ' end

Bp 8431 - 9346 Chicken Ovalbumin PolyA site (taken from GenBank Accession # J00895 (bp

8260-9176)

Bp 9347 - 14752 Puro/Mar Backbone (bp 5399-10804)

SEQ ID NO:39 VID# 365 - HPvsl/ CMViA/ CAss(-laa)/ hINF la-co / OPA

in kTN-10 (CMV-PURO) MAR Flanked BV

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - 148 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 149 - 747 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843)

Bp 748 - 822 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-918)

Bp 823 - 943 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 944 - 1769 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040-1865)

Bp 1770 - 1777 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 1778 - 1806 TN10 DNA, 3 'end from Genbank Accession #J01829 bp 79 - 107

Bp 1807 - 3015 Transposon, modified from TnlO GenBank Accession #J01829 Bp 108-1316

Bp 3016 - 3367 Putative PolyA from vector pNK2859

Bp 3368 - 3410 Lambda DNA from pNK2859

Bp 3411 - 3480 70bp of IS 10 left from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 3481 - 3651 pBluescriptll sk(-) base vector (Stratagene, INC)

Bp 3652 - 3674 Multiple cloning site from pBluescriptll sk(-)thru Notl, Bp 759-737

Bp 3675 - 5367 Chicken Lysozyme Matrix Attachment region (MAR)

Bp 5368 - 5381 Multiple Cloning Site Extension from pTn X-MCS, Xhol thru Ascl

Bp 5382 - 6223 Chicken Ovalbumin promoter from gDNA (Genbank Accession #J00895 bp

421-1261)

Bp 6224 - 6827 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843)with 5'

EcoRI RE site Bp 6828 - 6905 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-899, CTC, 900-918)

Bp 6906 - 7026 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 7027 - 7852 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040-1865) Bp 7853 - 7860 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 7861 - 7926 Conalbumin signal sequence (GenBank X02009 bp 74-133) with 5' Sail RE site

Bp 7927 - 8427 Human Interferon pia corresponding to GenBank NM 002176 bp 131-639, codon-optimized for chicken liver cells at bp 27 (CTA to CTT) and bp 451- 453 (CTA to TTG) of the sequence encoding hINFpi mature peptide.

Bp 8428 - 9349 Chicken Ovalbumin polyA from gDNA (GenBank Accession #J00895 bp

8260-9175)with 5' Agel RE site

Bp 9350 - 9396 MCS extension from pTN-MCS, Pad thru BsiWI

Bp 9397 - 9709 HSV-TK polyA from pS65TCl bp 3873-3561

Bp 9710 - 9738 Excess DNA from pMOD PURO (invivoGen)

BP 9739 - 10340 Puromycin resistance gene from pMOD PURO (invivoGen) bp 717-116 Bp 10341 - 10421 CMV promoter from vector pGWIZ, Gene Therapy Systems bp 844-918 with

5' MluI RE site

Bp 10422 - 12114 Chicken Lysozyme Matrix Attachment region (MAR) from gDNA

Bp 12115 - 12126 Synthetic DNA added during construction including a PspOMI RE site Bp 12127 - 12196 70 bp of IS10 from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 12197 - 12243 Lambda DNA from pNK2859

Bp 12244 - 14444 pBluescriptll sk(-) base vector (Stratagene, INC)

SEQ ID NO:40 Vector 5021-368 (Puro/Mar BV(CMV.Ovalpvs.l/Conss(-AA)/hIFN-oc

2a(N,N-gly)/OvpyA))

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - 148 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 149 - 747 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843) Bp 748 - 822 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-918)

Bp 823 - 943 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 944 - 1769

Bp 1770 - 1777

Systems)bp 1866-1873)

Bp 1778 - 1806

Bp 1807 - 3015

Bp 3016 - ^■ 3367

Bp 3368 - 3410

Bp 3411 - 3480

Bp 3481 - 3651

Bp 3652 - 3674

Bp 3675 - 5367

Bp 5368 - 5381

Bp 5382 - 6228

J00895 bp 421-1260), including synthetic DNA added during vector construction (EcoRI cut site) at 3 ' end

Bp 6229 - 6905 CMV enhancer/promoter (bp 245-899 of gWIZ blank vector), CTC, bp 900- 918 CMVpromoter from gWIZ blank vector

Bp 6906 - 7866 CMV intron A' (bp 919-1873 of gWIZ; includes CMV immediate-early gene,

Exonl; CMV intron A; CMV immediate-early gene, partial Exon 2), including synthetic DNA added during vector contruction (Sail cut site) on 3 ' end

Bp 7867 - 7926 Chicken Conalbumin Signal Sequence + Kozak sequence (from GenBank

Accession # X02009)

Bp 7927 - 8430 Human Interferon alpha-2a (IFN-a 2a) gene, taken from GenBank Accession #

J00207 (bp 580-1077); Start codon omitted; changed codon encoding ARG to LYS at bp 67-69 to create 2a sequence, changed codon encoding ASP to ASN at bp 211-213 to create N-glycosylation site, changed codon encoding LEU to ASN at bp 457-459 to create N-glycosylation site; including synthetic DNA added during vector construction (BamHI cut site used for ligation) on 3 ' end Bp 8431 - 9346 Chicken Ovalbumin PolyA site, taken from GenBank Accession # J00895 (bp 8260-9176)

Bp 9347 - 9393 MCS extension from pTN-MCS, Pad thru BsiWI

Bp 9394 - 9706 HSV-TK polyA from pS65TCl bp 3873-3561

Bp 9707 - 9735 Excess DNA from pMOD PURO (invivoGen)

BP 9736 - 10337 Puromycin resistance gene from pMOD PURO (invivoGen) bp 717-116 Bp 10338 - 10729 SV40 promoter from pS65TCl, bp 2232-2617 with 5' Mlul RE site

Bp 10730 - 12422 Chicken Lysozyme Matrix Attachment region (MAR) from gDNA

Bp 12423 - 12434 Synthetic DNA added during construction including a PspOMI RE site Bp 12435 - 12504 70 bp of IS10 from TnlO (GenBank Accession #J01829 Bp 70-1)

Bp 12505 - 12552 Lambda DNA from pNK2859

Bp 12553 - 14752 pBluescriptll sk(-) base vector (Stratagene, INC)

SEQ ID NO: 41 Vector 5037-369 (Tn-MarFBV(CMV-Puro)(CMV.Ovalpvs.l/Conss(- AA)/hIFN-oc2a(N,N-gly)/OvpyA))

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - 148 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 149 - 747 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843) Bp 748 - 822 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-918) Bp 823 - 943 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 944 - 1769 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040- 1865) Bp 1770 - 1777 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 1778 - 1806 TNI 0 DNA, 3 'end from Genbank Accession #J01829 bp 79 - 107

Bp 1807 - 3015 Transposon, modified from TnlO GenBank Accession #J01829 Bp 108-1316 Bp 3016 - 3367 Putative PolyA from vector pNK2859

Bp 3368 - 3410 Lambda DNA from pNK2859

Bp 3411 - 3480 70bp of IS 10 left from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 3481 - 3651 pBluescriptll sk(-) base vector (Stratagene, INC)

Bp 3652 - 3674 Multiple cloning site from pBluescriptll sk(-)thru Notl, Bp 759-737 Bp 3675 - 5367 Chicken Lysozyme Matrix Attachment region (MAR)

Bp 5368 - 5381 Multiple Cloning Site Extension from pTn X-MCS, Xhol thru Ascl

Bp 5382 - 6228 Chicken Ovalbumin enhanced promoter (taken from GenBank Accession #:

J00895 bp 421-1260), including synthetic DNA added during vector construction (EcoRI cut site) at 3 ' end

Bp 6229 - 6905 CMV enhancer/promoter (bp 245-899 of gWIZ blank vector), CTC, bp 900-

918 CMVpromoter from gWIZ blank vector

Bp 6906 - 7866 CMV intron A' (bp 919-1873 of gWIZ; includes CMV immediate-early gene,

Exonl; CMV intron A; CMV immediate-early gene, partial Exon 2), including synthetic DNA added during vector contruction (Sail cut site) on 3 ' end Bp 7867 - 7926 Chicken Conalbumin Signal Sequence + Kozak sequence (from GenBank

Accession X02009)

Bp 7927 - 8430 Human Interferon alpha-2a (IFN-a 2a) gene, taken from GenBank Accession #

J00207 (bp 580-1077); Start codon omitted; changed codon encoding ARG to LYS at bp 67-69 to create 2a sequence, changed codon encoding ASP to ASN at bp 211-213 to create N-glycosylation site, changed codon encoding LEU to ASN at bp 457-459 to create N-glycosylation site; including synthetic DNA added during vector construction (BamHI cut site used for ligation) on 3 ' end

Bp 8431 - 9346 Chicken Ovalbumin PolyA site, taken from GenBank Accession # J00895 (bp

8260-9176)

Bp 9347 - 9393 MCS extension from pTN-MCS, Pad thru BsiWI

Bp 9394 - 9706 HSV-TK polyA from pS65TCl bp 3873-3561

Bp 9707 - 9735 Excess DNA from pMOD PURO (invivoGen)

BP 9736 - 10337 Puromycin resistance gene from pMOD PURO (invivoGen) bp 717-116 Bp 10338 - 10418 CMV promoter from vector pGWIZ, Gene Therapy Systems bp 918-844 with

5' MluI RE site

Bp 10419 - 12111 Chicken Lysozyme Matrix Attachment region (MAR) from gDNA

Bp 12112 - 12123 Synthetic DNA added during construction including a PspOMI RE site Bp 12124 - 12193 70 bp of IS10 from TnlO (GenBank Accession #J01829 Bp 70-1)

Bp 12194 - 12239 Lambda DNA from pNK2859

Bp 12240 - 14441 pBluescriptll sk(-) base vector (Stratagene, INC) SEQ ID NO:42 ID# 381 - HPvsl/ CMViA/ CAss(-laa)/ hINFa-2b (4N77) / OPA in kTN-10 PURO-MAR Flanked BV

Bp 1 - 132 Remainder of Fl (-) ori of pBluescriptll sk(-) (Stratagene) bp 4-135

Bp 133 - 148 pGWIZ base vector (Gene Therapy Systems) bp 229-244

Bp 149 - 747 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843)

Bp 748 - 822 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-918)

Bp 823 - 943 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

Bp 944 - 1769 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040- 1865)

Bp 1770 - 1777 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 1778 - 1806 TNI 0 DNA, 3 'end from Genbank Accession #J01829 bp 79 - 107

Bp 1807 - 3015 Transposon, modified from TnlO GenBank Accession #J01829 Bp 108-1316

Bp 3016 - 3367 Putative PolyA from vector pNK2859

Bp 3368 - 3410 Lambda DNA from pNK2859

Bp 3411 - 3480 70 bp of IS 10 left from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 3481 - 3651 pBluescriptll sk(-) base vector (Stratagene, INC)

Bp 3652 - 3674 Multiple Cloning Site from pBluescriptll sk(-)thru Notl, Bp 759-737

Bp 3675 - 5367 Chicken Lysozyme Matrix Attachment region (MAR)

Bp 5368 - 5381 Multiple Cloning Site Extension from pTn X-MCS, Xhol thru Ascl

Bp 5382 - 6222 Chicken Ovalbumin promoter from gDNA (Genbank Accession #J00895 bp

421-1261)

Bp 6223 - 6827 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843)with 5'

EcoRI RE site

Bp 6828 - 6905 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-899, CTC,

900-918)

Bp 6906 - 7026 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

BP 7027 - 7852 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040-1865) Bp 7853 - 7860 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 7861 - 7926 Conalbumin Signal Peptide, GenBank NM 205304 bp 74-133 with 5' Sail RE site

Bp 7927 - 8424 Human Interferon alpha-2b Gene (IFN-a 2b) corresponding GenBank #J00207 bp 580-1077, with the following changes to create N-glycosylation sites: codon encoding PRO to ASN at bp 10-12 (amino acid 4), codon encoding ARG to ASN at bp 67-69 (amino acid 23), codon encoding ASP to ASN at bp 211-213 (amino acid 71) and codon encoding ASP to ASN at bp 229-231 (amino acid 77) of the hINFa 2b sequence

Bp 8425 - 9346 Chicken Ovalbumin polyA from gDNA (GenBank Accession #J00895 bp

8260-9175)with 5' Agel RE site

Bp 9347 - 9393 MCS extension from pTN-MCS, Pad thru BsiWI

Bp 9394 - 9706 HSV-TK polyA from pS65TCl bp 3873-3561

Bp 9707 - 9735 Excess DNA from pMOD PURO (invivoGen)

BP 9739 - 10337 Puromyacin resistance gene from pMOD PURO (invivoGen) bp 717-116

Bp 10338 - 10729 SV40 promoter from pS65TCl, bp 2232-2617 with 5' Mlul RE site

Bp 10730 - 12422 Chicken Lysozyme Matrix Attachment region (MAR) from gDNA

Bp 12423 - 12434 Synthetic DNA added during construction including a PspOMI RE site

Bp 12435 - 12504 70 bp of IS10 from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 12505 - 12551 Lambda DNA from pNK2859

Bp 12551 - 14752 pBluescriptll sk(-) base vector (Stratagene, INC)

SEQ ID NO: 43 ID# 382 - HPvsl/ CMViA/ CAss(-laa)/ hINFoc-2b (4N134) / OPA in kTN-10 PURO-MAR Flanked BV

Bp 1 - 132

Bp 133 - 148

Bp 149 - 747

Bp 748 - 822

Bp 823 - 943

bp 919-1039) Bp 944 - 1769 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040- 1865) Bp 1770 - 1777 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy Systems) bp 1866-1873)

Bp 1778 - 1806 TNI 0 DNA, 3 'end from Genbank Accession #J01829 bp 79 - 107

Bp 1807 - 3015 Transposon, modified from TnlO GenBank Accession #J01829 Bp 108-1316

Bp 3016 - 3367 Putative PolyA from vector pNK2859

Bp 3368 - 3410 Lambda DNA from pNK2859

Bp 3411 - 3480 70 bp of IS 10 left from TnlO (GenBank Accession #J01829 Bp 1-70) Bp 3481 - 3651 pBluescriptll sk(-) base vector (Stratagene, INC)

Bp 3652 - 3674 Multiple Cloning Site from pBluescriptll sk(-)thru Notl, Bp 759-737

Bp 3675 - 5367 Chicken Lysozyme Matrix Attachment region (MAR)

Bp 5368 - 5381 Multiple Cloning Site Extension from pTn X-MCS, Xhol thru Ascl

Bp 5382 - 6222 Chicken Ovalbumin promoter from gDNA (Genbank Accession #J00895 bp

421-1261)

Bp 6223 - 6827 CMV Enhancer (vector pGWIZ, Gene Therapy Systems bp 245-843)with 5'

EcoRI RE site

Bp 6828 - 6905 CMV Promoter (vector pGWIZ, Gene Therapy Systems bp 844-899, CTC,

900-918)

Bp 6906 - 7026 CMV Immediate Early Gene, Exon 1 (vector pGWIZ, Gene Therapy Systems bp 919-1039)

BP 7027 - 7852 CMV Intron A (vector pGWIZ, Gene Therapy Systems bp 1040-1865) Bp 7853 - 7860 CMV Immediate Early Gene, Partial Exon 2 (pGWIZ,Gene Therapy

Systems)bp 1866-1873)

Bp 7861 - 7926 Conalbumin Signal Peptide, GenBank NM 205304 bp 74-133 with 5' Sail RE site

Bp 7927 - 8424 Human Interferon alpha-2b (IFN-a 2b)Gene corresponding GenBank #J00207 bp 580-1077, with the following changes to create N-glycosylation sites: codon encoding PRO to ASN at bp 10-12 (amino acid 4), codon encoding ARG to ASN at bp 67-69 (amino acid 23), codon encoding ASP to ASN at bp 211-213 (amino acid 71), codon encoding LYS to ASN at bp 400-402 (amino acid 134) and encoding TYR to CYS at bp 403-405 (amino acid 135) of the hlNFoc 2b sequence

Bp 8425 - 9346 Chicken Ovalbumin polyA from gDNA (GenBank Accession #J00895 bp

8260-9175)with 5' Agel RE site

Bp 9347 - 9393 MCS extension from pTN-MCS, Pad thru BsiWI

Bp 9394 - 9706 HSV-TK polyA from pS65TCl bp 3873-3561

Bp 9707 - 9735 Excess DNA from pMOD PURO (invivoGen)

BP 9739 - 10337 Puromycin resistance gene from pMOD PURO (invivoGen) bp 717-116

Bp 10338 - 10729 SV40 promoter from pS65TCl, bp 2232-2617 with 5' Mlul RE site

Bp 10730 - 12422 Chicken Lysozyme Matrix Attachment region (MAR) from gDNA

Bp 12423 - - 12434 Synthetic DNA added during construction including a PspOMI RE site

Bp 12435 - - 12504 70 bp of IS 10 from TnlO (GenBank Accession #J01829 Bp 1-70)

Bp 12505 - 12551 Lambda DNA from pNK2859

Bp 12551 - 14752 pBluescriptll sk(-) base vector (Stratagene, INC)

In one embodiment, the present application provides a novel sequence comprising a promoter, a gene of interest, and a poly A sequence. Each of these novel sequences may be identified from the annotations for each expression vector shown above, and also as sequences within the sequence listing for each expression vector. The specific bases of these novel sequences are provided in Table 3 below for each expression vector SEQ ID NOs: 33, 34, and 35.

Table 3

The following examples will serve to further illustrate the present invention without, at the same time, however, constituting any limitation thereof. On the contrary, it is to be clearly understood that resort may be had to various embodiments, modifications and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the invention.

EXAMPLE 1

Preparation of Vectors for Expression of Interferon

Construction of Vector #324 SEP ID NO: 33

The pTopo containing the human interferon a 2b N,N-glycosylated (hIFN-α 2b (N,N- glycosylated)) cassettes driven by the hybrid promoter version 1 (SEQ ID NO: 16), were digested with restriction enzymes Asc I and Pac I (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research). To insert the hIFN-α 2b (N,N-glycosylated) cassette into the MCS of the p5021 vector (SEQ ID NO:6), the purified hIFN-a 2b(N,N- glycosylated) DNA and the p5021 vector (SEQ ID NO:6) were digested with Asc I and Pac I, purified as described above, and ligated using a Quick T4 DNA Ligase Kit (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen's protocol. Transformed bacteria were incubated in 0.25 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread to LB (Luria-Bertani media (broth or agar)) plates supplemented with 100 μg/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C and resulting colonies picked to LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 0.8% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). Column purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was done on a Beckman Coulter CEQ 8000 Genetic Analysis Systyem. Once a clone was identified that contained the hIFN-α 2b (Ν,Ν-glycosylated) gene, the DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid was grown in 250 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen EndoFree Plasmid Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500μί of Endotoxin free water and stored at -20°C until needed.

This gene encoding for N, N glycosylation encodes Asn at positions 71 and 153, and the amino acid sequence is shown at SEQ ID NO:33. This is different from naturally occurring interferon which has an Asp at residue 71 and Leu at residue 153.

Construction of Vectors SEQ ID NO:34 Mat.E.O.hIFN-a2a (N-glvcosylated and SEQ ID NO:35 E.O.hIFN-a2a (N.N-glvcosylated

The pTopo containing the expression optimized (E.O.) human interferon a 2a (E.O. hIFN-a 2a) cassette driven by the hybrid promoter version 1 (SEQ ID NO: 16), was digested with restriction enzymes Asc I and Pac I (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research). To insert the E.O. hIFN-a 2a cassette into the MCS of the p5021 vector (SEQ ID NO:6), the purified E.O. hIFN-a 2a DNA and the p5021 vector (SEQ ID NO:6) were digested with Asc I and Pac I, purified as described above, and ligated using a Quick T4 DNA Ligase Kit (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen's protocol. Transformed bacteria were incubated in 0.25ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread to LB (Luria-Bertani media (broth or agar)) plates supplemented with 100 μg/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C and resulting colonies picked to LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 0.8% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi- Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). Column purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was done on a Beckman Coulter CEQ 8000 Genetic Analysis Systyem.

Once a clone was identified that contained the E.O. hIFN-α 2a gene, the DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid was grown in 250 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen EndoFree Plasmid Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500μί of Endotoxin free water and stored at -20°C until needed.

The gene encoding for N glycosylation encodes Asn at position 71, and the amino acid sequence is shown at SEQ ID NO:34. This is different from naturally occurring interferon which has an Asp at residue 71.

The gene encoding for N, N glycosylation encodes for Asn at positions 71 and 153, and the amino acid sequence is shown at SEQ ID NO:35. This is different from naturally occurring interferon which has an Asp at residue 71 and Leu at residue 153.

EXAMPLE 2

Preparation of Vectors for Expression of Interferon

A vector was designed for inserting a desired coding sequence into the genome of eukaryotic cells, given below as SEQ ID NO:6. The vector of SEQ ID NO:6 was constructed and its sequence verified.

This vector is a modification of p5018 (SEQ ID NO:5) described above. The modification includes insertion of the puromycin (puro) gene into the multiple cloning site adjacent to one of the MAR insulator elements. To accomplish this, the 602 bp puromycin gene was amplified by polymerase chain reaction (PCR) from the vector pMOD Puro (Invitrogen Life Technologies, Carlsbad, CA). Amplified PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA). Purified DNA from the puromycin gene was digested with the restriction enzymes BsiWI and Mlul (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from agarose using a Zymo DNA Clean and Concentrator kit (Zymo Research). To insert the puro gene into the MCS of vector p5018 (SEQ ID NO:5), puro and the p5018 DNA (SEQ ID NO:5) were digested with BsiWI and Mlul, purified as described above, and ligated using Stratagene's T4 Ligase Kit (La Jolla, CA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed cells were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37° C then spread onto LB (Luria-Bertani) agar plates supplemented with 100 μg/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37° C. Resulting colonies were picked into LB/amp broth for overnight growth at 37° C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 1% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in a minimum of 250 ml of LB/amp broth. The plasmid DNA was harvested using a Qiagen Maxi-Prep Kit according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). The DNA was used as a sequencing template to verify changes made in the vector were desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis Systyem. Once a clone was identified that contained the puro gene, the DNA was isolated (see below) and used for cloning in specific genes of interest.

All plasmid DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid of interest was grown in 500 ml of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 of PCR-grade water and stored at -20°C until needed.

Construction of Vector 337 SEQ ID NO: 36

Invitrogen' s pTopo plasmid (Carlsbad, CA) containing the human interferon a 2b N,N- glycosylated [hIFN-α 2b (Ν,Ν-Gly)] cassette driven by the hybrid promoter version 4 (SEQ ID: 18), was digested with restriction enzymes AscI and Pad (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified using Zymo Research's DNA Clean and Concentrator kit (Orange, CA). To insert the hIFN-α 2b (N,N-Gly) cassette into the MCS of vector p5021 (SEQ ID NO:6), purified hIFN-a 2b(N,N-Gly) DNA and p5021 (SEQ ID NO:6) were digested with AscI and Pad, purified as described above, and ligated using a Quick T4 DNA Ligase Kit (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed bacterial cells were incubated in 0.25 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C then spread onto LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 0.8% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in a minimum of 250 ml of LB/amp broth. Plasmid DNA was harvested using Qiagen's Maxi-Prep Kit according to the manufacturer's protocol (Chatsworth, CA). The DNA was then used as a sequencing template to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System.

Once a clone was identified that contained the hIFN-α 2b (Ν,Ν-Gly) cassette, the DNA was isolated by standard procedures. Briefly, Escherichia coli bacteria containing the plasmid of interest was grown in 250 ml of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using Qiagen's EndoFree Plasmid Maxi-Prep kit (Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500μί of endotoxin free water and stored at -20°C until needed. The interferon produced by this vector is shown in SEQ ID NO: 44.

Construction of Vector 340 SEP ID NO: 37

Invitrogen' s pTopo plasmid (Carlsbad, CA) containing the human interferon a 2b N,N- glycosylated [hIFN-α 2b (Ν,Ν-Gly)] cassette driven by the hybrid promoter version 1 (SEQ ID NO: 16), was digested with restriction enzymes AscI and Pad (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified using Zymo Research's DNA Clean and Concentrator kit (Orange, CA). To insert the hIFN-α 2b (N,N-Gly) cassette into the MCS of vector p5021 (SEQ ID NO:6), purified hIFN-a 2b(N,N-Gly) DNA and p5021 (SEQ ID NO:6) were digested with AscI and Pad, purified as described above, and ligated using a Quick T4 DNA Ligase Kit (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed bacterial cells were incubated in 0.25 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C then spread onto LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 0.8% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in a minimum of 250 ml of LB/amp broth. Plasmid DNA was harvested using Qiagen's Maxi-Prep Kit according to the manufacturer's protocol (Chatsworth, CA). The DNA was then used as a sequencing template to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System.

Once a clone was identified that contained the hIFN-α 2b (Ν,Ν-Gly) cassette, the DNA was isolated by standard procedures. Briefly, Escherichia coli bacteria containing the plasmid of interest was grown in 250 ml of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using Qiagen's EndoFree Plasmid Maxi-Prep kit (Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500μί of endotoxin free water and stored at -20°C until needed. The interferon produced by this vector is shown in SEQ ID NO: 47.

Construction of Vector 341 SEP ID NO: 38

Invitrogen' s pTopo plasmid (Carlsbad, CA) containing the human interferon a 2b N,N- glycosylated [hIFN-α 2b (Ν,Ν-Gly)] cassette driven by the hybrid promoter version 1 (SEQ ID NO: 16), was digested with restriction enzymes AscI and Pad (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified using Zymo Research's DNA Clean and Concentrator kit (Orange, CA). To insert the hIFN-α 2b (N,N-Gly) cassette into the MCS of vector p5021 (SEQ ID NO:6), purified hIFN-a 2b(N,N-Gly) DNA and p5021 (SEQ ID NO:6) were digested with AscI and Pad, purified as described above, and ligated using a Quick T4 DNA Ligase Kit (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to the manufacturer's protocol. Transformed bacterial cells were incubated in 0.25 ml of SOC (GIBCO BRL, CAT# 15544-042) for 1 hour at 37°C then spread onto LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C. Resulting colonies were picked into LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 0.8% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in a minimum of 250 ml of LB/amp broth. Plasmid DNA was harvested using Qiagen's Maxi-Prep Kit according to the manufacturer's protocol (Chatsworth, CA). The DNA was then used as a sequencing template to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System.

Once a clone was identified that contained the hIFN-α 2b (Ν,Ν-Gly) cassette, the DNA was isolated by standard procedures. Briefly, Escherichia coli bacteria containing the plasmid of interest was grown in 250 ml of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight in a shaking incubator. Plasmid DNA was isolated from the bacteria using Qiagen's EndoFree Plasmid Maxi-Prep kit (Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500μί of endotoxin free water and stored at -20°C until needed. The interferon produced by this vector is shown in SEQ ID NO: 48.

Construction of Vector 365 (SEP ID NO: 39)

The pTopo vector containing the human interferon β la (hlFN-pia) cassette driven by the hybrid promoter version 1 (SEQ ID NO: 16) was digested with restriction enzymes AscI and Pa (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research, Orange, CA). To insert the hlFN-pia cassette into the MCS of the p5037 vector (SEQ ID NO: 15), the purified hlFN-pia cassette DNA and the p5037 vector (SEQ ID NO: 15) were digested with AscI and Pad, purified as described above, and ligated using a Quick T4 DNA Ligase Kit (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen' s protocol. Transformed bacteria were incubated in 0.25 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread to LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp). These plates were incubated overnight at 37°C, and resulting colonies picked to LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 0.8% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). Column purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System.

Once a clone was identified that contained the hlFN-pia gene, the DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid was grown in 250 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen EndoFree Plasmid Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500μί of Endotoxin free water and stored at -20°C until needed.

Construction of Vector 368 (SEP ID NO: 40)

The pTopo containing the human interferon a 2a N,N-glycosylated (hIFN-α 2a (N,N- glycosylated)) cassette driven by the hybrid promoter version 1 (SEQ ID NO: 16) was digested with restriction enzymes AscI and Pad (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research, Orange, CA). To insert the hIFN-α 2a (N,N- glycosylated) cassette into the MCS of the p5021 vector (SEQ ID NO:6), the purified hIFN-a 2a (Ν,Ν-glycosylated) DNA and the p5021 vector (SEQ ID NO: 6) were digested with Asc I and Pac I, purified as described above, and ligated using a Quick T4 DNA Ligase Kit (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen' s protocol. Transformed bacteria were incubated in 0.25 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread to LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp). These plates were incubated overnight at 37°C, and resulting colonies picked to LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 0.8% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). Column purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis Systyem.

Once a clone was identified that contained the hIFN-α 2a (Ν,Ν-glycosylated) gene, the DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid was grown in 250 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen EndoFree Plasmid Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500μί of Endotoxin free water and stored at -20°C until needed.

This gene encoding for N, N glycosylation encodes Asn at positions 71 and 153 and the amino acid sequence is shown at SEQ ID NO:50. This is different from naturally occurring interferon which has an Asp at residue 71 and Leu at residue 153. Construction of Vector 369 (SEP ID NO:41)

The pTopo containing the human interferon a 2a N,N-glycosylated (hIFN-α 2a (N,N- glycosylated)) cassette driven by the hybrid promoter version 1 (SEQ ID NO: 16) was digested with restriction enzymes AscI and Pad (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research, Orange, CA). To insert the hIFN-α 2a (N,N- glycosylated) cassette into the MCS of the p5037 vector (SEQ ID NO: 15), the purified hIFN-a 2a (Ν,Ν-glycosylated) DNA and the p5037 vector (SEQ ID NO: 15) were digested with AscI and Pad, purified as described above, and ligated using a Quick T4 DNA Ligase Kit (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen' s protocol. Transformed bacteria were incubated in 0.25 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread to LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp). These plates were incubated overnight at 37°C, and resulting colonies picked to LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 0.8% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). Column purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis Systyem.

Once a clone was identified that contained the hIFN-α 2a (Ν,Ν-glycosylated) gene, the DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid was grown in 250 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen EndoFree Plasmid Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500μί of Endotoxin free water and stored at -20°C until needed. This gene encoding for N, N glycosylation encodes Asn at positions 71 and 153 and the amino acid sequence is shown at SEQ ID NO:51. This is different from naturally occurring interferon which has an Asp at residue 71 and Leu at residue 153.

Construction of Vector 381 (SEQ ID NO:42

The pTopo containing the human interferon 2b 4N-glycosylated (hIFN-α 2b (4N- glycosylated)) cassette driven by the hybrid promoter version 1 (SEQ ID NO: 16) was digested with restriction enzymes AscI and Pad (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research, Orange, CA). To insert the hIFN-α 2b (4N- glycosylated) cassette into the MCS of the p5021 vector (SEQ ID NO:6), the purified hIFN-a 2b (4N-glycosylated) DNA and the p5021 vector (SEQ ID NO: 6) were digested with AscI and Pad, purified as described above, and ligated using a Quick T4 DNA Ligase Kit (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen' s protocol. Transformed bacteria were incubated in 0.25 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread to LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C and resulting colonies picked to LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 0.8% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). Column purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System.

Once a clone was identified that contained the hIFN-α 2b (4N-glycosylated) gene, the DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid was grown in 250 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen EndoFree Plasmid Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500μί of Endotoxin free water and stored at -20°C until needed.

This gene encoding 4N-glycosylation encodes Asn at positions 4, 23, 71, and 77 in the amino acid sequence that is shown at SEQ ID NO:52. This is different from naturally occurring interferon which has a Pro at residue 4, Arg at residue 23, and Asp at residues 71 and 77.

Construction of Vector 382 (SEQ ID NO:43

The pTopo containing the human interferon a 2b 4N-glycosylated [hIFN-α 2b (4N- glycosylated)] cassette driven by the hybrid promoter version 1 (SEQ ID NO: 16) was digested with restriction enzymes AscI and Pad (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research, Orange, CA). To insert the hIFN-α 2b (4N- glycosylated) cassette into the MCS of the p5021 vector (SEQ ID NO:6), the purified hIFN-a 2b (4N-glycosylated) DNA and the p5021 vector (SEQ ID NO: 6) were digested with AscI and Pad, purified as described above, and ligated using a Quick T4 DNA Ligase Kit (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Ligated product was transformed into E. coli Top 10 competent cells (Invitrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invitrogen's protocol. Transformed bacteria were incubated in 0.25 ml of SOC (GIBCO BRL, CAT# 15544-042) medium for 1 hour at 37°C before being spread to LB (Luria-Bertani) agar plates supplemented with 100 g/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37°C, and resulting colonies picked to LB/amp broth for overnight growth at 37°C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al, 1989), electrophoresed on a 0.8% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA). Column purified DNA was used as template for sequencing to verify the changes made in the vector were the desired changes and no further changes or mutations occurred. All sequencing was performed using Beckman Coulter's CEQ 8000 Genetic Analysis System.

Once a clone was identified that contained the hIFN-α 2b (4N-glycosylated) gene, the DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid was grown in 250 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen EndoFree Plasmid Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500μί of Endotoxin free water and stored at -20°C until needed.

This gene encoding for 4N glycosylation encodes Asn at positions 4, 23, 71, and 134, and encodes Cys at position 135 of the amino acid sequence and is shown at SEQ ID NO:53. This is different from naturally occurring interferon which has a Pro at residue 4, Arg at residue 23, Asp at residue 71, Lys at residue 134, and Tyr at residue 135.

EXAMPLE 3

Production of Glycosylated Interferon In Vitro

LMH2A cells were separately transfected with vectors represented as SEQ ID NOs:33, 34, 36, 37, or 38 using methods described above. Cell culture medium was collected and interferon levels measured with ELISA using methods described above. These cells produced interferon at levels of about 4 μg/ml (SEQ ID NO: 33), 3.77 μ /ml (SEQ ID NO: 34), 7 μg/ml (SEQ ID NO: 36), 4.4 μg/ml (SEQ ID NO: 37), and 4.8 μg/ml (SEQ ID NO: 38), respectively, based on ELISA results.

Western blots demonstrated that the interferon proteins engineered to have a glycosylation site not present in wild type interferon migrate as a larger molecular weight protein, demonstrating the glycosylation of the interferon. Analysis of the interferon produced by cells transfected with these vectors, using PNGase treatment, demonstrated that the larger molecular weight proteins were glycosylated.

The PBL iLite Human interferon Alpha kit (PBL Interferon Source, Piscattaway, NJ) was used to analyze biological activity of interferon produced by cells transfected with SEQ ID NO: 33 and SEQ ID NO: 34. Interferon from these cells showed high levels of biological activity when compared to the kit controls and the commercially available interferon. All patents, publications and abstracts cited above are incorporated herein by reference in their entirety. It should be understood that the foregoing relates only to preferred embodiments of the present invention and that numerous modifications or alterations may be made therein.

SEQUENCE LISTING

A sequence listing including 53 sequences is submitted along with this application as an ASCII text file entitled 51687-822677_ST25.txt. This file was created on October 17, 2011 and is 412 kilobytes.

Claims

1. An isolated interferon protein comprising two or more glycosylation sites that are not present in wild type interferon.

2. The isolated interferon protein of claim 1, wherein the interferon protein is interferon a 2a (IFN-a 2a), interferon a 2b (IFN-a 2b), or interferon la (IFN-β la).

3. The isolated interferon protein of claim 1 or 2, comprising a sequence selected from the group consisting of SEQ ID NOs:44 and 46-53.

4. The isolated interferon protein of any one of the preceding claims, comprising four glycosylation sites that are not present in wild type interferon.

5. The isolated interferon protein of any one of the preceding claims, wherein the interferon is human interferon.

6. An isolated nucleic acid sequence encoding an interferon protein comprising two or more glycosylation sites that are not present in wild type interferon.

7. The isolated nucleic acid sequence of claim 6, wherein the interferon protein is interferon a 2a (IFN-a 2a), interferon a 2b (IFN-a 2b), or interferon la (IFN-β la).

8. The isolated nucleic acid sequence of claim 6 or 7, wherein the nucleic acid sequence encodes an interferon protein having an amino acid sequence selected from the group consisting of SEQ ID NOs:44 and 46-53.

9. The isolated nucleic acid sequence of any one of claims 6-8, wherein the nucleic acid sequence encodes an interferon protein comprising four glycosylation sites that are not present in wild type interferon.

10. The isolated nucleic acid sequence of any one of claims 6-9, wherein the nucleic acid sequence encodes a human interferon.

11. A transposon-based vector comprising:

a modified transposase gene operably linked to a first promoter, wherein the nucleotide sequence 3 ' to the first promoter comprises a modified Kozak sequence, and wherein a plurality of the first twenty codons of the transposase gene are modified from the wild-type sequence by changing the nucleotide at the third base position of the codon to an adenine or thymine without modifying the amino acid encoded by the codon;

one or more genes encoding an interferon operably-linked to one or more additional promoters, wherein the one or more genes encoding the interferon and their operably-linked promoters are flanked by transposon insertion sequences recognized by a transposase encoded by the modified transposase gene, and wherein the one or more genes encoding the interferon comprises two or more sequences encoding glycosylation sites that are not present in wild type interferon; and

one or more insulator elements located between the transposon insertion sequences and the one or more genes of interest encoding interferon.

12. The vector of claim 11, wherein the vector comprises any one of SEQ ID NOs:33 and 35- 43.

13. The vector of claim 11 or 12, wherein the interferon is interferon a 2a (IFN-a 2a), interferon a 2b (IFN-a 2b), and interferon ia (IFN-β la).

14. The vector of any one of claims 11-13, wherein the interferon is a human interferon.

15. The vector of any one of claims 11-14, wherein the one or more insulator elements comprises an HS4 element, a lysozyme replicator element, a combination of a lysozyme replicator element and an HS4 element, or a matrix attachment region element.

16. A method of producing glycosylated interferon comprising:

transfecting a cell with a vector comprising a modified gene encoding for a transposase, a promoter, and a gene encoding an interferon comprising two or more sequences encoding glycosylation sites that are not present in wild type interferon;

culturing the transfected cell in culture medium;

permitting the cell to release interferon into the culture medium; collecting the culture medium; and,

isolating the interferon.

17. The method of claim 16, wherein the vector comprises any one of SEQ ID NOs: 33 and 35-43.

18. The method of claim 16 or 17, wherein the interferon is interferon a 2a (IFN-a 2a), interferon a 2b (IFN-a 2b), or interferon ia (IFN-β la).

19. The method of any one of claims 16-18, wherein the interferon is a human interferon.

20. The method of any one of claims 16-19, wherein the vector comprises one or more insulator elements located between the transposon insertion sequences and the one or more genes of interest encoding interferon, wherein the one or more insulator elements comprise an HS4 element, a lysozyme replicator element, a combination of a lysozyme replicator element and an HS4 element, or a matrix attachment region element.

21. A cell comprising the vector of any one of claims 11-15.

22. A cell comprising the isolated nucleic acid of any one of claims 6-10.