WO2003024199A2

WO2003024199A2 - Production of transgenic avians using sperm-mediated transfection

Info

Publication number: WO2003024199A2
Application number: PCT/US2002/030156
Authority: WO
Inventors: Jeffrey C. Rapp; Pramod Sutrave
Original assignee: Avigenics, Inc.
Priority date: 2001-09-21
Filing date: 2002-09-23
Publication date: 2003-03-27
Also published as: AU2002341795A1; WO2003024199A3

Abstract

The present invention relates to novel methods of producing transgenic avians, preferably chickens, wherein the incorporated transgene may be expressed as a constituent protein of the white of a hard-shell egg. The present invention provides sperm-mediated transfer for the introduction to an avian egg of a transgene encoding a heterologous polypeptide. The avian sperm may be irradiated before the transgenic gene is incorporated therein. Transgenic genes may be incorporated into avian sperm by lipofection, electroporation, restriction enzyme mediated integration (REMI) or similar methods. The modified avian sperm may then be delivered to an avian oocyte by microinjection, intracytoplasmic sperm injection (ICSI) or artificial insemination, or by natural coitus after the modified avian sperm are returned to a male bird. Heterologous nucleic acid may be integrated directly into the genomic nucleic acid of the oocyte or after first integrating the heterologous nucleic acid into the nucleic acid of a male germ cell and subsequent delivery of the transgenic male germ cell to an oocyte. Alternatively, the heterologous nucleic acid may be a episome within the sperm, or within the derivative zygote formed by the fusion of the sperm and the recipient oocyte, and may replicate independently of the zygote genome. Co-segregation of the episome with the replicated ooctye genome into all of the daughter cells may be induced by the heterologous nucleic acid having a centromeric body derived from, for example, a chromosome of a chicken.

Description

PRODUCTION OF TRANSGENIC AVIANS USING SPERM-MEDIATED

TRANSFECTION

This application claims the benefit of United States Provisional Patent Application Serial No. 60/323,961, filed September 21, 2001, and United States Provisional Patent Application Serial No. 60/324,001, filed September 21, 2001, both of which are incorporated by reference herein in their entireties.

1. FIELD OF THE INVENTION The present invention relates to methods of producing a transgenic avian by introducing a nucleic acid encoding a heterologous protein into the genome of an avian oocyte by sperm-mediated transfection. The present invention further relates generally to a transgenic avian capable of expressing a heterologous polypeptide, which, preferably is deposited into the white of an avian egg, said avian generated by sperm-mediated transgenesis. The invention further provides vectors containing coding sequences for heterologous proteins, the expression of which is under the control of a promoter and other regulatory elements that cause expression of the heterologous protein and preferably, lead to deposition of the protein in the avian egg. Also included in the invention are avian eggs derived from the transgenic avians and the heterologous proteins isolated therefrom.

2. BACKGROUND

The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and the phenomena of gene activation, expression, and interaction. The technology has also been used to produce models for various diseases in humans and other animals and is amongst the most powerful tools available for the study of genetics and the understanding of genetic mechanisms and function. From an economic perspective, however, the use of transgenic technology for the production of specific proteins or other substances of pharmaceutical interest offers significant advantages over more conventional methods of protein production by gene expression. (Gordon et al, 1987, Biotechnology 5: 1183-1187; Wilmut et al, 1990, Theriogenology 33: 113-123).

In particular, the production of monoclonal antibodies by traditional methods is labor-intensive and costly. The purification of monoclonal antibodies from serum is a slow and low-yielding process. The use of hybridomas cell lines which were developed by fusing a B lymphocyte with a myeloma cell to propagate indefinitely in vivo as ascites, or in vitro in tissue culture requires major expenditures in tissue culture facilities or mice breeding. (Kohler and Milstein, 1975, Nature 256: 495-497). Although various strategies have been proposed to overcome the deficiencies in antibody yield (e.g., engineering single-chain antibodies (scAb) comprising immunoglobulin heavy and light chain variable regions), no method has been proven entirely satisfactory in elevating antibody yields to the levels desired for adequate commercial production.

The industry has been experimenting with transgenic animals that can express, for example, an exogenous protein such as an antibody under conditions that offer high yield of the protein in an active form while incorporating post-translational modifications, such as glycosylation, typically required for full functionality of the antibody. In this context, heterologous nucleic acids have been engineered so that an expressed protein may be joined to a protein or peptide that will allow secretion of the transgenic expression product into milk or urine, from which the protein may then be recovered. These procedures have had limited success, however, and may require lactating animals, with the attendant costs of maintaining individual animals or herds of large species, including cows, sheep or goats.

Avian Transgenics

One transgenic system that holds potential is the avian reproductive system. The exogenous protein can be produced in the white of an avian egg from which it may be readily purified. (MacArthur, PCT Publication WO 97/47739). The production of an avian egg begins with formation of a large yolk in the ovary of the hen. The unfertilized oocyte or ovum is positioned on top of the yolk sac. After ovulation, the ovum passes into the infundibulum of the oviduct where it is fertilized, if sperm are present, and then moves into the magnum of the oviduct, lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin, into the lumen of the magnum where they are deposited onto the avian embryo and yolk.

The hen oviduct, for example, can serve as an excellent protein bioreactor because of the high levels of protein production, the promise of proper folding and post-translation modification of the target protein, the ease of product recovery, and the shorter developmental period of chickens compared to other potential animal species. The economic advantage of breeding flocks of transgenic birds laying eggs expressing exogenous proteins would be significant when compared to more traditional animals, such as cows, sheep or goats, producing heterologous protein in milk. What is needed, however, is an efficient method of introducing a heterologous nucleic acid into a recipient avian embryonic cell. Vectors

Genetic information has been transferred to avian embiyos using vectors. Bosselman et al. in U.S. Patent No. 5,162,215 describes a method for introducing a replication-defective retroviral vector into a pluripotent stem cell of an unincubated chick embryo, and further describes chimeric chickens whose cells express a heterologous vector nucleic acid sequence. However, the percentage of GI transgenic offspring (progeny from vector-positive male GO birds) was low and varied between 1% and approximately 8%. In addition, the use of viral vectors poses limitations, including limitations on transgene size and potential viral infection of the offspring, thus, posing significant regulatory issues for production of biologies.

Similarly, Jaenisch reported that while retroviral vectors did transfer genetic information to embryos, the resulting animals were mosaics with gene insertions at various loci in the genomic nucleic acid. (1976, Proc. Natl Acad. Sci. USA 73: 1260-1264). The transgenes were also differentially expressed in the different tissues of each animal. (Jaenisch, 1980, Cell 19: 181-188).

Nuclear Transfer

Nuclear transfer from cultured cell populations is another route to produce transgenics, wherein donor cells may be sexed, optionally genetically modified, and then selected in culture before their use. The resultant transgenic animal originates from a single transgenic nucleus and therefore, mosaics are avoided. Nuclear transfer from cultured somatic cells also provides a route for directed genetic manipulation of animal species, including the addition or "knock-in" of genes, and the removal or inactivation or "knockout" of genes or their associated control sequences (Polejaeva et al, 2000, Theriogenology 53: 117-26).

Two types of recipient cells are commonly used in nuclear transfer procedures: oocytes arrested at the metaphase of the second meiotic division (Mil) and which have a metaphase plate with the chromosomes arranged on the meiotic spindle, and pronuclear zygotes. In agricultural mammals, however, development does not always occur when pronuclear zygotes are used, and, therefore, Mll-arrested oocytes are the preferred recipient cells. Enucleated two-cell stage blastomeres of mice have also been used as recipients. After enucleation and introduction of donor genetic material, the reconstructed embryo is cultured to the morula or blastocyte stage, and transferred to a recipient animal, either in vitro or in vivo, and developed to term. (Eyestone and Campbell, 1999, J. Reprod. Fertil. Suppl. 54: 489-97). Double nuclear transfer has been reported in which an activated, previously transferred nucleus is removed from the host unfertilized egg and transferred again into an enucleated fertilized embryo. Activation (initiation of development) is most often induced chemically. Cultured cells can also be frozen and stored indefinitely for future use. Although gene targeting techniques combined with nuclear transfer hold tremendous promise for nutritional and medical applications, current approaches suffer from several limitations, including long generation times between the founder animal and production transgenic herds, and extensive husbandry and veterinary costs. It is therefore desirable to use a system where cultured somatic cells for nuclear transfer are more efficiently employed.

Sperm-Mediated Transfection Mechanism

A promising method for producing transgenic animals is the stable transfection of male germ cells in vitro and their transfer to a recipient oocyte. PCT Publication WO 87/05325 discloses a method of transferring organic and/or inorganic material into sperm or egg cells by using liposomes. Bachiller et al. used Lipofectin-based liposomes to transfer DNA into mice sperm, and provided evidence that the liposome transfected DNA was overwhelmingly contained within the sperm's nucleus. (1991, Mol. Reprod. Develop. 30: 194-200). However, no transgenic mice could be produced by this technique. Similarly, Nakanishi and Iritani used Lipofectin-based liposomes to associate heterologous DNA with chicken sperm, which were in turn used to artificially inseminate hens. (1993, Mol. Reprod. Develop. 36: 258-261). Although the heterologous DNA was detectable in many of the resultant fertilized eggs, there was no evidence of genomic integration of the heterologous DNA either in the DNA-liposome treated sperm or in the resultant chicks.

Heterologous DNA may also be transferred into sperm cells by a process called electroporation that creates temporary, short-lived pores in the cell membrane of living cells by exposing them to a sequence of brief electrical pulses of high field strength. The pores allow cells to take up heterologous material such as DNA, while only slightly compromising cell viability. Gagne et al discloses the use of electroporation to introduce heterologous DNA into bovine sperm subsequently used to fertilize ova. (1991, Mol. Reprod. Develop. 29: 6-15). However, there was no evidence of integration of the electroporated DNA either in the sperm nucleus or in the nucleus of the egg subsequent to fertilization by the sperm.

Yet another method initially developed for integrating heterologous DNA into yeasts and slime molds, and later adapted to avian sperm, is restriction enzyme mediated integration (REMI), which utilizes a linear DNA derived from a plasmid DNA by cutting that plasmid with a restriction enzyme that generates single-stranded cohesive ends. (Shemesh et al, PCT International Publication WO 99/42569). The linear, cohesive-ended DNA together with the restriction enzyme used to produce the cohesive ends is then introduced into the target cells by electroporation or liposome transfection. The restriction enzyme is then thought to cut the genomic DNA at sites that enable the heterologous DNA to integrate via its matching cohesive ends. (Schiestl and Petes, 1991, Proc. Natl. Acad. Sci. USA 88: 7585-7589). Although Shemesh described transgenic birds that were resistant to Infectious Bursal Disease, there was no evidence of expression or deposition of a heterologous protein in the oviduct for deposition onto egg whites.

What is needed, therefore, is an efficient method of generating a transgenic avian capable of expressing a heterologous protein coded by a transgene, particularly in the oviduct for deposition into egg whites.

3. SUMMARY OF THE INVENTION

The invention provides methods for the stable introduction by sperm-mediated transfection of heterologous coding sequences into the genome of an avian, preferably a chicken, and expressing those heterologous coding sequence to produce desired proteins and/or to alter the phenotype of the transgenic avian. Synthetic vectors and gene promoters useful in the methods are also provided by the present invention, as are transgenic avians that express a heterologous protein and avian eggs, preferably chicken eggs, containing a heterologous protein. In a preferred embodiment, the vectors useful in methods of the invention are not eukaryotic viral, more preferably not retroviral, vectors (although the vectors may contain transcriptional regulatory elements, such as promoters, from eukaryotic viruses). In other embodiments, however, the vectors are retroviral vectors.

One aspect of the present invention is a method of producing a transgenic avian, preferably a chicken, by introducing in an avian oocyte at least one transgene encoding at least one heterologous polypeptide by sperm-mediated transfection. The method comprises first, isolating an avian sperm, second, incorporating a transgene into the avian sperm, and third, delivering the modified avian sperm to an avian oocyte. In one embodiment, the avian sperm is irradiated with gamma rays before the transgene is incorporated therein.

In one embodiment, the transgene is injected directly into the testis of a male avian and incorporated in the avian sperm. The modified sperm is then delivered to the avian oocyte by mating the male avian with a wild type or transgenic female avian. In another embodiment, the transgene is incorporated in the avian sperm in vitro by lipofection, electroporation, restriction enzyme mediated integration (REMI) or similar methods. In a preferred embodiment, the modified avian sperm is then delivered to the avian oocyte by natural coitus after the modified avian sperm are returned to the testis of a male avian. In another preferred embodiment, the modified avian sperm is delivered to the avian oocyte by microinjection (e.g., intracytoplasmic sperm injection (ICIS) or standard artificial fertilization methods). The resulting transgenic embryo can then be transferred to the oviduct of a recipient hen for development and to be laid as a shelled egg (or, alternatively, cultured ex vivo). The shelled egg is incubated to hatch a transgenic avian that has incorporated, preferably integrated into its genome, the selected nucleic acid. In preferred embodiments, the avian sperm is first irradiated before incorporated with the transgene.

In certain embodiments, a transgene comprising a heterologous nucleic acid may be integrated directly into the genomic nucleic acid of an avian sperm and subsequently delivered to an avian oocyte. When the heterologous nucleic acid is directly integrated into the genome of the avian sperm which then fertilizes an avian oocyte, the resulting transgenic embryo will include the transgenic heterologous nucleic acid in all of its cells. In preferred embodiments, the transgenic heterologous nucleic acid is incorporated into at least one embryonic cell, preferably the germinal disk of an early stage embryo, that then develop into a transgenic avian.

Alternatively, the heterologous nucleic acid may be an episome within the modified avian sperm, or within the derivative zygote formed by the fusion of the modified avian sperm and the avian oocyte. The episome may replicate independently of the zygote genome. When the heterologous nucleic acid is episomal with respect to the genome of the transgenic zygote, and the episomal nucleic acid has a centromeric body, most, if not all, of the cells of the transgenic embryo will include the heterologous nucleic acid. Accordingly, in preferred embodiments, the transgene further comprises centromere and/or telomere sequences of an avian chromosome.

The invention further provides method for incorporating at least one transgene into the genome of a spermatozoon cell or a precursor thereof isolated from a donor male avian, and returning the modified cell to the testis of a recipient male avian, preferably the donor male avian, so that a genetically modified male gamete is produced by the male avian. Breeding the male avian with a female of its species will generate a transgenic progeny carrying the at least one transgene in its genome. The invention also provides methods for introducing a heterologous nucleic acid to an avian oocyte in addition to those described in United States Application Serial No. 09/877,374, filed June 8, 2001, entitled "Production of Monoclonal Antibody By a Transgenic Chicken", by Jeffrey C. Rapp; and United States Application Serial No. , filed September 18, 2002, entitled "Production of a Transgenic Avian By

Cytoplasmic Injection", by Jeffrey C. Rapp and Leandro Christmann, both of which are incorporated by reference herein in their entireties. In certain embodiments, the avian oocyte is removed from the ovaries of a donor female avian to facilitate in vitro fertilization by the modified avian sperm of the invention. In other certain embodiments, the modified avian sperm is delivered to an avian oocyte in vivo by natural coitus. The fertilized ova is then, preferably, returned to or maintained in the oviduct of the donor female avian or a surrogate female avian to be laid as a hard-shell egg or, as an alternative, cultured ex vivo. The hard-shell egg is incubated and hatched, producing a transgenic chick that expresses a heterologous protein and/or that can be bred to generate a line of transgenic avians expressing a heterologous protein.

Preferably, the avian sperm or the reproductive system of a male avian, preferably the seminiferous tubules and/or site of sperm production, development, and/or storage in the testis, is irradiated by gamma rays before transgene incorporation. More preferably, the transgene is integrated directly into the genome of the avian sperm. Most preferably, the transgene further comprises centromere and/or telomere sequences.

In particular embodiments, the level of mosaicism of the transgene (percentage of cells containing the transgene) in avians hatched from sperm-mediated transfected embryos (t.e., the GOs) is greater than 5%, 10%, 25%, 50%, 75% or 90%, or is the equivalent of one copy per one genome, two genomes, five genomes, seven genomes or eight genomes, as determined by any number of techniques known in the art and described infra. In additional particular embodiments, the percentage of GOs that transmit the transgene to progeny (Gls) is greater than 5%, preferably, greater than 10%, 20%, 30%, 40%, and, most preferably, greater than 50%.

In certain other embodiments, the level of transgenics that result from mating with a wild type or transgenic avian avians hatched from sperm-mediated transfected embryos (i.e., the GOs) is greater than 5%, 10%, 25%, 50%, 75% or 90%.

In another embodiment, the present invention provides methods for producing heterologous proteins in avians. Transgenes are introduced by sperm-mediated transfection into the genome of an avian oocyte which becomes fertilized and then develops into a transgenic avian. The heterologous protein(s) of interest may be expressed in the tubular gland cells of the magnum of the oviduct, secreted into the lumen, and most preferably, deposited within the egg white onto the egg yolk or expressed, for example, in the serum of the avian. In preferred embodiments, the level of expression of the heterologous protein in the egg white of eggs laid by GO and/or GI chicks and/or their progeny is greater than 5 μg, 10 μg, 50 μg, 100 μg, 250 μg, 500 μg or 750 μg, more preferably greater than 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 700 mg, 1 gram, 2 grams, 3 grams, 4 grams or 5 grams.

The transgenic avians can also be bred to identify those avians that carry the transgene in their germ line. The exogenous gene coding for the heterologous proteins can therefore be transmitted by sperm-mediated transfection of the exogenous gene into the avian oocytes, and by subsequent stable transmission of the exogenous gene to the avian' s offspring in a Mendelian fashion. More information on Mendelian inheritance can be found in Haiti and Jones, 2001, Genetics: Analysis of Genes and Genomes, 5th ed., Jones & Bartlett Publishers, Inc., the content of which is incorporated by reference herein in its entirety.

Another aspect of the invention provides for the isolation of heterologous proteins in transgenic avians and the use thereof in pharmaceutical products including but not limited to vaccines, biologies and, particularly, therapeutically or diagnostically useful antibodies. The expressed heterologous protein(s) of interest may be collected and processed using standard techniques from the avian eggs, preferably the egg white, the serum, or other tissues from the transgenic avian.

The present invention further provides methods for producing a heterologous protein in an avian oviduct. The method comprises, as a first step, providing a vector containing a coding sequence and a promoter that functions in avians, preferably in the avian magnum, operably linked to the coding sequence, so that the promoter can effect expression of the nucleic acid in the tubular gland cells of the magnum of an avian oviduct and/or in any other desired tissue of the avian. In a preferred embodiment, the vector containing the transgene is not a eukaryotic viral vector (preferably, not a retroviral vector, such as but not limited to reticuloendotheliosis virus (REV), ALV or MMLN) or derived from a eukaryotic virus (but, in certain embodiments, may contain promoter and/or other gene expression regulatory sequences from a eukaryotic virus, such as, but not limited to, a cytomegalovirus promoter). Next, the vector is introduced into avian sperm in vitro by lipofection, electroporation, restriction enzyme mediated integration (REMI) or similar methods, or in vivo by directly injecting into the testis, so that the vector sequence may be incorporated into the avian sperm. In preferred embodiments, the avian sperm or precursor cells are irradiated by gamma rays before the vector sequence is incorporated therein. In another preferred embodiment, the vector sequence further comprises centromere and/or telomere sequence. Then, the modified avian sperm are delivered to an avian oocyte by natural coitus or in vitro by microinjection or artificial insemination to form a transgenic embryonic cell. In certain embodiments, the recipient avian oocyte is wild type unmodified or preferably, modified in a manner that facilitates the delivery of transgene by the modified avian sperm. In certain other embodiments, the recipient avian oocyte is derived from a first-generation or preferably, second-generation transgenic avian whose germ-line carries the transgene. Finally, a mature transgenic avian that expresses the exogenous protein in its oviduct is derived from the transgenic embryonic cell or by breeding a transgenic avian derived from the transgenic embryonic cell.

The present invention further provides promoters useful for expression of the heterologous protein in the egg. For example, the transgene may comprise regions of at least two promoters derived from an avian including, but not limited to, an oviduct-specific promoter such as ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin promoter or any other promoter that directs expression of a gene in an avian, particularly in a specific tissue of interest, such as the magnum, and a protamine promoter, or a fragment thereof which is sufficient to drive the expression of a marker gene such as Green Fluorescent Protein (GFP). Alternatively, the promoter used in the expression vector may be derived from that of the lysozyme gene that is expressed in both the oviduct and macrophages. In particular embodiments, the gene regulatory sequences are flanked by matrix attachment regions (MARs), preferably, but not limited to those associated with the lysozyme gene in chickens or other avians. The nucleic acid encoding the polypeptide may be operably linked to a transcription promoter and/or a transcription terminator. Other embodiments of the invention provide for transgenic avians, such as chickens or quail, carrying a transgene in the genetic material of their germ-line tissue, preferably where the transgene was not introduced into the avian genome using a eukaryotic viral promoter. The transgene incorporated into the genomic DNA of a recipient avian can encode at least one polypeptide that may be, for example, but is not limited to, a cytokine, a growth factor, enzyme, structural protein, immunoglobulin, or any other polypeptide of interest that is capable of being expressed by an avian cell or tissue. Preferably, the heterologous protein is a mammalian, preferably a human, protein or derived from a mammalian, or preferably a human, protein (e.g. , a derivative or variant thereof). In particular embodiments, the invention provides heterologous proteins isolated or purified from an avian tissue, preferably serum, more preferably eggs, most preferably egg whites, and pharmaceutical compositions comprising such heterologous proteins. In a more preferred embodiment, the heterologous protein is an antibody that is human (including antibodies produced from human immunoglobulin sequences in mice or in antibody libraries or synthetically produced but having variable domain framework regions that are the same as or homologous to human framework regions) or humanized.

The present invention further relates to nucleic acid vectors (preferably, not derived from eukaryotic viruses, except, in certain embodiments, for eukaryotic viral promoters and/ or enhancers) and transgenes inserted therein that incorporate multiple polypeptide- encoding regions, wherein a first polypeptide-encoding region is operatively linked to a transcription promoter and a second polypeptide-encoding region is operatively linked to an Internal Ribosome Entry Sequence (IRES). For example, the vector may contain coding sequences for two different heterologous proteins (e.g. , the heavy and light chains of an immunoglobulin) or the coding sequences for all or a significant part of the genomic sequence for the gene from which the promoter driving expression of the transgene is derived, and the heterologous protein desired to be expressed (e.g., a construct containing the genomic coding sequences, including introns, of the avian lysozyme gene when the avian lysozyme promoter is used to drive expression of the transgene, an IRES, and the coding sequence for the heterologous protein desired to be expressed downstream (i.e., 3' on the RNA transcript of the IRES). Thus, in certain embodiments, the nucleic acid encoding the heterologous protein is introduced into the 5' untranslated or 3' untranslated regions of an endogenous gene, such as but not limited to, ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin, with an IRES sequence directing translation of the heterologous sequence.

Such nucleic acid constructs, when inserted into the genome of an avian and expressed therein, will generate individual polypeptides that may be post-translationally modified, for example, glycosylated or, in certain embodiments, form complexes, such as heterodimers with each other in the white of the avian egg. Alternatively, the expressed polypeptides may be isolated from an avian egg and combined in vitro, or expressed in a non-reproductive tissue such as serum. In other embodiments, for example, but not limited to, when expression of both heavy and light chains of an antibody is desired, two separate constructs, each containing a coding sequence for one of the heterologous proteins operably linked to a promoter (either the same or different promoters), are introduced into embryonic cells by sperm-mediated transfection to generate transgenic avians that harbor both transgenes in their genomes and expressing both heterologous proteins are identified. Alternatively, two transgenic avians each containing one of the two heterologous proteins (e.g., one transgenic avian having a transgene encoding the light chain of an antibody and a second transgenic avian having a transgene encoding the heavy chain of the antibody) can be bred by Mendelian genetics to obtain an avian containing both transgenes in its germline and expressing both transgene encoded proteins, preferably in eggs. (See Haiti and Jones, 2001, Genetics: Analysis of Genes and Genomes, 5th ed., Jones & Bartlett Publishers, Inc., the content of which is incorporated by reference herein in its entirety).

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

Additional objects and aspects of the present invention will become more apparent upon review of the detailed description set forth below when taken in conjunction with the accompanying figures, which are briefly described as follows.

3.1 DEFINITIONS

The term "animal" as used herein refers to all vertebrate animals, including birds. It also includes an individual animal in all stages of development, including embryonic and fetal stages.

The term "avian" as used herein refers to any species, subspecies or race of organism of the taxonomic class aves, such as, but not limited to, chicken, quail, turkey, duck, goose, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary. The term includes the various known strains of Gallus gallus, or chickens, (for example, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, Italian Partridge-colored), as well as strains of turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred in commercial quantities. The term "male germ cells" as used herein refers to sperm, sperm cells, spermatozoa

(i.e., male gametes) and developmental precursors thereof. Male germ cells with the capacity to swim and transfer nucleic acid to an ovum are herein referred to as "viable male germ cells." In fetal development, primordial germ cells are thought to arise from the embryonic ectoderm, and are first seen in the epithelium of the endodermal yolk sac at the E8 stage. From there they migrate through the hindgut endoderm to the genital ridges. In the sexually mature male vertebrate animal, there are several types of cells that are precursors of spermatozoa, and which can be genetically modified, including the primitive spermatogonial stem cells, known as AO/As, which differentiate into type B spermatogonia. The latter further differentiate to form primary spermatocytes, and enter a prolonged meiotic prophase during which homologous chromosomes pair and recombine. Useful precursor cells at several morphological/developmental stages are also distinguishable: preleptotene spermatocytes, leptotene spermatocytes, zygotene spermatocytes, pachytene spermatocytes, secondary, spermatocytes, and the haploid spermatids. The latter undergo further morphological changes during spermatogenesis, including the reshaping of their nucleus,

5 the formation of aerosome, and assembly of the tail. The final changes in the spermatozoon (i.e., male gamete) take place in the genital tract of the female, prior to fertilization.

The terms "ovum" and "oocyte" are used interchangeably herein. Although only one ovum matures at a time, an animal is born with a finite number of ova. In avian species, such as a chicken, ovulation, which is the shedding of an egg from the ovarian

10 follicle, occurs when the brain's pituitary gland releases a luteinizing hormone, LH. Mature follicles form a stalk or pedicel of connective tissue and smooth muscle. Immediately after ovulation the follicle becomes a thin-walled sac, the post-ovulatory follicle. The mature ovum erupts from its sac and starts its journey through the oviduct. Eventually, the ovum enters the infundibulum where fertilization occurs. Fertilization must take place within 15

15 minutes of ovulation, before the ovum becomes covered by albumen. During fertilization, sperm (avians have polyspermic fertilization) penetrate the blastodisc. When the sperm lodges within this germinal disk, an embryo begins to form as a "blastoderm" or "zygote."

The term "embryonic cells" as used herein refers to cells that are typically single cell embryos, fertilized or unfertilized, or the equivalent thereof, and is meant to encompass

20 dividing embryos, such as two-cell, four-cell, or even later stages as described by Eyal- Giladi and Kochav (1976, Dev. Biol. 49: 321-337) and ova 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 hours after the preceding lay. The embryonic cells may be isolated freshly, maintained in culture, or reside within an embryo.

The term "fragment" as used herein to refers to an at least 10, 20, 50, 75, 100, 150,

25 200, 250, 300, 500, 1000, 2000 or 5000 nucleotide long portion of a nucleic acid (e.g., cDNA) that has been constructed artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or enzymatically, for example, by PCR or any other polymerizing technique known in the art, or expressed in a host cell by recombinant nucleic acid technology known to one of skill

30 in the art. The term "fragment" as used herein may also refer to an at least 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, 1000, 2000 or 5000 amino acid portion of a polypeptide, which portion is cleaved from a naturally occurring polypeptide by proteolytic cleavage by at least one protease, or is a portion of the naturally occurring polypeptide synthesized by chemical methods or using recombinant DNA technology (e.g., expressed

35 from a portion of the nucleotide sequence encoding the naturally occurring polypeptide) known to one of skill in the art.

The term "isolated nucleic acid" as used herein refers to a nucleic acid that has been removed from other components of the cell containing the nucleic acid or from other components of chemical/synthetic reaction used to generate the nucleic acid. In specific embodiments, the nucleic acid is 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% pure. The "isolated nucleic acid" is neither (a) identical to that of any naturally occurring nucleic acid nor (b) identical to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes, and includes DNA, RNA, or derivatives or variants thereof. The term covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic molecule but is not flanked by at least one of the coding sequences that flank that part of the molecule in the genome of the species in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic nucleic acid of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any vector or naturally occurring genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis, or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein; and (e) a recombinant nucleotide sequence that is part of a hybrid sequence that is not naturally occurring. The techniques used to isolate and characterize the nucleic acids and proteins of the present invention are well known to those of skill in the art and standard molecular biology and biochemical manuals may be consulted to select suitable protocols without undue experimentation. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press (2001); the content of which is herein incorporated by reference in its entirety.

By the use of the term "enriched" in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. Enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased, for example, by 1 fold, 2 fold, 5 fold, 10 fold, 50 fold, 100 fold, 500 fold, 1000 fold, 10,000 fold, 100,000 fold or 1,000,000 fold. The other DNA may, for example, be derived from a yeast or bacterial genome, or a cloning vector, such as a plasmid or a viral vector. The term "transcription regulatory sequences" as used herein refers to nucleotide sequences that are associated with a gene nucleic acid sequence and that regulate the transcriptional expression of the gene. The "transcription regulatory sequences" may be isolated and incorporated into a vector nucleic acid to enable regulated transcription in appropriate cells of portions of the vector DNA. Exemplary transcription regulatory sequences include enhancer elements, hormone response elements, steroid response elements, negative regulatory elements, and the like. The "transcription regulatory sequences" may be isolated and incorporated into a vector nucleic acid to enable regulated transcription in appropriate cells of portions of the vector DNA. The "transcription regulatory sequence" may precede, but is not limited to, the region of a nucleic acid sequence that is in the region 5' of the end of a protein coding sequence that may be transcribed into mRNA. Transcriptional regulatory sequences may also be located within a protein coding region, in regions of a gene that are identified as "intron" regions, or may be in regions of nucleic acid sequence that are in the region of nucleic acid. The term "promoter" as used herein refers to the DNA sequence that determines the site of transcription initiation by an RNA polymerase. A "promoter-proximal element" may be a regulatory sequence within about 200 base pairs of the transcription start site. A "magnum-specific" promoter, as used herein, is a promoter that is primarily or exclusively active in the tubular gland cells of the avian magnum. Useful promoters also include exogenously inducible promoters. These are promoters that can be "turned on" in response to an exogenously supplied agent or stimulus, which is generally not an endogenous metabolite or cytokine. Examples include an antibiotic-inducible promoter, such as a tetracycline-inducible promoter, a heat-inducible promoter, a light-inducible promoter, or a laser inducible promoter. (See, e.g., Halloran et al, 2000, Development 127: 1953-1960; Gemer et al, 2000, Int. J. Hyperthermia 16: 171-81; Rang and Will, 2000, Nucleic Acids Res. 28: 1120-5; Hagihara et al, 1999, Cell Transplant 8: 4314; Huang et al, 1999, Mol. Med. 5: 129-37; Forster et al, 1999, Nucleic Acids Res. 27: 708-10; Liu et al, 1998, Biotechniques 24: 624-8, 630-2; the contents of which have been incorporated herein by reference in their entireties). To facilitate manipulation and handling of the nucleic acid to be administered, the nucleic acid is preferably inserted into a cassette where it is operably linked to a promoter. The promoter should be capable of driving expression in the desired cells. The selection of appropriate promoters can be readily accomplished. For some applications, a high expression promoter is preferred such as the cytomegalovirus (CMN) promoter. Other promoters useful in the present invention include the Rous Sarcoma Virus (RSN) promoter (Davis et al, 1993, Hum. Gene Therap. 4:151). In other embodiments, all or a portion of the, for example, lysozyme, ovomucoid, albumin, conalbumin or ovotransferrin promoters, which direct expression of proteins present in egg white, are used, as detailed infra, or synthetic promoters such as the MDOT promoter described infra. The term "expressed" or "expression" as used herein refers to the transcription from a gene to give an RNA nucleic acid molecule complementary at least in part to a region of one of the two nucleic acid strands of the gene. The term "expressed" or "expression" as used herein also refers to the translation from said RNA nucleic acid molecule to give a protein or polypeptide or a portion thereof. The term "matrix attachment regions" as used herein refers to DNA sequences having an affinity or intrinsic binding ability for the nuclear scaffold or matrix. The MAR elements of the chicken lysozyme locus were described by Phi- Van et al, 1996, E.M.B.O. J. 76:665-664 and Phi-Van, L. and Srratling, W.H., 1996, Biochem. 35:10735-10742; incorporated herein by reference in their entireties. The term "nucleic acid vector" as used herein refers to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule, or any other nucleic acid molecule, such as but not limited to YACs, BACs, bacteriophage-derived artificial chromosome (BBPAC), cosmid or PI derived artificial chromosome (PAC), that can be transfected or transformed into cells and replicate independently of, or within, the host cell genome. A circular double stranded vector can be linearized by treatment with an appropriate restriction enzyme based on the nucleotide sequence of the vector. A nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the pieces together. The nucleic acid molecule can be RNA or DNA.

The term "expression vector" as used herein refers to a nucleic acid vector that comprises regulatory sequences operably linked to a nucleotide sequence coding for at least one polypeptide. As used herein, the term "regulatory sequences" includes promoters, enhancers, and other elements that may control expression. Standard molecular biology textbooks such as Sambrook et al, (supra) and Lodish et al, eds "Molecular Cell Biology" Freeman (2000) and incorporated herein by reference in their entireties, may be consulted to design suitable expression vectors, promoters, and other expression control elements. It should be recognized, however, that the choice of a suitable expression vector depends upon multiple factors including the choice of the host cell to be transformed and/or the type of protein to be expressed. Also useful for various applications are tissue-selective (i.e., tissue- specific) promoters, i.e., promoters from which expression occurs preferentially in cells or a particular kind of tissue, compared to one or more other types of tissue. For example, chicken oviduct-specific promoters naturally associated with the proteins of avian egg whites including, but not limited to, lysozyme, ovomucoid, albumin, conalbumin, and ovotransferrin may be used.

The term "recombinant cell" refers to a cell that has a new combination of nucleic acid segments that are not covalently linked to each other in nature in that particular configuration. A new combination of nucleic acid segments can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art. A recombinant cell can be a single eukaryotic cell, or a single prokaryotic cell, or a mammalian cell. The recombinant cell may harbor a vector that is extragenomic. An extragenomic nucleic acid vector does not insert into the cell's genome. A recombinant cell can further harbor a vector or a portion thereof (e.g. , the portion containing the regulatory sequences and the coding sequence) that is intragenomic. The term "intragenomic" defines a nucleic acid construct incorporated within the recombinant cell's genome. The terms "recombinant nucleic acid" and "recombinant DNA" as used herein refer to a combination of at least two nucleic acid sequences that is not naturally found in a eukaryotic or prokaryotic cell in that particular configuration. The nucleic acid sequences may include, but are not limited to, nucleic acid vectors, gene expression regulatory elements, origins of replication, sequences that when expressed confer antibiotic resistance, and protein-encoding sequences. The term "recombinant polypeptide" is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location, purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature. As used herein, the term "transgene" refers to a nucleic acid sequence (encoding, for example, a human interferon polypeptide) that is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene also includes a regulatory sequence designed to be inserted into the genome such that it regulates the expression of an endogenous coding sequence, e.g., to increase expression and or to change the timing and or tissue specificity of expression, etc. (e.g., to effect "gene activation"). The term "transgenic animal" as used herein refers to an animal, including an avian species such as a chicken, in which one or more of the cells of the animal contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art or by the methods of the present invention. As used herein, a "transgenic avian" is any avian species, including but not limited to, chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary, in which one or more of the cells of the avian may contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques known in the art, and particularly, as described herein. The nucleic acid is introduced into a cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical crossbreeding, or in vitro fertilization (although it does include fertilization with sperm into which a transgene has been introduced, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the typical transgenic avian, the transgene causes cells to express a recombinant form of the subject polypeptide, e.g. either agonistic or antagonistic forms, or a form in which the gene has been disrupted.

The terms "chimeric animal" or "mosaic animal" are used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal. The term "tissue-specific chimeric animal" indicates that the polypeptide encoding gene is present and expressed in some tissues, but not others.

The term "knock-in animal" refers to an animal that carries a specific nucleic acid sequence such as a "knock-in sequence" in a predetermined coding or noncoding region, wherein the knock-in sequence is introduced through methods of recombination, such as homologous recombination. The recombination event comprises replacing all or part of a gene of the animal by a functional homologous gene or gene segment of another animal, where the respective knock-in sequence is placed in the genomic sequence.

The term "chromosomal positional effect (CPE)" as used herein refers to the variation in the degree of gene transcription as a function of the location of the transcribed locus within the cell genome. Random transgenesis may result in a transgene being inserted at different locations in the genome so that individual cells of a population of transgenic cells may each have at least one transgene, each at a different location and therefore each in a different genetic environment. Each cell, therefore, may express the transgene at a level specific for that particular cell and dependant upon the immediate genetic environment of the transgene. In a transgenic avian, as a consequence, different tissues may exhibit different levels of transgene expression.

The term "cytokine" as used herein refers to any secreted polypeptide that affects the functions of cells and is a molecule that modulates interactions between cells in the immune, inflammatory or hematopoietic response. A cytokine includes, but is not limited to, monokines and lymphokines regardless of which cells produce them. For instance, a monokine is generally referred to as being produced and secreted by a mononuclear cell, such as a macrophage and/or monocyte. Many other cells however also produce monokines, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes and B-lymphocytes.

Lymphokines are generally referred to as being produced by lymphocyte cells. Examples of cytokines include, but are not limited to, Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor Necrosis Factor-alpha (TNF-alpha) and Tumor Necrosis Factor β (TNF-β). The term "antibody" as used herein refers to polyclonal and monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof. The term "antibody" refers to a homogeneous molecular entity, or a mixture such as a polyclonal serum product made up of a plurality of different molecular entities, and may further comprise any modified or derivatised variant thereof that retains the ability to specifically bind an epitope. A monoclonal antibody is capable of selectively binding to a target antigen or epitope.

Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, disulfide-linked Fvs (sdFv) fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, intrabodies, synthetic antibodies, and epitope-binding fragments of any of the above.

The term "immunoglobulin polypeptide" as used herein refers to a polypeptide derived from a constituent polypeptide of an immunoglobulin. An "immunoglobulin polypeptide" may be, but is not limited to, an immunoglobulin (preferably an antibody) heavy or light chain and may include a variable region, a diversity region, joining region and a constant region or any combination, variant or truncated form thereof. The term

"immunoglobulin polypeptide" further includes single-chain antibodies comprised of, but not limited to, an immunoglobulin heavy chain variable region, an immunoglobulin light chain variable region and optionally a peptide linker.

The term "origin of replication" (ori) as used herein refers to unique regions of a nucleic acid sequence containing multiple short repeated sequences, recognized by multimeric origin of replication binding proteins that organize the assembly of multiple enzymes and proteins required for the replication of the nucleic acid. The origin of replication derived from E. coli may be included in a plasmid for replication of the plasmid in a bacterial host. The SV40 viral ori is a 65 bp region derived from the SV40 viral chromosome that when included in a nucleic acid sequence will allow replication of the nucleic acid in an animal cell. Inclusion of the SN40 ori region in a plasmid that also has the E. coli ori element will allow the plasmid to be replicated in both a bacterial host and in an animal cell.

The term "centromere" as used herein refers to a small, specialized region of a chromosome recognized as a constriction in a condensed chromosome. A kinetochore lies within the centromeric region and is attached to microtubules extending to the poles of a dividing cell.

The term "telomere" as used herein refers to repetitive oligomeric nucleic acid sequences located at the ends of linear eukaryotic chromosomes. Telomeres are required to prevent shortening of chromosomal DΝA during replication of the linear nucleic acid.

Recombinant expression vectors can be designed for the expression of the encoded proteins eukaryotic cells. Useful vectors may comprise constitutive or inducible promoters to direct expression of either fusion or non-fusion proteins. With fusion vectors, a number of amino acids are usually added to the expressed target gene sequence such as, but not limited to, a protein sequence for thioredoxin. A proteolytic cleavage site may further be introduced at a site between the target recombinant protein and the fusion sequence. Additionally, a region of amino acids, such as a polymeric histidine region, may be introduced to allow binding of the fusion protein to metallic ions such as nickel bonded to a solid support, and thereby allow purification of the fusion protein. Once the fusion protein has been purified, the cleavage site allows the target recombinant protein to be separated from the fusion sequence. Enzymes suitable for use in cleaving the proteolytic cleavage site include, but are not limited to, Factor Xa and thrombin. Fusion expression vectors that may be useful in the present invention include pGex (Amrad Corp., Melbourne, Australia), pRIT5 (Pharmacia, Piscataway, ΝJ) and pMAL (New England Biolabs, Beverly, MA), that fuse glutathione S-transferase, protein A, or maltose E binding protein, respectively, to the target recombinant protein.

Expression of a foreign gene can be obtained using eukaryotic host cells such as, but not limited to, mammalian or avian cells. The use of eukaryotic host cells permit partial or complete post-translational modification such as, but not only, glycosylation and/or the formation of the relevant inter- or intra-chain disulfide bonds. Examples of vectors useful for expression in the chicken Gallus gallus include pYepSecl as in Baldari et al , E.M.B.O.J. 6, 229-234 (1987) and pYES2 (Invitrogen Corp., San Diego, CA), incorporated herein by reference in their entireties. Once the isolated DNA molecule of the present invention has been cloned into an expression system, it is ready to be incorporated into a host cell.

The terms "transformation" and "transfection" as used herein refer to the process of inserting a nucleic acid into a cell. Many techniques are well known to those skilled in the art to facilitate transformation or transfection of a nucleic acid into a prokaryotic or eukaryotic organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt such as, but not only, a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection, to render the host cell competent for the uptake of the nucleic acid molecules, and by such methods as sperm-mediated and restriction-mediated integration.

The term "transfecting agent" as used herein refers to a composition of matter added to the genetic material for enhancing the uptake of heterologous DNA segment(s) into a eukaryotic cell, preferably an avian cell, and more preferably a chicken male germ cell. The enhancement is measured relative to the uptake in the absence of the transfecting agent. Examples of transfecting agents include adenovirus-transferrin-polylysine-DNA complexes. These complexes generally augment the uptake of DNA into the cell and reduce its breakdown during its passage through the cytoplasm to the nucleus of the cell. These complexes can be targeted to the male germ cells using specific ligands that are recognized by receptors on the cell surface of the germ cell, such as the c-kit ligand or modifications thereof.

Other preferred transfecting agents include, but are not limited to> lipofectin, lipfectamine, DIMRIE C, Supeffect, and Effectin (Qiagen), unifectin, maxifectin, DOTMA, DOGS (Transfectam; dioctadecylamidoglycylspermine), DOPE (l,2-dioleoyl-sn-glycero-3- phosphoethanolamine), DOTAP (l,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyl dioctadecytammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N- dihydroxyethyl ammonium bromide), HDEAB (N-n-hexadecylN,N- dihydroxyethylammonium bromide), polybrene, or poly(ethylenimine) (PEI). These nonviral agents have the advantage that they can facilitate stable integration of xenogeneic DNA sequences into the vertebrate genome, without size restrictions commonly associated with virus-derived transfecting agents.

The terms "intracytoplasmic sperm injection" and "ICSI" as used herein refer to delivering an exogenous nucleic acid to a recipient cell by associating the exogenous nucleic acid with the head of a sperm cell and then delivering the sperm cell head to the recipient cell by microinjection. The exogenous nucleic acid may be integrated into the endogenous genomic nucleic acid of the sperm, non-integrated as an episomal element of the nucleic acid complement of the sperm head, or linked internally or externally to the head of the sperm. The terms "chlCSI" and "CHICSI™" as used herein refer to intracytoplasmic sperm injection into a chicken cell.

The terms "sub-zonal injection" and "SUZI" refer to delivering viable spermatozoa to an oocyte by microinjection, wherein the sperm are microinjected between the zona pellucida and the cytoplasmic membrane of an oocyte. The term "gene delivery (or transfection) mixture" as used herein, in the context of the methods of sperm mediated transfer described herein, refers to selected genetic material in an appropriate vector mixed, for example, with an effective amount of lipid transfecting agent, for example, a cationic or polycationic lipid, such as polybrene. The amount of each component of the mixture is chosen so that the genetic modification, e.g., by transfection or transduction, of a specific species of male germ cell is optimized. Such optimization requires no more than routine experimentation. The ratio of DNA to lipid is broad, preferably about 1:1, although other proportions can also be utilized depending on the type of lipid transfecting agent used.

This application uses gene nomenclature accepted by the Cucurbit Genetics Cooperative as it appears in the Cucurbit Genetics Cooperative Report 18:85 (1995); herein incorporated by reference in its entirety. Using this gene nomenclature, genes are symbolized by italicized Roman letters. If a mutant gene is recessive to the normal type, then the symbol and name of the mutant gene appear in italicized lower case letters.

3.2 ABBREVIATIONS

Abbreviations used in the present specification include the following: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; mRNA, messenger RNA; tRNA, transfer RNA; nt, nucleotide(s); SSC, sodium chloride-sodium citrate; MAR, matrix attachment region; DMSO, dimethyl sulfoxide; TPLSM, two photon laser scanning microscopy; REMI, restriction enzyme mediated integration; WEFs, whole embryo fibroblasts.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 A-E illustrate the nucleotide sequence (SEQ ID NO: 6) comprising the chicken lysozyme gene expression control region (SEQ ID NO: 7), the nucleotide sequence encoding the chicken expression optimized human interferon α2b (IFNMAGMAX; SEQ ID NO: 5) and a SN40 polyadenylation signal sequence (SEQ ID NO: 8).

FIG. 2 illustrates the nucleotide sequence SEQ ID NO: 5 encoding the chicken expression optimized human interferon α2b (IFNMAGMAX).

FIGS. 3A-E illustrate the nucleotide sequence SEQ ID NO: 7 encoding the chicken lysozyme gene expression control region.

FIG. 4 illustrates the nucleotide sequence SEQ ID NO: 8 encoding the SV40 polyadenylation signal sequence.

FIGS. 5 A-C illustrate the nucleotide sequence SEQ ID NO: 9 encoding the chicken lysozyme 3' domain.

FIGS. 6A-J illustrate the nucleotide sequence SEQ ID NO: 10 encoding the lysozyme gene expression control region (SEQ ID NO: 7) linked to the insert having the nucleotide sequence of SEQ ID NO: 5 encoding the chicken expression-optimized human interferon α2b (IFNMAGMAX) and the chicken lysozyme 3' domain SEQ ID NO: 9.

FIG. 7 illustrates the nucleotide sequence SEQ ID NO: 11, of the combinatorial promoter MDOT.

FIGS. 8A-B illustrate the oligonucleotides and primers (SEQ ID NOS: 14-31) used in the formation of the chicken codon optimized human interferon α2b-encoding nucleic acid.

FIG. 9 illustrates the primers (SEQ ID NOS: 32-35) used in the synthesis of the MDOT promoter.

FIG. 10 illustrates the level of human monoclonal antibodies IgG expressed in the serum of transgenic chick using ELISA.

FIG. 11 illustrates the detection of EGFP positive bands from transgenic sperm. 5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of introducing nucleic acids into avian oocytes by sperm-mediated transfection to produce a transgenic chicken or quail, or other avian species, carrying the transgene in the genetic material in all or most of its tissue, including germ-line tissue. The methods and vectors of the present invention further generate transgenic avians that express heterologous genes in the serum of the avian and/or are deposited into an avian egg, preferably in the egg white. Vectors containing promoters that direct high level of expression of the heterologous protein in the avian, particularly in the magnum for deposition into the avian egg are provided. Additional regulatory elements, such as MARs, IRES's, enhancers, polyadenlyation signals, etc., may be included in the vectors of the invention to improve expression and efficiency.

5.1 METHODS OF TRANSGENESIS 5.1.1 SPERM-MEDIATED INTEGRATION OF HETEROLOGOUS TRANSGENES

The transgenic avians of the present invention are most preferably generated using sperm-mediated transfection of nucleic acid into avian oocytes. Specifically, the present invention provides methods for introducing nucleic acids containing a transgene, preferably, a nucleic acid vector of the invention as described in Section 5.2, infra, into an avian oocyte by sperm-mediated transfection. In preferred embodiments, the nucleic acid is first introduced into an avian sperm in vitro by lipofection, electroporation, restriction enzyme mediated integration (REMI) or similar methods, or in vivo by microinjection into the testis, and the modified avian sperm is then delivered to an avian oocyte by natural coitus after the modified avian sperm are returned to the testis of a male avian or in the method in which the nucleic acid has been injected directly into the testis or in vitro by microinjection, intracytoplasmicosperm injection (ICSI) or artificial insemination of oocytes isolated from an ovulating female bird, thereby generating a transgenic zygote and chick. In certain embodiments, the male germ cells are irradiated, more preferably irradiated by gamma rays, before the heterologous nucleic acid is incorporated therein. In other embodiments, the testis is depopulated of sperm prior to introduction of the transfected sperm.

The present invention contemplates that any technique capable of transferring heterologous material into sperm could be used so long as the technique preserves enough of the sperm's fertilization functions, such that the resultant sperm will be able to fertilize the oocyte. It is understood that the heterologous nucleic acid may be integrated into the genome of a recipient cell such as a spermatogonial cell or a spermatogonial precursor cell for subsequent transfer to an embryo or the testicular material of the recipient male animal, preferably a chicken. It is further understood that the heterologous nucleic acid may not be integrated into the genome of the recipient cell but delivered as an episome which may or may not be integrated into the genome of the resulting zygote or chick.

5.1.1.1 PREPARATION OFTRANSGENIC CONSTRUCT

One aspect of the present invention relates to the preparation of a transgene which is to be incorporated into the genome of an avian sperm. In certain embodiments, the transgene comprises at least one heterologous nucleic acid. It is contemplated to be within the scope of the present invention for the heterologous nucleic acid to comprise an expression vector such as, but not limited to, viral vectors, plasmid vectors, or linearized nucleic acid vectors or a combination thereof. (See section 5.2, infra, for details on vectors, and the preparation thereof). The expression vector may particularly be any suitable nonviral vector including plasmid DNA, bacteria artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc. The expression vector may also be any suitable viral vector, for example, retroviral vectors, adenoviral vectors, transferrin-polylysine enhanced adenoviral vectors, human immunodeficiency virus vectors, lentiviral vectors, Moloney murine leukemia virus-derived vectors, and virus-derived DNAs that facilitate polynucleotide uptake by and release into the cytoplasm of germs cells. Transcriptional promoters of an expression vector of the present invention may be a constitutively active promoter such as the cytomegaloviral promoter or Rous sarcoma virus promoter, or a tissue-specific promoter, preferably a tissue-specific promoter operable in oviduct cells of an avian species including, but not limited to, the promoters of the genes encoding ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin. Optionally, the transcriptional promoter of an expression vector may be a regulatable promoter. The expression vector may further comprise a region encoding a transcriptional terminator, such as a bovine growth hormone transcriptional terminator.

In preferred embodiments, a transgene construct comprises at least two separate or independent elements. A first element could comprise an oviduct-specific promoter, such as, but not limited to ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin, which would drive expression of a gene coding for a protein of interest in the oviduct. A second element can be located either upstream or downstream for the first element and comprises a protamine promoter, or a segment thereof that is sufficient to drive the expression of a marker gene such as the Green Fluorescent Protein (GFP) to facilitate identification of transfected sperm. In one embodiment of the present invention, the heterologous nucleic acid comprises cohesive ends characterized as capable of hybridizing to cohesive ends generated by a restriction endonuclease. The cohesive ends on the nucleic acid may be generated by restriction endonuclease cleavage of a circular or linear nucleic acid, by the chemical addition of nucleotides to the ends of a linear nucleic acid, or by a combination of chemical and enzymatic methods.

In another embodiment of the present invention, the heterologous nucleic acid is linearized and has at least one blunt end. The blunt end of the nucleic acid may be generated, by an exonuclease digestion of cohesive ends, such as SI nuclease. In the methods of generating transgenic cells according to the present invention, the genomic nucleic acid of the recipient cell, male germ cell or oocyte can be cleaved to receive the integrating heterologous nucleic acid. Any method may be selected that will generate limited, random cleavage that will allow integration of the heterologous nucleic acid into the genome of the recipient cell or oocyte. When the integrating heterologous nucleic acid has cohesive ends, the recipient genomic nucleic acid may be cleaved with a restriction endonuclease generating cohesive ends capable of hybridizing to the cohesive ends of the heterologous nucleic end. When the heterologous nucleic acid has blunt ends, the genomic nucleic acid can be cleaved by any method that will generate blunt ends at the cleavage site, including restriction endonuclease cleavage, or irradiation of the cell with high-energy irradiation. Suitable radiations that may be applied to the methods of the present invention include, for example, gamma rays, x-rays, ultraviolet light or ultrasound. It is contemplated that the cleavage of genomic nucleic acid and integration of a heterologous nucleic acid therein will result in a viable recipient cell that can be used to fertilize an avian oocyte, or will not yield a viable cell. A non-viable sperm cell may, however, be used to deliver the transgene to an oocyte using, for example, the ICSI (CHICSI™) method.

The heterologous nucleic acid of the present invention may further comprise a centromere element and at least one telomere element. In one embodiment, the centromere and the at least one telomeres are derived from the chicken. While the ori site alone will allow replication of the heterologous nucleic acid when transfected into an oocyte or zygote thereof, segregation of the replicates into each daughter cell will require the optional centromeric element. In the absence of this centromeric element, segregation will be random between daughter cells with some daughter cells not receiving one copy of the transgenic nucleic acid. A mosaic transgenic animal would, therefore, result. In one embodiment of the present invention, therefore, the heterologous nucleic acid is an artificial chromosome comprising a heterologous transgenic element having the properties desired to be expressed by a transgenic animal, an origin of replication (ori) site, and a centromere. In this embodiment, the heterologous nucleic acid may be a circular nucleic acid or a linear nucleic acid. In another embodiment, the heterologous nucleic acid is a linear nucleic acid further comprising telomeres.

In another aspect of the methods according to the present invention, the transgenic oocyte or ovum of the present invention is incubated for development of the zygote therein to a fetus, and subsequently to a chick for hatching. In one embodiment of the present invention, therefore, the zygote is incubated in a surrogate avian female, wherein the method comprises the steps of fistulating an avian female, delivering the avian oocyte to the infundibulum of the female bird, allowing the avian female to incubate the avian oocyte to an embryo within an egg, allowing the avian female to lay the egg, and allowing the embryo to hatch as a viable chick, wherein the chick is a transgenic chick having an exogenous nucleic acid incorporated therein.

5.1.1.2 SPERM TRANSGENESIS

The heterologous nucleic acid may be delivered to an avian male germ cell (i.e., sperm, spermatozoon cell or a precursor cell) by a method such as by contacting the male germ cell with a gene delivery mixture comprising a nucleic acid, either a eukaryotic viral vector or a vector that is not derived from a eukaryotic virus, at about or below the avian' s body temperature and for an effective period of time such that the nucleic acid is incorporated into the cell, and preferably into the genome of the cell, optionally isolating or selecting the genetically modified cell with the aid of a genetic selection marker expressed in the genetically modified cell, transferring the isolated or selected genetically modified germ cell to a testis of a recipient male avian such that the cell lodges in a seminiferous tubule of the testis. A genetically modified male gamete may be produced therein, and breeding the recipient male avian with a female avian of its species will generate transgenic progeny that carry the heterologous transgenic nucleic acid in its genome. I certain embodiments, the avian male germ cells are isolated and removed from a male avian. The avian male germ cells is then transfected by introducing the heterologous nucleic acid into the genome of the avian male germ cells by lipofection, electroporation, restriction enzyme mediated infection (REMI) or similar methods. In certain other embodiments, the heterologous nucleic acid is injected directly into the testis of the male avian for transfection. Male germ cells can be extracted to determine whether transfection has occurred or the extent of transfection. The male avian can be mated with a female avian to produce transgenic offsprings or the sperm can be used for INF.

The precursor cell may be selected from the group consisting of spermatogonial stem cells, type B spermatogonia, primary spermatocytes, preleptotene spermatocytes, leptotene spermatocytes, zygotene spermatocytes, pachytene spermatocytes, secondary spermatocytes, and spermatids. The embodiment further comprises the steps of incorporating the heterologous transgene into the genome of the spermatozoon cell or the precursor cell, so that a genetically modified male gamete is produced by the male avian, and breeding the male avian with a female of the same species such that a transgenic progeny is thereby produced that carries the polynucleotide in its genome.

In certain embodiments, the heterologous genetic material may be introduced into the genome of an avian male germ cell, such that a polynucleotide is delivered using known gene delivery systems to male germ cells in situ in the testis of the male avian (e.g., by in vivo transfection or transduction). In one embodiment, the invention relates to an in vitro method of incorporating heterologous genetic material into the genome of a male avian by isolating male germ cells ex corpora, delivering a polynucleotide thereto, and then returning the transfected cells to the testes of a recipient male bird. In yet another embodiment, the in vitro method involves microinjecting the recombinant male germ cells into a recipient fertilized oocyte, whereupon the sperm head enters the oocyte nucleus to deliver the heterologous nucleic acid thereto.

In a preferred embodiment, the invention relates to an in vivo method that injects a gene delivery mixture, preferably into the seminiferous tubules, or into the testis, and most preferably into the vas efferens or vasa efferentia using, for example, a micropipette and a picopump delivering a precise measured volume under controlled amounts of pressure. The modified germ cells differentiate in their own milieu. Progeny animals exhibiting the nucleic acid's integration into its germ cells (i.e., transgenic animals) are selected. The selected progeny can then be mated, or their sperm utilized for insemination or in vitro fertilization, to produce further generations of transgenic progeny or for microinjection into isolated oocytes. In another preferred embodiment, the invention relates to an in vitro method wherein male germ cells are obtained or collected from a donor male avian, by any means known in the art such as, for example, transection of the testes. The male germ cells are then exposed to a gene delivery mixture, preferably within several hours of collection, or cryopreserved for later use. When the male germ cells are obtained from the donor avian by transection of the testes, the cells can be incubated in an enzyme mixture known for gently breaking up the tissue matrix and releasing undamaged cells. Suitable enzymes to disrupt the integrity of a tissue include, but are not limited to, pancreatic trypsin, collagenase type I, pancreatic DNAse type I, as well as bovine serum albumin and a modified DMEM medium. After washing the cells, they can be placed in an incubation medium such as DMEM or the like, and plated on a culture dish for genetic modification by exposure to a gene delivery mixture. In other embodiments, a transgene can be incorporated into an avian sperm by lipofection, electroporation, restriction enzyme mediated integration (REMI), intracytoplasmic sperm injection (ICSI) or similar methods.

Liposome

In a preferred embodiment, a transgene is incorporated into an avian sperm by liposomes. The male germ cells, which may be intact and viable spermatozoa, or the non- viable heads thereof, may be transfer to a recipient oocyte using liposome-mediated delivery. PCT Publication WO 87/05325, which is incorporated by reference herein in its entirety, discloses a method of transferring organic and/or inorganic material into sperm or egg cells by using liposomes. The heterologous nucleic acid can also be incorporated into a male sperm using Lipofectin-based liposomes. (See, e.g., Bachiller et al, 1991, Mol. Reprod. Develop. 30: 194-200; Nakanishi and Iritani, 1993, Mol. Reprod. Develop. 36: 258- 261).

Electroporation

In another preferred embodiment, a transgene is incorporated into an avian sperm by electroporation. The application of electrical current has been shown to enhance the uptake of exogenous DNA fragments by cultured cells. Enhancement of nuclear uptake of the heterologous DNA will promote earlier chromosomal integration of the exogenous DNA molecules, thus reducing the degree of genetic mosaicism observed in transgenic avian founders.

In one embodiment, the male germ cells is placed in a cuvette and a solution of the transgenic nucleic acid coding the protein of interest is added. A direct current pulse is discharged in the cuvette suspension. The current pulse creates temporary, short-lived pores in the cell membrane and allow the male germ cells to take up the transgene while only slightly compromising cell viability. More description on the use of electroporation to incorporate DNA can be found in Gagne et al, 1991, Mol. Reprod. Develop. 29: 6-15, which is incorporated herein by reference in its entirety. Restriction Enzyme Mediated Integration (REMI)

In yet another preferred embodiment, a transgene is incorporated into an avian sperm by restriction enzyme mediated integration (REMI). The heterologous nucleic acid to be integrated into, for example, the sperm nuclear DNA is converted to a linear double stranded DNA possessing single-stranded cohesive ends by contacting the heterologous DNA with a type II restriction enzyme that upon scission, generates such ends. The nucleic acid to be cut can be a circular nucleic acid such as in a plasmid or a viral vector or a linear nucleic acid that possesses at least one recognition and cutting site outside of the genes or regulatory regions critical to the desired post-integration function of the nucleic acid, and no recognition and cutting sites within the critical regions.

Alternatively the heterologous DNA to be integrated into the sperm nuclear DNA can be prepared by chemically and/or enzymatically adding cohesive ends to a linear DNA. The added cohesive ends must be able to hybridize to the cohesive ends characteristic of a nucleic acid cleaved by a type II restriction endonuclease. Alternatively, the cohesive ends can be added by combining the methods based on type II restriction enzyme cutting and chemical and/or enzymatic addition. It is also within the scope of the present invention for the linearized nucleic acid to have one end that is a blunt end without unpaired nucleotides. Such blunt ends can be generated by restriction endonuclease digestion, exonuclease digestion of cohesive ends or fill-in of cohesive ends by polynucleotide synthesis, using methods as described, for example, in Sambrook et al, (supra), incorporated herein by reference in its entirety.

It is also to be understood that a nucleic acid to be delivered to a recipient cell may be cleaved with two different restriction endonucleases that may generate the same or different cohesive termini, or at least one blunt-end terminus. Neither restriction endonucleases will have a recognition site within the nucleic acid sequence required to be a transgene in the recipient cell.

When a restriction endonuclease is used to cleave the genomic nucleic acid of the recipient cell, the endonuclease may be co-delivered to the recipient cell such as a sperm cell with the heterologous nucleic acid, or sequentially delivered. If a nucleic acid is cleaved with at least two restriction endonucleases, thereby generating at least one cohesive terminus, the at least two endonucleases may be delivered to a recipient cell either together or sequentially. The transfected nucleic acid may be mixed with at least one of the endonucleases or delivered to a recipient cell before or after at least one endonuclease is delivered thereto. At least one terminus of a linearized nucleic acid to be delivered to a recipient cell may be a blunt end terminus, generated by endonuclease cleavage, chemical synthesis, enzyme directed nucleic acid digestion or synthesis, or any combination thereof. A recipient cell genome such as a sperm cell genome, may therefore be cleaved before, during or after delivery of the linearized nucleic acid to the cell, by delivery of a blunt-end generating restriction endonuclease to the recipient cell, or by radiation-induced cleavage. Suitable radiations that may be applied to, for example, a sperm cell include, but are not limited to, gamma radiation, x-rays, ultraviolet light and ultrasound. The dose and duration of the radiation applied to a cell sample are determined for each sample, for levels of cleavage that will allow integration of the transfected nucleic acid into the cell genome, while maintaining viability of the cells for use in artificial insemination or recolonization of an avian testes. Viability of a recipient sperm may not be required when the transfected sperm are delivered to a recipient avian oocyte by such procedures as ICSI or CHICSI™. Cleavage of the genomic nucleic acid by irradiation or ultrasound can be either before, during or after delivery of the heterologous nucleic acid to the recipient cell.

While not wishing to be bound by any one theory, the transfected nucleic acid may be integrated into a cleavage site of the genomic nucleic acid. Integration may be facilitated by the cohesive ends on the heterologous nucleic acid that hybridize to the like cohesive ends of the cleaved genomic nucleic acid. The integrated heterologous nucleic acid will then replicate and segregate with the genome of the recipient cell.

Alternatively, the heterologous nucleic acid may not be integrated into a recipient genome, but will remain as an extrachromosomal episome. The heterologous nucleic acid of the present invention may circularize by hybridization of the cohesive ends of the nucleic acid, rather than be integrated into the genome. When the heterologous nucleic acid comprises any natural or synthetic origin of replication (ori element) the nucleic acid will be capable of replicating independently of the recipient genome. In one embodiment of the present invention the ori site included with a heterologous nucleic acid is derived from the SN40 virus. Episomal replication and segregation of daughter copies of the episome is facilitated by the linearized viral ori site and/or a centromere isolated from, for example, a chicken chromosome, thereby generating a chicken artificial chromosome. In another embodiment, the linearized heterologous nucleic acid will not be integrated into the genome of the recipient cell but remain as a separate unit that, because of a centromeric structure incorporated therein, will segregate into daughter cells during mitotic division. In this case, the unincorporated episomal heterologous nucleic acid is a chicken artificial chromosome (CAC). The REMI method for stably integrating heterologous DNA into the genomic DNA of a recipient cell is described by Shemesh et al. in PCT Publication No. WO 99/42569 and incorporated herein by reference in its entirety. This REMI method comprises in part an adaptation of the REMI technique disclosed by Schiest and Petes (Proc. Nat. Acad. Sci. U.S.A. 88, 7585-7589 (1991)) and Kuspa and Loomis (Proc. Nat. Acad. Sci. U.S.A., 89, 8803-8807 (1992)) both incorporated herein by reference in their entireties.

In preferred embodiments, the avian sperm are irradiated before being exposed to gene delivery mixture or having a transgene incorporated therein. The male germ cells can be irradiated with a suitable dose of gamma irradiation, preferably, 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy, 12 Gy, 15 Gy or 20 Gy, without compromising the viability and/or mobility of the sperms. (See Wooster et al., 1977, Can. J. Genet. Cytol. 19: 437-446).

Whether employed in the in vivo, in situ or in vitro method, the gene delivery mixture, once in contact with the male germ cells, facilitates the uptake and transport of heterologous genetic material into the appropriate cell location for integration into the genome and expression. A number of known gene delivery methods can be used for the uptake of nucleic acid sequences into the cell and facilitate the integration of the heterologous nucleic acid into the genome of the recipient cell. Such methods include, but are not limited to viral vectors, liposomes, electroporation, REMI, and ICSI. A gene delivery mixture suitable for use in the in vivo, in situ or in vitro methods of sperm-mediated transfection comprises a nucleic acid encoding a desired trait or product, and a suitable promoter sequence such as, for example, a tissue-specific promoter, or an IRES. The transgenic nucleic acids of the present invention may further comprise an origin of replication. For example, an origin of replication may be the SV40 ori, or a centromere derived from the chicken. A linear nucleic acid may further comprise a telomere at one or both ends of the nucleic acid.

Optionally, agents that increase the uptake of, or comprise non-eukaryotic viral vectors, e.g., plasmids, BACs, YACs, etc., the nucleic acid sequence, such as liposomes, retroviral vectors, adenoviral vectors, adenovirus enhanced gene delivery systems, or combinations thereof may be included in the gene delivery mixture. A reporter construct, including a genetic selection marker, such as the gene encoding for Green Fluorescent Protein, may also be added to the gene delivery mixture. Targeting molecules, such as c-kit ligand, can be added to the gene delivery mixture to enhance the transfer of genetic material into the male germ cell. An immunosuppressing agent, such as cyclosporin or a corticosteroid may also be added to the gene delivery mixture as known in the art. Any of a number of commercially available gene delivery mixtures can be used, to which the polynucleotide encoding a desire trait or product is further admixed. The final gene delivery mixture comprising the polynucleotide can then be admixed with the male gamete cells and allowed to interact for a period of between about 2 hours to about 16 hours, at a temperature of about 33 ° C to about 37 ° C . After this period, the cells are preferably placed at a lower temperature of about 33 °C to about 34 °C, for about 4 hours to about 20 hours, preferably about 16 to about 18 hrs.

Isolating and/or selecting genetically transgenic germ cells (and transgenic somatic cells, and of transgenic vertebrates) is by any suitable means, such as, but not limited to, physiological and/or morphological phenotypes of interest using any suitable means, such as biochemical, enzymatic, immunochemical, histologic, electrophysiologic, biometric or like methods, and analysis of cellular nucleic acids, for example the presence or absence of specific DNAs or RNAs of interest using conventional molecular biological techniques, including hybridization analysis, nucleic acid amplification including, but not limited to, polymerase chain reaction, transcription-mediated amplification, reverse transcriptase- mediated ligase chain reaction, and/or electrophoretic technologies.

One preferred method of isolating or selecting male germ cell populations comprises obtaining specific male germ cell populations, such as spermatogonia, from a mixed population of testicular cells by extrusion of the cells from the seminiferous tubules and enzyme digestion. The spermatogonia, or other male germ cell populations, can be isolated from a mixed cell population by methods such as the utilization of a promoter sequence that is specifically or selectively active in cycling male germ line stem cell populations. Suitable promoters include B-Myb or a specific promoter, such as the c-kit promoter region, c-raf-1 promoter, ATM (ataxia-telangiectasia) promoter, vasa promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, cyclin Al promoter, or FRMI (from Fragile X site) promoter and the like. A selected promoter may be linked to a reporter construct, for example, a construct comprising a gene encoding Green Fluorescent Protein (or EGFP), Yellow Fluorescent Protein, Blue Fluorescent Protein, a phycobiliprotein, such as phycoerythrin or phycocyanin, or any other protein which fluoresces under suitable wave-lengths of light, or encoding a light-emitting protein, such as luciferase or apoaequorin. The unique promoter sequences drive the expression of the reporter construct only during specific stages of male germ cell development (e.g., Mailer et al, 1999, J. Biol. Chem. 276(16), 11220-28; Schrans-Stassen et al, 1999, Endocrinology 140, 5894-5900, both of which are incorporated herein by reference in their entireties). In the case of a fluorescent reporter construct, the cells can be sorted with the aid of, for example, a FACS set at the appropriate wavelength(s), or they can be selected by chemical methods.

Male germ cells that have the DNA modified in the desired manner are isolated or selected, and transferred to the testis of a suitable recipient avian, preferably the donor male avian of the male germ cells. Further selection can be attempted after biopsy of one or both of the recipient male's testes, or after examination of the animal's ejaculate amplified by the polymerase chain reaction to confirm that the desired nucleic acid sequence had been incorporated.

The genetically modified germ cells isolated or selected as described above are transferred to a testis of a suitable male avian, preferably a chicken, that can be, but need not be, the same donor animal. Before transferring the genetically modified male germ cells to the recipient animal, the testes of the recipient are depopulated of endogenous germ cells, thereby facilitating the colonization of the recipient testis by the genetically modified germ cells. Depopulation of the testis has commonly been accomplished by exposing the whole animal to gamma irradiation or by localized irradiation of the testis. The basic rigid architecture of the gonad should not be destroyed, nor significantly damaged. Disruption of tubules may lead to impaired transport of testicular sperm and result in infertility. Sertoli cells should not be irreversibly damaged, as they provide a base for development of the germ cells during maturation, and for preventing the host immune defense system from destroying grafted foreign spermatogonia.

Suitable denuding methods, include irradiation by gamma-rays, x-rays, ultrasound, ultraviolet light, by chemical treatment, by means of infectious agents such as viruses, or by autoimmune depletion or by combinations thereof, preferably by a combined treatment of the vertebrate with an alkylating agent and gamma irradiation as taught in WO 00/69257, incorporated herein by reference in its entirety.

Gamma radiation-induced spermatogonial degeneration probably related to the process of apoptosis. (Hasegawa et α/. , 1998, Radiat. Res. 149: 263-70). Alternatively, a composition containing an alkylating agent such as busulfan (MYLERAN™) can be used to depopulate. (Jiang F.X., 1998, Anat. Embryol. 198(1): 53-61; Russell and Brinster, 1996, J. Androl. 17(6): 615-27; Boujrad et al, 1995, Andrologia 27(4): 223-28; Linder et al, 1992, Reprod. Toxicol. 6(6): 491-505; Kasuga and Takahashi, 1986, Endocrinol. Jpn 33( 1): 105- 15). Other cytotoxic alkylating agent, may be, but is not limited to, chlorambucil, cyclophosphamide, melphalan, or ethyl ethanesulfonic acid, and may be combined with gamma irradiation, to be administered in either sequence. The dose of the alkylating agent and the dose of gamma radiation are in an amount sufficient to substantially depopulate the testis. The alkylating agent can be administered by any pharmaceutically acceptable delivery system, including but not limited to, intraperitoneal, intravenous, or intramuscular injection, intravenous drip, implant, transdermal or transmucosal delivery systems.

The isolated or selected genetically modified germ cells are transferred into the recipient testis by direct injection using a suitable micropipette. Support cells, such as Leydig or Sertoli cells, that can be unmodified or genetically modified, can be transferred to a recipient testis along with the modified germ cells.

5.1.1.3 DELIVERY OF TRANSGENIC SPERM TO OOCYTES

The transfected male avian germ cells may be used to deliver a heterologous nucleic acid to an avian oocyte by implanting the transfected male germ cells such as transfected spermatogonial precursor cells, into the testicular tissue of host male birds previously denuded of viable spermatogonial cells or sperm. The implanted transfected male avian germ cells may colonize the testicular tissue, proliferate therein, and generate viable transgenic sperm that may be harvested for use in artificial insemination procedures, or transferred to a recipient oocyte by natural coitus.

In certain embodiments, therefore, the transgenic avian may be produced by the sperm-mediated transfer of at least one heterologous transgene. The transgene may be incorporated into the genomic nucleic acid of a spermatozoon cell or a precursor thereof, so that a genetically modified avian sperm is produced by the male avian. Breeding the male avian with a female of its species will generate a transgenic progeny carrying the at least one transgene in its genome. A union of male and female gametes to form a transgenic zygote is brought about by copulation of the male and female vertebrates of the same species, or by in vitro or in vivo artificial means. If artificial means are chosen, then incorporating into the genome a genetic selection marker that is expressed in male germ cells is particularly useful.

Suitable artificial means include, but are not limited to, artificial insemination, in vitro fertilization (INF) and/or other artificial reproductive technologies, such as intracytoplasmic sperm injection (ICSI), subzonal insemination (SUZI), or partial zona dissection (PZD). Also others, such as cloning and embryo transfer, cloning and embryo splitting, and the like, can be employed.

In a preferred embodiment, a transgene is incorporated into an avian sperm by intracytoplasmic sperm injection (ICSI). The male germ cells, which may be intact and viable spermatozoa, or the non- viable heads thereof, may be microinjected into the cytoplasm or the nucleus of an isolated oocyte such as an avian oocyte, preferably a chicken oocyte, by any method known to one of skill in the art, including, for example, combining a confocal microscope and micromanipulator, or the like to visualize and monitor the microinjection of an opaque avian egg.

The transgenic vertebrate progeny can, in turn, be bred by natural mating, artificial insemination, or by in vitro fertilization (INF) and/or other artificial reproductive technologies, such as intracytoplasmic sperm injection (ICSI) and chicken intracytoplasmic sperm injection (CHICSI™), subzonal insemination (SUZI), or partial zona dissection (PZD), to obtain further generations of transgenic progeny. Although the genetic material is originally inserted solely into the germ cells of a parent animal, it will ultimately be present in the germ cells of future progeny and subsequent generations thereof. In addition, the genetic material will also be present in cells of the progeny other than germ cells, i.e., somatic cells. The methods of the present invention may further comprise returning a transfected fertilized oocyte to a surrogate mother, especially a female chicken, for the continued incubation and development of the transgenic zygote. With chickens, the developed embryo is laid as a hard-shell egg that will hatch as a viable chick. When the heterologous nucleic acid is directly integrated into the genome of the oocyte, the transgenic chick will include the transgenic heterologous nucleic acid in all of its cells. Where the heterologous nucleic acid is episomal with respect to the genome of the transgenic zygote and chick, and the episomal nucleic acid comprises a centromeric body, most, if not all, of the cells of the zygote and chick will comprise the heterologous nucleic acid. When the episomal nucleic acid does not include a centromeric body, however, the transgenic zygote and chick can be a mosaic wherein expression of the exogenous transgene will only occur in some, but not all cells or tissues of the transgenic animal.

5.1.2 BREEDING AND MAINTENANCE OF TRANSGENIC AVIAN

Another aspect of the present invention is a transgenic avian produced by the methods of the present invention and producing a heterologous polypeptide in an egg, wherein the transgenic avian comprises at least one heterologous nucleic acid sequence encoding the polypeptide and wherein the heterologous polypeptide is delivered to the white of an avian egg by a female of the avian.

The invention relates to a method of producing transgenic avians that express significant quantities of useful heterologous proteins, e.g., therapeutic and diagnostic proteins, including immunoglobulins, industrially useful proteins and other biologies etc. in the avian egg white. The heterologous protein can then be readily purified from the avian egg. The methods of the invention provide improved efficiencies of transgenesis, transmission of the transgene and/or level of heterologous protein expression. Another

5 aspect of the invention is a method of producing a transgenic avian capable of expressing a heterologous protein. Therefore, the present invention relates to methods of producing transgenic avians, preferably chickens, wherein the incorporated transgene may be expressed as a constituent protein of the white of a hard-shell egg.

Although the genetic material is originally inserted solely into the germ cells of a

10 parent animal, it will ultimately be present in the germ cells of future progeny and subsequent generations thereof. In addition, the genetic material will also be present in cells of the progeny other than germ cells, i.e., somatic cells.

Using the methods of the invention for producing transgenic avians, particularly methods using vectors that are not derived from eukaryotic viruses, and, preferably, the

1 methods of cytoplasmic micro-inj ection described herein, the level of mosaicism of the transgene (percentage of cells containing the transgene) in avians hatched from microinjected embryos (i.e., the G₀s) is greater than 5%, 10%, 25%, 50%), 75% or 90%), or is the equivalent of one copy per one genome, two genomes, five genomes, seven genomes or eight genomes, as determined by any number of techniques known in the art and described

20 infra.

In additional particular embodiments, the percentage of GOs that transmit the transgene to progeny (Gls) is greater than 5%>, preferably, greater than 10%>, 20%, 30%>, 40%, and, most preferably, greater than 50%, 60%, 70%, 80%, 90%. In other embodiments, the transgene is detected in 10%>, 20%>, 30%>, 40%, and most preferably, greater than 50%,

25 60%), 70%, 80%), 90%) of chicks hatching from embryos into which nucleic acids have been introduced using methods of the invention.

5.2 VECTORS

A variety of vectors useful in carrying out the methods of the present invention are 30 described herein. These vectors may be used for stable introduction of a selected heterologous polypeptide-coding sequence (and/or regulatory sequences) into the genome of an avian, in particular, to generate transgenic avians that produce exogenous proteins in specific tissues of an avian, and in the oviduct in particular, or in the serum of an avian. In still further embodiments, the vectors are used in methods to produce avian eggs containing 35 exogenous protein. In particular embodiments, preferably for use in the sperm-mediated transgenesis methods described herein, the vectors of the invention are not derived from eukaryotic viral vectors or retroviral vectors (except in certain embodiments for containing eukaryotic viral regulatory elements such as promoters, origins of replication, etc). In particular

5 embodiments, the vector is not an REV, ALV or MuLV vector. In particular, useful vectors include, bacteriophages such as lambda derivatives, such as λgtl 1, λgt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV40, PBLUESCRIPT® II SK +/- or KS +/- (see "Sfratagene Cloning Systems" Catalog (1993) from STRATAGENE®, La Jolla,

10 Calif, which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier, F.W. et. al, 1990, "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes" Gene Expression Technology 185, which is hereby incorporated by reference) and any derivatives thereof, cosmid vectors and, in preferred embodiments, artificial chromosomes, such as, but not limited to, YACs, BACs, BBPACs or PACs. Such artificial

15 chromosomes are useful in that a large nucleic acid insert can be propagated and introduced into the avian cell.

In other particular embodiments, as detailed above in section 5.2, infra, the vectors of the invention are derived from eukaryotic viruses, preferably avian viruses, and can be replication competent or, preferably, replication deficient. In particular embodiments, the

20 vectors are derived from REV, ALV or MuLV. Nucleic acid sequences or derivative or truncated variants thereof, may be introduced into viruses such as vaccinia virus. Methods for making a viral recombinant vector useful for expressing a protein under the control of the lysozyme promoter are analogous to the methods disclosed in U.S. Patent Nos. 4,603,112; 4,769,330; 5,174,993; 5,505,941; 5,338,683; 5,494,807; 4,722,848; Paoletti, E,

25 1996, Proc. Natl. Acad. Sci. 93: 11349-11353; Moss, 1996, Proc. Natl Acad. Sci. 93:

11341-11348; Roizman, 1996, Proc. Natl Acad. Sci. 93: 11307-11302; Frolov et al, 1996, Proc. Natl. Acad. Sci. 93: 11371-11377; Grunhaus et al, 1993, Seminars in Virology 3: 237-252 and U.S. Patent Nos. 5,591,639; 5,589,466; and 5,580,859 relating to DNA expression vectors, inter alia; the contents of which are incorporated herein by reference in

30 their entireties.

Recombinant viruses can also be generated by transfection of plasmids into cells infected with virus.

Preferably, vectors can replicate (i.e., have a bacterial origin of replication) and be manipulated in bacteria (or yeast) and can then be introduced into avian cells. Preferably, 5 the vector comprises a marker that is selectable and/or detectable in bacteria or yeast cells and, preferably, also in avian cells, such markers include, but are not limited to, Amp^r, tef, LacZ, etc. Preferably, such vectors can accommodate (i.e., can be used to introduce into cells and replicate) large pieces of DNA such as genomic sequences, for example, large pieces of DNA consisting of at least 25 kb, 50 kb, 75 kb, 100 kb, 150 kb, 200 kb or 250 kb, such as BACs, YACs, cosmids, etc.

The insertion of a DNA fragment into a vector can, for example, be accomplished by ligating the DNA fragment into a vector that has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and the transgene may be modified by homopolymeric tailing. The vector can be cloned using methods known in the art, e.g., by the methods disclosed in Sambrook et al , (supra); Ausubel et al , 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., both of which are hereby incorporated by reference in their entireties. Preferably, the vectors contain cloning sites, for example, restriction enzyme sites that are unique in the sequence of the vector and insertion of a sequence at that site would not disrupt an essential vector function, such as replication.

As discussed above, vectors used in certain methods of the invention preferably can accommodate, and in certain embodiments comprise, large pieces of heterologous DNA such as genomic sequences, particularly avian genomic sequences. Such vectors can contain an entire genomic locus, or at least sufficient sequence to confer endogenous regulatory expression pattern, e.g. , high level of expression in the magnum characteristic of ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin, etc, and to insulate the expression of the transgene sequences from the effect of regulatory sequences surrounding the site of integration of the transgene in the genome. Accordingly, as detailed below, in preferred embodiments, the transgene is inserted in an entire genomic loci or significant portion thereof.

To manipulate large genomic sequences contained in, for example, a BAC, nucleotide sequences coding for the heterologous protein to be expressed and/or other regulatory elements may be inserted into the BAC by directed homologous recombination in bacteria, e.g., the methods of Heintz WO 98/59060; Heintz et al, WO 01/05962; Yang et al, 1991, Nature Biotechnol. 15: 859-865; Yang et al, 1999, Nature Genetics 22: 321 -35; which are incorporated herein by reference in their entireties.

Alternatively, the BAC can also be engineered or modified by "E-T cloning," as described by Muyrers et al. (1999, Nucleic Acids Res. 27(6): 1555-57, incorporated herein

5 by reference in its entirety). Using these methods, specific DNA may be engineered into a BAC independently of the presence of suitable restriction sites. This method is based on homologous recombination mediated by the recE and recT proteins ("ET-cloning") (Zhang et al, 1998, Nat. Genet. 20(2): 123-28; incorporated herein by reference in its entirety). Homologous recombination can be performed between a PCR fragment flanked by short

1 homology arms and an endogenous intact recipient such as a BAC. Using this method, homologous recombination is not limited by the disposition of restriction endonuclease cleavage sites or the size of the target DNA. A BAC can be modified in its host strain using a plasmid, e.g. , pBAD-αβγ, in which recE and recT have been replaced by their respective functional counterparts of phage lambda (Muyrers et al, 1999, Nucleic Acids Res. 27(6):

15 1555-57). Preferably, a BAC is modified by recombination with a PCR product containing homology arms ranging from 27-60 bp. In a specific embodiment, homology arms are 50 bp in length.

In another embodiment, a transgene is inserted into a yeast artificial chromosome (YAC) (Burke et al, 1987, Science 236: 806-12; and Peterson et al, 1997, Trends Genet.

20 13:61, both of which are incorporated by reference herein in their entireties) .

In other embodiments, the transgene is inserted into another vector developed for the cloning of large segments of genomic DNA, such as a cosmid or bacteriophage PI (Sternberg et al, 1990, Proc. Natl Acad. Sci. USA 87: 103-07). The approximate maximum insert size is 30-35 kb for cosmids and 100 kb for bacteriophage PI. In another

25 embodiment, the transgene is inserted into a P-l derived artificial chromosome (PAC) (Mejia et al, 1997, Genome Res 7:179-186). The maximum insert size is 300 kb.

Vectors containing the appropriate heterologous sequences may be identified by any method well known in the art, for example, by sequencing, restriction mapping, hybridization, PCR amplification, etc.

30 The vectors of the invention comprise one or more nucleotide sequences encoding a heterologous protein desired to be expressed in the transgenic avian, as well as regulatory elements such as promoters, enhancers, MARs, IRES's and other translation control elements, transcriptional termination elements, polyadenylation sequences, etc, as discussed infra. In particular embodiments, the vector of the invention contains at least two

35 nucleotide sequences coding for heterologous proteins, for example, but not limited to, the heavy and light chains of an immunoglobulin.

In a preferred embodiment, the nucleotide sequence encoding the heterologous protein is inserted into all or a significant portion of a nucleic acid containing the genomic sequence of an endogenous avian gene, preferably an avian gene that is expressed in the magnum, e.g., ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin, etc. For example, the heterologous gene sequence may be inserted into or replace a portion of the 3' untranslated region (UTR) or 5' untranslated region (UTR) or an intron sequence of the endogenous gene genomic sequence. Preferably, the heterologous gene coding sequence has its own IRES. For descriptions of IRES' s, see, e.g., Jackson et al, 1990, Trends Biochem Sci. 15(12):477-83; Jang et al, 1988, J. Virol 62(8):2636-43; Jang et al, 1990, Enzyme 44(l-4):292-309; and Martinez-Salas, 1999, Curr. Opin. Biotechnol. 10(5):458-64; Palmenberg et al, United States Patent No. 4,937,190, which are incorporated by reference herein in their entireties. In another embodiment, the heterologous protein coding sequence is inserted at the 3' end of the endogenous gene coding sequence. In another preferred embodiment, the heterologous gene coding sequences are inserted using 5' direct fusion wherein the heterologous gene coding sequences are inserted in-frame adjacent to the initial ATG sequence (or adjacent the nucleotide sequence encoding the first two, three, four, five, six, seven or eight amino acids) of the endogenous gene or replacing some or all of the sequence of the endogenous gene coding sequence. In yet another specific embodiment, the heterologous gene coding sequence is inserted into a separate cistron in the 5' region of the endogenous gene genomic sequence and has an independent IRES sequence.

The present invention further relates to nucleic acid vectors (preferably, not derived from eukaryotic viruses, except, in certain embodiments, for eukaryotic viral promoters and/ or enhancers) and transgenes inserted therein that incorporate multiple polypeptide- encoding regions, wherein a first polypeptide-encoding region is operatively linked to a transcription promoter and a second polypeptide-encoding region is operatively linked to an IRES. For example, the vector may contain coding sequences for two different heterologous proteins (e.g. , the heavy and light chains of an immunoglobulin) or the coding sequences for all or a significant part of the genomic sequence for the gene from which the promoter driving expression of the transgene is derived, and the heterologous protein desired to be expressed (e.g., a construct containing the genomic coding sequences, including introns, of the avian lysozyme gene when the avian lysozyme promoter is used to drive expression of the transgene, an IRES, and the coding sequence for the heterologous protein desired to be expressed downstream (i.e., 3' on the RNA transcript of the IRES). Thus, in certain embodiments, the nucleic acid encoding the heterologous protein is introduced into the 5' untranslated or 3' untranslated regions of an endogenous gene, such as but not limited to, ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin, with an IRES sequence directing translation of the heterologous sequence.

Such nucleic acid constructs, when inserted into the genome of a bird and expressed therein, will generate individual polypeptides that may be post-translationally modified, for example, glycosylated or, in certain embodiments, form complexes, such as heterodimers with each other in the white of the avian egg. Alternatively, the expressed polypeptides may be isolated from an avian egg and combined in vitro, or expressed in a non-reproductive tissue such as serum. In other embodiments, for example, but not limited to, when expression of both heavy and light chains of an antibody is desired, two separate constructs, each containing a coding sequence for one of the heterologous proteins operably linked to a promoter (either the same or different promoters), are introduced by microinjection into cytoplasm of one or more embryonic cells and transgenic avians harboring both transgenes in their genomes and expressing both heterologous proteins are identified. Alternatively, two transgenic avians each containing one of the two heterologous proteins (e.g., one transgenic avian having a transgene encoding the light chain of an antibody and a second transgenic avian having a transgene encoding the heavy chain of the antibody) can be bred to obtain an avian containing both transgenes in its germline and expressing both transgene encoded proteins, preferably in eggs.

Recombinant expression vectors can be designed for the expression of the encoded proteins in eukaryotic cells. Useful vectors may comprise constitutive or inducible promoters to direct expression of either fusion or non-fusion proteins. With fusion vectors, a number of amino acids are usually added to the expressed target gene sequence such as, but not limited to, a protein sequence for thioredoxin, a polyhistidine, or any other amino acid sequence that facilitates purification of the expressed protein. A proteolytic cleavage site may further be introduced at a site between the target recombinant protein and the fusion sequence. Additionally, a region of amino acids such as a polymeric histidine region may be introduced to allow binding of the fusion protein to metallic ions such as nickel bonded to a solid support, and thereby allow purification of the fusion protein. Once the fusion protein has been purified, the cleavage site allows the target recombinant protein to be separated from the fusion sequence. Enzymes suitable for use in cleaving the proteolytic cleavage site include, but are not limited to, Factor Xa and thrombin. Fusion expression vectors that may be useful in the present invention include pGex (AMRAD® Corp., Melbourne, Australia), pRIT5 (PHARMACIA®, Piscataway, NJ) and pMAL (NEW ENGLAND BIOLABS®, Beverly, MA), fusing glutathione S-transferase, protein A, or maltose E binding protein, respectively, to the target recombinant protein.

Once a promoter and a nucleic acid encoding a heterologous protein of the present invention have been cloned into a vector system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. It is contemplated that the incorporation of the DNA of the present invention into a recipient cell may be by any suitable method such as, but not limited to, viral transfer, electroporation, gene gun insertion, sperm- mediated transfer to an ovum, microinjection and the like. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, and the like. In particular, the present invention contemplates the use of recipient avian cells, such as chicken cells or quail cells.

Another aspect of the present invention, therefore, is a method of expressing a heterologous polypeptide in a eukaryotic cell by transfecting an avian cell with a recombinant DNA comprising an avian tissue-specific promoter operably linked to a nucleic acid insert encoding a polypeptide and, optionally, a polyadenylation signal sequence, and culturing the transfected cell in a medium suitable for expression of the heterologous polypeptide under the control of the avian lysozyme gene expression control region. Yet another aspect of the present invention is a eukaryotic cell transformed with an expression vector according to the present invention and described above. In one embodiment of the present invention, the transformed cell is a chicken oviduct cell and the nucleic acid insert comprises the chicken lysozyme gene expression control region, a nucleic acid insert encoding a human interferon α2b and codon optimized for expression in an avian cell, and an SV40 polyadenylation sequence.

In another embodiment, the transformed cell is a quail oviduct cell and the nucleic acid insert comprises the artificial avian promoter construct MDOT (SEQ ID NO.:l 1) operably linked to an interferon-encoding sequence, as described in Example 23 below. In yet another embodiment of the present invention, a quail oviduct cell is transfected with the nucleic acid insert comprising the MDOT artificial promoter construct operably linked to an erythropoietin (EPO)-encoding nucleic acid, wherein the transfected quail produces heterologous erythropoietin. 5.2.1 PROMOTERS

The vectors of the invention contain promoters that function in avian cells, preferably, that are tissue-specific and, in preferred embodiments, direct expression in the magnum or serum or other tissue such that expressed proteins are deposited in eggs, more preferably, that are specific for expression in the magnum. Alternatively, the promoter directs expression of the protein in the serum of the transgenic avian. Introduction of the vectors of the invention, preferably, generate transgenics that express the heterologous protein in tubular gland cells where it is secreted into the oviduct lumen and deposited, e.g., into the white of an egg. In preferred embodiments, the promoter directs a level of expression of the heterologous protein in the egg white of eggs laid by GO and/or GI chicks and/or their progeny that is greater than 5 μg, 10 μg, 50 μg, 100 μg, 250 μg, 500 μg or 750 μg, more preferably greater than 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 700 mg, 1 gram, 2 grams, 3 grams, 4 grams or 5 grams. Such levels of expression can be obtained using the promoters of the invention. In preferred embodiments, the promoters of the invention are derived from genes that express proteins present in significant levels in the egg white and/or the serum. For example, the promoter comprises regions of an ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin or ovomucin promoter or any other promoter that directs expression of a gene in an avian, particularly in a specific tissue of interest, such as the magnum or in the serum. Alternatively, the promoter used in the expression vector may be derived from that of the lysozyme gene that is expressed in both the oviduct and macrophages. Portions of two or more of these, and other promoters that function in avians, may be combined to produce effective synthetic promoter.

The promoter may optionally be a segment of the ovalbumin promoter region that is sufficiently large to direct expression of the coding sequence in the tubular gland cells. Other exemplary promoters include the promoter regions of the ovalbumin, lysozyme, ovomucoid, conalbumin, ovotransferrin or ovomucin genes (for example, but not limited to, as disclosed in co-pending United States Patent Application Nos. 09/922,549, filed August 3, 2001 and 10/114,739, filed April 1, 2002, both entitled "Avian Lysozyme Promoter", by Rapp, and United States Patent Application No. 09/998,716, filed November 30, 2001, entitled "Ovomucoid Promoter and Methods of Use," by Harvey et al, all of which are incorporated by reference herein in their entireties). Alternatively, the promoter may be a promoter that is largely, but not entirely, specific to the magnum, such as the lysozyme promoter. Other suitable promoters may be artificial constructs such as a combination of nucleic acid regions derived from at least two avian gene promoters. One such embodiment of the present invention is the MDOT construct (SEQ ID NO: 11) comprising regions derived from the chicken ovomucin and ovotransferrin promoters, including but not limited to promoters altered, e.g., to increase expression, and inducible promoters, e.g., the tet^r system. The ovalbumin gene encodes a 45 kD protein that is also specifically expressed in the tubular gland cells of the magnum of the oviduct (Beato, 1989, Cell 56:335-344). Ovalbumin is the most abundant egg white protein, comprising over 50 percent of the total protein produced by the tubular gland cells, or about 4 grams of protein per large Grade A egg (Gilbert, "Egg albumen and its formation" in Physiology and Biochemistry of the Domestic Fowl, Bell and Freeman, eds., Academic Press, London, New York, pp. 1291- 1329). The ovalbumin gene and over 20 kb of each flanking region have been cloned and analyzed (Lai et al, 1978, Proc. Natl. Acad. Sci. USA 75:2205-2209; Gannon et al, 1979, Nature 278:428-424; Roop et /.,1980, Cell 19:63-68; and Royal et α/.,1975, Nature 279:125-132). The ovalbumin gene responds to steroid hormones such as estrogen, glucocorticoids, and progesterone, which induce the accumulation of about 70,000 ovalbumin mRNA transcripts per tubular gland cell in immature chicks and 100,000 ovalbumin mRNA transcripts per tubular gland cell in the mature laying hen (Palmiter, 1973, J. Biol. Chem. 248:8260-8270; Palmiter, 1975, Cell 4:189-197). The 5' flanking region contains four DNAse I-hypersensitive sites centered at -0.25, -0.8, -3.2, and -6.0 kb from the transcription start site. These sites are called HS-I, -II, -III, and -IV, respectively. Promoters of the invention may contain one, all, or a combination of HS-I, HS-II, HS-III and HS0IV. Hypersensitivity of HS-II and -III are estrogen-induced, supporting a role for these regions in hormone-induction of ovalbumin gene expression. HS-I and HS-II are both required for steroid induction of ovalbumin gene transcription, and a 1.4 kb portion of the 5' region that includes these elements is sufficient to drive steroid-dependent ovalbumin expression in explanted tubular gland cells (Sanders and McKnight, 1988, Biochemistry 27: 6550-6557). HS-I is termed the negative-response element ("NRE") because it contains several negative regulatory elements which repress ovalbumin expression in the absence of hormone (Haekers et α/. , 1995, Mol. Endo. 9:1113- 1126). Protein factors bind these elements, including some factors only found in oviduct nuclei suggesting a role in tissue-specific expression. HS-II is termed the steroid-dependent response element ("SDRE") because it is required to promote steroid induction of transcription. It binds a protein or protein complex known as Chirp-I. Chirp-I is induced by estrogen and turns over rapidly in the presence of cyclohexamide (Dean et al. , 1996, Mol Cell Biol. 16:2015-2024). Experiments using an explanted tubular gland cell culture system defined an additional set of factors that bind SDRE in a steroid-dependent manner, including aNFκB-like factor (Nordstrom et al, 1993, J Biol. Chem. 268:13193-13202; Schweers and Sanders, 1991, J. Biol. Chem. 266: 10490-10497). Less is known about the function of HS-III and HS-IV. HS-III contains a functional estrogen response element, and confers estrogen inducibility to either the ovalbumin proximal promoter or a heterologous promoter when co-transfected into HeLa cells with an estrogen receptor cDNA. These data imply that HS-III may play a functional role in the overall regulation of the ovalbumin gene. Little is known about the function of HS-IV, except that it does not contain a functional estrogen-response element (Kato et al, 1992, Cell 68: 731-742).

In an alternative embodiment of the invention, transgenes containing constitutive promoters are used, but the transgenes are engineered so that expression of the transgene effectively becomes magnum-specific. Thus, a method for producing an exogenous protein in an avian oviduct provided by the present invention involves generating a transgenic avian having two transgenes in its tubular gland cells. One transgene comprises a first coding sequence operably linked to a constitutive promoter. The second transgene comprises a second coding sequence that is operably linked to a magnum-specific promoter, where expression of the first coding sequence is either directly or indirectly dependent upon the cellular presence of the protein expressed by the second coding sequence.

Additional promoters useful in the present invention include inducible promoters, such as the tet operator and the metallothionein promoter which can be induced by treatment with tetracycline and zinc ions, respectively (Gossen et al, 1992, Proc. Natl. Acad. Sci. 89: 5547-5551 and Walden et al, 1987, Gene 61: 317-327; incorporated herein by reference in their entireties).

5.2.1.1 CHICKEN LYSOZYME GENE EXPRESSION CONTROL

REGION NUCLEIC ACID SEQUENCES

The chicken lysozyme gene is highly expressed in the myeloid lineage of hematopoietic cells, and in the tubular glands of the mature hen oviduct (Hauser et al , 1981, Hematol and Blood Transfusion 26: 175-178; Schutz et al, 1978, Cold Spring Harbor Symp. Quart. Biol. 42: 617-624) and is therefore a suitable candidate for an efficient promoter for heterologous protein production in transgenic animals. The regulatory region of the lysozyme locus extends over at least 12 kb of DNA 5' upstream of the transcription start site, and comprises a number of elements that have been individually isolated and characterized. The known elements include three enhancer sequences at about -6.1 kb, -3.9 kb, and -2.7 kb (Grewal et al, 1992, Mol. Cell Biol 12: 2339-2350; Bonifer et al, 1996, J Mol Med. 74: 663-671), a hormone responsive element (Hecht et al, 1988, E.M.B. O.J. 7: 2063-2073), a silencer element and a complex proximal promoter. The constituent elements of the lysozyme gene expression control region are identifiable as DNAase 1 hypersensitive chromatin sites (DHS). They may be differentially exposed to nuclease digestion depending upon the differentiation stage of the cell. For example, in the multipotent progenitor stage of myelomoncytic cell development, or in erythroblasts, the silencer element is a DHS. At the myeloblast stage, a transcription enchancer located -6.1 kb upstream from the gene transcription start site is a DHS, while at the later monocytic stage another enhancer, at -2.7 kb becomes DNAase sensitive (Huber et al, 1995, DNA and Cell Biol. 14: 391 -402).

This invention also envisions the use of promoters other than the lysozyme promoter, including but not limited to, a cytomegalovirus promoter, an ovomucoid, conalbumin or ovotransferrin promoter or any other promoter that directs expression of a gene in an avian, particularly in a specific tissue of interest, such as the magnum.

Another aspect of the methods of the present invention is the use of combinational promoters comprising an artificial nucleic acid construct having at least two regions wherein the regions are derived from at least two gene promoters, including but not limited to a lysozyme, ovomucoid, conalbumin or ovotransferrin promoter. In one embodiment of the present invention, the promoter may comprise a region of an avian ovomucoid promoter and a region of an avian oxotransferrin promoter, thereby generating the MDOT avian artificial promoter construct. The avian MDOT promoter construct of the present invention has the nucleic acid sequence SEQ ID NO: 11 and is illustrated in Figure 7. This promoter is useful for allowing expression of a heterologous protein in chicken oviduct cells and may be operably linked to any nucleic acid encoding a heterologous polypeptide of interest including, for example, a cytokine, growth hormone, growth factor, enzyme, structural protein or the like.

5.2.2 MATRIX ATTACHMENT REGIONS

In preferred embodiments of the invention, the vectors contain matrix attachment regions (MARs) that preferably flank the transgene sequences to reduce position effects on expression when integrated into the avian genome. In fact, 5' MARs and 3' MARs (also referred to as "scaffold attachment regions" or SARs) have been identified in the outer boundaries of the chicken lysozyme locus (Phi- Van et al, 1988, E.M.B. O.J. 7: 655-664; Phi-Van, L. and Staffing, W.H., 1996, Biochem. 35: 10735-10742). Deletion of a 1.32 kb or a 1.45 kb halves region, each comprising half of a 5' MAR, reduces positional variation in the level of transgene expression (Phi- Van and Stratling, supra).

The 5' matrix-associated region (5' MAR), located about -11.7 kb upstream of the chicken lysozyme transcription start site, can increase the level of gene expression by limiting the positional effects exerted against a transgene (Phi- Van et al, 1988, supra). At least one other MAR is located 3' downstream of the protein encoding region. Although MAR nucleic acid sequences are conserved, little cross-hybridization is seen, indicating significant overall sequence variation. However, MARs of different species can interact with the nucleomatrices of heterologous species, to the extent that the chicken lysozyme MAR can associate with the plant tobacco nucleomatrix as well as that of the chicken oviduct cells (Mlynarona et al, 1994, Cell 6: 417-426; von Kries et al, 1990, Nucleic Acids Res. 18: 3881-3885).

Gene expression must be considered not only from the perspective of cis-regulatory elements associated with a gene, and their interactions with trans-acting elements, but also with regard to the genetic environment in which they are located. Chromosomal positioning effects (CPEs), therefore, are the variations in levels of transgene expression associated with different locations of the transgene within the recipient genome. An important factor governing CPE upon the level of transgene expression is the chromatin structure around a transgene, and how it cooperates with the cis-regulatory elements. The cis-elements of the lysozyme locus are confined within a single chromatin domain (Bonifer et al, 1996, supra; Sippel et al, pgs. 133-147 in Eckstein F. & Lilley D.M.J. (eds), "Nucleic Acids and Molecular Biology", Vol. 3, 1989, Springer.

The lysozyme promoter region of chicken is active when transfected into mouse fibroblast cells and linked to a reporter gene such as the bacterial chloramphenicol acetyltransferase (CAT) gene. The promoter element is also effective when transiently transfected into chicken promacrophage cells. In each case, however, the presence of a 5' MAR element increased positional independency of the level of transcription (Stief et al, 1989, Nature 341: 343-345; Sippel et al, pgs. 257 - 265 in Houdebine L.M. (ed), "Transgenic Animals: Generation and Use").

The ability to direct the insertion of a transgene into a site in the genome of an animal where the positional effect is limited offers predictability of results during the development of a desired transgenic animal, and increased yields of the expressed product. Sippel and Steif disclose, in U.S. Patent No. 5,731,178, which is incorporated by reference herein in its entirety, methods to increase the expression of genes introduced into eukaryotic cells by flanking a transcription unit with scaffold attachment elements, in particular the 5' MAR isolated from the chicken lysozyme gene. The transcription unit disclosed by Sippel and Steif was an artificial construct that combined only the -6.1 kb enhancer element and the proximal promoter element (base position -579 to +15) from the lysozyme gene. Other promoter associated elements were not included. However, although individual cis- regulatory elements have been isolated and sequenced, together with short regions flanking DNA, the entire nucleic acid sequence comprising the functional 5' upstream region of the lysozyme gene has not been determined in its entirety and therefore not employed as a functional promoter to allow expression of a heterologous transgene. Accordingly, vectors of the invention comprise MARs, preferably both 5' and 3'

MARs that flank the transgene, including the heterologous protein coding sequences and the regulatory sequences.

5.2.3 CODON-OPTIMIZED GENE EXPRESSION Another aspect of the present invention provides nucleic acid sequences encoding heterologous polypeptides that are codon-optimized for expression in avian cells, and derivatives and fragments thereof. When a heterologous nucleic acid is to be delivered to a recipient cell for expression therein, the sequence of the nucleic acid sequence may be modified so that the codons are optimized for the codon usage of the recipient species. For example, if the heterologous nucleic acid is transfected into a recipient chicken cell, the sequence of the expressed nucleic acid insert is optimized for chicken codon usage. This may be determined from the codon usage of at least one, and preferably more than one, protein expressed in a chicken cell. For example, the codon usage may be determined from the nucleic acid sequences encoding the proteins ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin of chicken. Briefly, the DNA sequence for the target protein may be optimized using the BACKTRANSLATE® program of the Wisconsin Package, version 9.1 (Genetics Computer Group, Inc., Madison, Wl) with a codon usage table compiled from the chicken (Gallus gallus) ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin proteins. The template and primer oligonucleotides are then amplified, by any means known in the art, including but not limited to PCR with Pfu polymerase (STRATAGENE®, La Jolla CA).

In one exemplary embodiment of a heterologous nucleic acid for use by the methods of the present invention, a nucleic acid insert encoding the human interferon α2b polypeptide optimized for codon-usage by the chicken is microinjected into the cytoplasm of a stage 1 embryo. Optimization of the sequence for codon usage is useful in elevating the level of translation in avian eggs.

It is contemplated to be within the scope of the present invention for any nucleic acid encoding a polypeptide to be optimized for expression in avian cells. It is further contemplated that the codon usage may be optimized for a particular avian species used as a source of the host cells. In one embodiment of the present invention, the heterologous polypeptide is encoded using the codon-usage of a chicken.

5.2.4 SPECIFIC VECTORS OF THE INVENTION In a preferred embodiment, a transgene of the invention comprises a chicken, or other avian, lysozyme control region sequence which directs expression of the coding sequence within the transgene. A series of PCR amplifications of template chicken genomic DNA are used to isolate the gene expression control region of the chicken lysozyme locus. Two amplification reactions used the PCR primer sets 5pLMAR2 (5'- TGCCGCCTTCTTTGATATTC-3') (SEQ ID NO: 1) and LE-6.1kbrevl (5'- TTGGTGGTAAGGCCTTTTTG-3') (SEQ ID NO: 2) (Set 1) and lys-6.1 (5'- CTGGCAAGCTGTCAAAAACA-3') (SEQ ID NO: 3) and LysElRev (5'- CAGCTCACATCGTCCAAAGA-3') (SEQ ID NO: 4) (Set 2). The amplified PCR products were united as a contiguous isolated nucleic acid by a third PCR amplification step with the primers SEQ ID NOS: 1 and 4.

The isolated PCR-amplified product, comprising about 12 kb of the nucleic acid region 5' upstream of the native chicken lysozyme gene locus, was cloned into the plasmid pCMV-LysSPIFNMM. pCMV-LysSPIFNMM comprises a modified nucleic acid insert encoding a human interferon α2b sequence and an SV40 polyadenylation signal sequence (SEQ ID NO: 8) 3' downstream of the interferon encoding nucleic acid. The sequence SEQ ID NO: 5 of the nucleic acid insert encoding human interferon α2b was in accordance with avian cell codon usage, as determined from the nucleotide sequences encoding chicken ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin.

The nucleic acid sequence (SEQ ID NO: 6) (GenBank Accession No. AF405538) of the insert in pAVIJCR-Al 15.93.1.2 is shown in Figures 1A-E. The modified human interferon α2b encoding nucleotide sequence SEQ ID NO: 5 (GenBank Accession No. AF405539) and the novel chicken lysozyme gene expression control region SEQ ID NO: 7 (GenBank Accession No. AF405540), shown in Figures 2 and 3 A-E, respectively. A polyadenylation signal sequence that is suitable for operably linking to the polypeptide- encoding nucleic acid insert is the SV40 signal sequence SEQ ID NO: 8, as shown in Figure 4.

The plasmid pAVIJCR-Al 15.93.1.2 was restriction digested with enzyme Fseϊ to isolate a 15.4 kb DNA containing the lysozyme 5' matrix attachment region (MAR) and the -12.0 kb lysozyme promoter during the expression of the interferon-encoding insert, as described in Example 17, below. Plasmid plllilys was restriction digested with Mlu\ and Xho to isolate an approximately 6 kb nucleic acids, comprising the 3' lysozyme domain, the sequence of which (SEQ ID NO: 9) is shown in Figures 5 A-C. The 15.4 kb and 6 kb nucleic acids were ligated and the 21.4 kb nucleic acid comprising the nucleic acid sequence SEQ ID NO: 10 as shown in Figures 6A-J was transformed into recipient STBL4 cells.

The inclusion of the novel isolated avian lysozyme gene expression control region of the present invention upstream of a codon-optimized interferon-encoding sequence in pAVIJCR-Al 15.93.1.2 allowed expression of the interferon polypeptide in avian cells transfected by sperm-mediated transfection. The 3' lysozyme domain SEQ ID NO: 9, when operably linked downstream of a heterologous nucleic acid insert, also allows expression of the nucleic acid insert as described in Example 18, below. For example, the nucleic acid insert may encode a heterologous polypeptide such as the α2b interferon encoded by the sequence SEQ ID NO: 5.

It is further contemplated that any nucleic acid sequence encoding a polypeptide may be operably linked to the novel isolated avian lysozyme gene expression control region (SEQ ID NO: 7) and optionally operably linked to the 3' lysozyme domain SEQ ID NO. 9 so as to be expressed in a transfected avian cell. The plasmid construct pAVIJCR-Al 15.93.1.2 can be introduced into cultured quail oviduct cells by transfection. ELISA assays of the cultured media showed that the transfected cells synthesized a polypeptide detectable with anti-human interferon α2b antibodies.

The isolated chicken lysozyme gene expression control region (SEQ ID NO: 7) for use in the methods of the present invention comprises the nucleotide elements that are positioned 5' upstream of the lysozyme-encoding region of the native chicken lysozyme locus and which are necessary for the regulated expression of a downstream polypeptide- encoding nucleic acid. While not wishing to be bound by any one theory, the inclusion of at least one 5' MAR sequence of or reference element in the isolated control region may confer positional independence to a transfected gene operably linked to the novel lysozyme gene expression control region.

The isolated lysozyme gene expression control region (SEQ ID NO: 7) of the present invention is useful for reducing the chromosomal positional effect of a transgene operably linked to the lysozyme gene expression control region and transfected into a recipient avian cell. By isolating a region of the avian genome extending from a point 5' upstream of a 5' MAR of the lysozyme locus to the junction between the signal peptide sequence and a polypeptide-encoding region, cis-regulatory elements are also included that may allow gene expression in a tissue-specific manner. The lysozyme promoter region of the present invention, therefore, will allow expression of an operably linked heterologous nucleic acid insert in a transfected avian cell such as, for example, an oviduct cell.

It is further contemplated that a recombinant DNA of the present invention may further comprise the chicken lysozyme 3' domain (SEQ. ID NO: 9) linked downstream of the nucleic acid insert encoding a heterologous polypeptide. The lysozyme 3' domain (SEQ ID NO: 9) includes a nucleic acid sequence encoding a 3' MAR domain that may cooperate with a 5' MAR to direct the insertion of the construct of the present invention into the chromosome of a transgenic avian, or may act independently of the 5' MAR.

Fragments of a nucleic acid encoding a portion of the subject lysozyme gene expression control region may also be useful as an autonomous gene regulatory element that may itself be operably linked to a polypeptide-encoding nucleic acid. Alternatively, the fragment may be combined with fragments derived from other gene promoters, such as an avian ovalbumin, vomucoid, ovotransferrin, conalbumin or ovomucin promoter, thereby generating novel promoters having new properties or a combination of properties. As used herein, a fragment of the nucleic acid encoding an active portion of a lysozyme gene expression control region refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the entire nucleic acid sequence of the lysozyme gene expression control region, but at least 200 nucleotides.

The present invention also contemplates the use of antisense nucleic acid molecules that are designed to be complementary to a coding strand of a nucleic acid (i.e., complementary to an endogenous DNA or an mRNA sequence) or, alternatively, complimentary to a 5' or 3' untranslated region of the mRNA and therefore useful for regulating the expression of a gene by the lysozyme promoter.

Synthesized oligonucleotides can be produced in variable lengths when for example, non-naturally occurring polypeptide sequences are desired. The number of bases synthesized will depend upon a variety of factors, including the desired use for the probes or primers. Additionally, sense or anti-sense nucleic acids or oligonucleotides can be chemically synthesized using modified nucleotides to increase the biological stability of the molecule or of the binding complex formed between the anti-sense and sense nucleic acids. For example, acridine substituted nucleotides can be synthesized. Protocols for designing isolated nucleotides, nucleotide probes, and/or nucleotide primers are well-known to those of ordinary skill, and can be purchased commercially from a variety of sources (e.g., SIGMA GENOSYS®, The Woodlands, TX or The Great American Gene Co., Ramona, CA).

5.2.5 RECOMBINANT EXPRESSION VECTORS

A useful application of the novel promoters of the present invention, such as the avian lysozyme gene expression control region (SEQ ID NO: 7) or the MDOT promoter construct (SEQ ID NO: 11) is the possibility of increasing the amount of a heterologous protein present in a bird, especially a chicken, by gene transfer. In most instances, a heterologous polypeptide-encoding nucleic acid insert transferred into the recipient animal host will be operably linked with a gene expression control region to allow the cell to initiate and continue production of the genetic product protein. A recombinant DNA molecule of the present invention can be transferred into the extra-chromosomal or genomic DNA of the host.

Expression of a foreign gene in an avian cell permits partial or complete post- translational modification such as, but not only, glycosylation, and/or the formation of the relevant inter- or intra-chain disulfide bonds. Examples of vectors useful for expression in the chicken Gallus gallus include pYepSecl (Baldari et al, 1987, E.M.B.O.J., 6: 229-234; incorporated herein by reference in its entirety) and pYES2 (INVITROGEN® Corp., San Diego, CA).

The present invention contemplates that the injected cell may transiently contain the injected DNA, whereby the recombinant DNA or expression vector may not be integrated into the genomic nucleic acid. It is further contemplated that the injected recombinant DNA or expression vector may be stably integrated into the genomic DNA of the recipient cell, thereby replicating with the cell so that each daughter cell receives a copy of the injected nucleic acid. It is still further contemplated for the scope of the present invention to include a transgenic animal producing a heterologous protein expressed from an injected nucleic acid according to the present invention. Heterologous nucleic acid molecules can be delivered to oocytes using the sperm- mediated transfection methods of the present invention. The nucleic acid molecule may be inserted into a cell to which the nucleic acid molecule (or promoter coding region) is heterologous (i.e., not normally present). Alternatively, the recombinant DNA molecule may be introduced into cells which normally contain the recombinant DNA molecule or the particular coding region, as, for example, to correct a deficiency in the expression of a polypeptide, or where over-expression of the polypeptide is desired.

Another aspect of the present invention, therefore, is a method of expressing a heterologous polypeptide in an avian cell by transfecting the avian cell with a selected heterologous nucleic acid comprising an avian promoter operably linked to a nucleic acid insert encoding a polypeptide and, optionally, a polyadenylation signal sequence. The transfected cell, which may be an avian embryonic cell microinjected with a heterologous nucleic acid, will generate a transgenic embryo that after introduction into a recipient hen will be laid as a hard-shell egg and develop into a transgenic chick. In another embodiment of the present invention, the nucleic acid insert comprises the chicken lysozyme gene expression control region, a nucleic acid insert encoding a human interferon α2b and codon optimized for expression in an avian cell, and a chicken 3' domain, i.e., downstream enhancer elements.

In one embodiment of the present invention, the transgenic animal is an avian selected from a turkey, duck, goose, quail, pheasant, ratite, and ornamental bird or a feral bird. In another embodiment, the avian is a chicken and the heterologous polypeptide produced under the transcriptional control of the avian promoter is produced in the white of an egg. In yet another embodiment of the present invention, the heterologous polypeptide is produced in the serum of a bird.

5.3 HETEROLOGOUS PROTEINS PRODUCED BY TRANSGENIC

AVIANS

Methods of the present invention, providing for the production of heterologous protein in the avian oviduct (or other tissue leading to deposition of the protein into the egg) and the production of eggs containing heterologous protein, involve providing a suitable vector coding for the heterologous protein and introducing the vector into oocytes by sperm- mediated transfection such that the vector is integrated into the genome of the resulting transgenic embryo. A subsequent step involves deriving a mature transgenic avian from the transgenic embryo produced in the previous steps by transferring the injected cell or cells into the infundibulum of a recipient hen; producing a hard shell egg from that hen; and allowing the egg to develop and hatch to produce a transgenic bird.

A transgenic avian so produced from transgenic embryonic cells is known as a founder. Such founders may be mosaic for the transgene (in certain embodiments, the founder has 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100% of the cells containing the transgene. The invention further provides production of heterologous proteins in other tissues of the transgenic avians. Some founders will carry the transgene in the tubular gland cells in the magnum of their oviducts. These birds will express the exogenous protein encoded by the transgene in their oviducts. If the exogenous protein contains the appropriate signal sequences, it will be secreted into the lumen of the oviduct and into the white of an egg.

Some founders are germ-line founders. A germ-line founder is a founder that carries the transgene in genetic material of its germ-line tissue, and may also carry the transgene in oviduct magnum tubular gland cells that express the exogenous protein. Therefore, in accordance with the invention, the transgenic bird may have tubular gland cells expressing the exogenous protein and the offspring of the transgenic bird will also have oviduct magnum tubular gland cells that express the exogenous protein. Alternatively, the offspring express a phenotype determined by expression of the exogenous gene in a specific tissue of the avian. In preferred embodiments, the heterologous proteins are produced from transgenic avians that were not (or the founder ancestors were not) using a eukaryotic viral vector, or a retroviral vector.

The present invention can be used to express, in large yields and at low cost, a wide range of desired proteins including those used as human and animal pharmaceuticals, diagnostics, and livestock feed additives. Proteins such as growth hormones, cytokines, structural proteins and enzymes, including human growth hormone, interferon, lysozyme, and β-casein, are examples of proteins that are desirably expressed in the oviduct and deposited in eggs according to the invention. Other possible proteins to be produced include, but are not limited to, albumin, α-1 antitrypsin, antithrombin III, collagen, factors VIII, IX, X (and the like), fibrinogen, hyaluronic acid, insulin, lactoferrin, protein C, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), tissue-type plasminogen activator (tPA), feed additive enzymes, somatotropin, and chymotrypsin Immunoglobulins and genetically engineered antibodies, including immunotoxins that bind to surface antigens on human tumor cells and destroy them, can also be expressed for use as pharmaceuticals or diagnostics. It is contemplated that immunoglobulin polypeptides expressed in avian cells following transfection by the methods of the present invention may include monomeric heavy and light chains, single-chain antibodies or multimeric immunoglobulins comprising variable heavy and light chain regions, i.e., antigen-binding domains, or intact heavy and light immunoglobulin chains. 5.3.1 MULTIMERIC PROTEINS

The invention, in preferred embodiments, provides methods for producing multimeric proteins, preferably immunoglobulins, such as antibodies, and antigen binding fragments thereof.

5 In one embodiment of the present invention, the multimeric protein is an immunoglobulin, wherein the first and second heterologous polypeptides are an immunoglobulin heavy and light chains respectively. Illustrative examples of this and other aspects and embodiments of the present invention for the production of heterologous multimeric polypeptides in avian cells are fully disclosed in U.S. Patent Application No.

10 09/877,374, filed June 8, 2001, by Rapp, which is incorporated herein by reference in its entirety. In one embodiment of the present invention, therefore, the multimeric protein is an immunoglobulin wherein the first and second heterologous polypeptides are an immunoglobulin heavy and light chain respectively. Accordingly, the invention provides immunoglobulin and other multimeric proteins that have been produced by transgenic

15 avians of the invention.

In the various embodiments of this aspect of the present invention, an immunoglobulin polypeptide encoded by the transcriptional unit of at least one expression vector may be an immunoglobulin heavy chain polypeptide comprising a variable region or a variant thereof, and may further comprise a D region, a J region, a C region, or a

20 combination thereof. An immimoglobulin polypeptide encoded by the transcriptional unit of an expression vector may also be an immunoglobulin light chain polypeptide comprising a variable region or a variant thereof, and may further comprise a J region and a C region. It is also contemplated to be within the scope of the present invention for the immunoglobulin regions to be derived from the same animal species, or a mixture of species including, but 5 not only, human, mouse, rat, rabbit and chicken. In preferred embodiments, the antibodies are human or humanized.

In other embodiments of the present invention, the immunoglobulin polypeptide encoded by the transcriptional unit of at least one expression vector comprises an immunoglobulin heavy chain variable region, an immimoglobulin light chain variable

30 region, and a linker peptide thereby forming a single-chain antibody capable of selectively binding an antigen.

Another aspect of the present invention provides a method for the production in an avian of an heterologous protein capable of forming an antibody suitable for selectively binding an antigen comprising the step of producing a transgenic avian incorporating at 5 least one transgene, wherein the transgene encodes at least one heterologous polypeptide selected from an immunoglobulin heavy chain variable region, an immunoglobulin heavy chain comprising a variable region and a constant region, an immunoglobulin light chain variable region, an immunoglobulin light chain comprising a variable region and a constant region, and a single-chain antibody comprising two peptide-linked immimoglobulin variable regions. Preferably, the antibody is expressed such that it is deposited in the white of the developing eggs of the avian. The hard shell avian eggs thus produced can be harvested and the heterologous polypeptide capable of forming or which formed an antibody can be isolated from the harvested egg. It is also understood that the heterologous polypeptides may also be expressed under the transcriptional control of promoters that allow for release of the polypeptides into the serum of the transgenic animal. Exemplary promoters for non- tissue specific production of a heterologous protein are the CMV promoter and the RSV promoter.

In one embodiment of this method of the present invention, the transgene comprises a transcription unit encoding a first and a second immunoglobulin polypeptide operatively linked to a transcription promoter, a transcription terminator and, optionally, an internal ribosome entry site (IRES) (see, for example, U.S. Patent No. 4,937,190 to Palmenberg et al, the contents of which is incorporated herein by reference in its entirety).

In an embodiment of this method of the present invention, the isolated heterologous protein is an antibody capable of selectively binding to an antigen. In this embodiment, the antibody may be generated within the serum of an avian or within the white of the avian egg by combining at least one immunoglobulin heavy chain variable region and at least one immunoglobulin light chain variable region, preferably cross-linked by at least one disulfide bridge. The combination of the two variable regions will generate a binding site capable of binding an antigen using methods for antibody reconstitution that are well known i the art.

It is, however, contemplated to be within the scope of the present invention for immunoglobulin heavy and light chains, or variants or derivatives thereof, to be expressed in separate transgenic avians, and therefore isolated from separate media including serum or eggs, each isolate comprising a single species of immunoglobulin polypeptide. The method may further comprise the step of combining a plurality of isolated heterologous immunoglobulin polypeptides, thereby producing an antibody capable of selectively binding to an antigen. In this embodiment, two individual transgenic avians may be generated wherein one transgenic produces serum or eggs having an immunoglobulin heavy chain variable region, or a polypeptide comprising such, expressed therein. A second transgenic animal, having a second transgene, produces serum or eggs having an immunoglobulin light chain variable region, or a polypeptide comprising such, expressed therein. The polypeptides may be isolated from their respective sera and eggs and combined in vitro to generate a binding site capable of binding an antigen.

Examples of therapeutic antibodies that can be used in methods of the invention include but are not limited to HERCEPTIN® (Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REOPRO® (abciximab) (Centocor) which is an anti-glycoprotein Ilb/IIIa receptor on the platelets for the prevention of clot formation; ZENAPAX® (daclizumab) (Roche Pharmaceuticals, Switzerland) which is an immunosuppressive, humanized anti- CD25 monoclonal antibody for the prevention of acute renal allograft rejection;

PANOREX™ which is a murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™ which is a humanized anti-αVβ3 integrin antibody (Applied Molecular Evolution/Medlmmune); Campath 1H/LDP-03 which is a humanized anti CD52 IgGl antibody (Leukosite); Smart Ml 95 which is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimeric anti-CD20 IgGl antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is a humanized anti- CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primatied anti-CD80 antibody (IDEC Pharm/Mitsubishi);

ZEVALIN™ is a radiolabelled murine anti-CD20 antibody (IDEC/Schering AG); IDEC- 131 is a humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CAT/BASF); CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4 IgGl antibody (IDEC Pharm SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti- _α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody (Elan); and CAT-152 is a human anti-TGF-β₂ antibody (Cambridge Ab Tech). 5.3.2 PROTEIN RECOVERY

The protein of the present invention may be produced in purified form by any known conventional technique. For example, chicken cells may be homogenized and centrifuged. The supernatant can then be subjected to sequential ammonium sulfate precipitation and heat treatment. The fraction containing the protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC. In another embodiment, an affinity column is used, wherein the protein is expressed with a tag.

Accordingly, the invention provides proteins that are produced by transgenic avians of the invention. In a preferred embodiment, the protein is produced and isolated from an avian egg. In another embodiment, the protein is produced and isolated from avian serum.

5.4 PHARMACEUTICAL COMPOSITIONS

The present invention further provides pharmaceutical compositions, formulations, dosage units and methods of administration comprising the heterologous proteins produced by the transgenic avians using methods of the invneion. Preferably, compositions of the invention comprise a prophylactically or therapeutically effective amount of a the heterologous protein, and a pharmaceutically acceptable carrier.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound of the invention is administered. Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. When administered to a patient, the compounds of the invention and pharmaceutically acceptable vehicles are preferably sterile. Water is a preferred vehicle when the compound of the invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propyleneglycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable vehicle is a capsule (see e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical vehicles are described in "Remington: the Science and Practice of Pharmacy", 20th ed., by Mack Publishing Co. 2000.

In a preferred embodiment, the heterologous proteins are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compounds of the invention for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilizing agent. Compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the heterologous protein of the invention is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition of the invention is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. Compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions may contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds of the invention. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade.

Further, the effect of the heterologous proteins may be delayed or prolonged by proper formulation. For example, a slowly soluble pellet of the compound may be prepared

5 and incorporated in a tablet or capsule. The technique may be improved by making pellets of several different dissolution rates and filling capsules with a mixture of the pellets. Tablets or capsules may be coated with a film which resists dissolution for a predictable period of time. Even the parenteral preparations may be made long-acting, by dissolving or suspending the compound in oily or emulsified vehicles which allow it to disperse only

10 slowly in the serum.

5.5 TRANSGENIC AVIANS

Another aspect of the present invention concerns transgenic avians, preferably chicken or quail, produced by methods of the invention described in section 5.1 infra,

15 preferably by introducing a nucleic acid comprising a transgene into an avian oocyte by the sperm-mediated transfection methods of the present invention. In one embodiment, a heterologous nucleic acid introduced to an avian oocyte by sperm-mediated transfection, resulting in a transgenic embryo which is then allowed to develop, preferably, transferred into the reproductive tract of a recipient hen where it is encapsulated by natural egg white

20 proteins and a natural egg shell, then it is incubated and hatched to produce a transgenic chick. The heterologous polypeptide or polypeptides encoded by the transgenic heterologous nucleic acid may be secreted into the oviduct lumen of the mature transgenic chicken and deposited as a constituent component of egg white. The resulting transgenic avian chick (i.e, the GO) will carry one or more desired transgene(s) some or all of its cells,

25 preferably in its germ line. These GO transgenic avians can be bred using methods well known in the art to generate second generation (i.e., Gls) transgenic avians that carry the transgene, i.e., achieve germline transmission of the transgene. In preferred embodiments, the methods of the invention result in germline transmission, i.e., percentage of GOs that transmit the transgene to progeny (Gls), that is greater than 5%>, preferably, greater than

30 10%, 20%, 30%, 40%, and, most preferably, greater than 50%, 60%, 70%, 80%, 90% or even 100%>. In other embodiments, the efficiency of transgenesis (i.e., number of GOs containing the transgene) is greater than 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 99%.

The egg can be harvested after laying and before hatching of a chick, or further

35 incubated to generate a cloned chick, optionally genetically modified. The cloned chick may carry a transgene in all or most of its cells. After maturation, the transgenic avian may lay eggs that contain one or more desired heterologous protein(s).

The cloned chick may also be a knock-in chick expressing an alternative phenotype or capable of laying eggs having an heterologous protein therein. The reconstructed egg may also be cultured to term using the ex ovo method described by Perry et al. (supra).

Following maturation, the transgenic avian and/or transgenic progeny thereof, may lay eggs containing one or more desired heterologous protein(s) expressed therein and that can be easily harvested therefrom. The GI chicks, when sexually mature, can then be bred to produce progeny that are homozygous or heterozygous for the transgene. A transgenic avian of the invention may contain at least one transgene, at least two transgenes, at least 3 transgenes, at least 4 transgenes, at least 5 transgenes, and preferably, though optionally, may express the subject nucleic acid encoding a polypeptide in one or more cells in the animal, such as the oviduct cells of the chicken. In embodiments of the present invention, the expression of the transgene may be restricted to specific subsets of cells, tissues, or developmental stages utilizing, for example, cis-acting sequences that control expression in the desired pattern. Toward this end, it is contemplated that tissue- specific regulatory sequences, or tissue-specific promoters, and conditional regulatory sequences may be used to control expression of the transgene in certain spatial patterns. Moreover, temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences. The inclusion of a 5' MAR region, and optionally the 3' MAR on either end of the sequence, in the expression cassettes suitable for use in the methods of the present invention may allow the heterologous expression unit to escape the chromosomal positional effect (CPE) and therefore be expressed at a more uniform level in transgenic tissues that received the transgene by a route other than through germ line cells.

The transgenes may, in certain embodiments, be expressed conditionally, e.g., the heterologous protein coding sequence is under the control of an inducible promoter, such as a prokaryotic promoter or operator that requires a prokaryotic inducer protein to be activated. Operators present in prokaryotic cells have been extensively characterized in vivo and in vitro and can be readily manipulated to place them in any position upstream from or within a gene by standard techniques. Such operators comprise promoter regions and regions that specifically bind proteins such as activators and repressors. One example is the operator region of the lexA gene of E. coli to which the LexA polypeptide binds. Other exemplary prokaryotic regulatory sequences and the corresponding trans-activating prokaryotic proteins are disclosed by Brent and Ptashne in U.S. Patent No. 4,833,080 (the contents of which is herein incorporated by reference in its entirety). Transgenic animals can be created which harbor the subject transgene under transcriptional control of a prokaryotic sequence or other activator sequence that is not appreciably activated by avian proteins. Breeding of this transgenic animal with another animal that is transgenic for the corresponding trans-activator can be used to activate of the expression of the transgene. . Moreover, expression of the conditional transgenes can also be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g., a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell- type specific manner. Transactivators in these inducible or repressible transcriptional regulation systems are designed to interact specifically with sequences engineered into the transgene. Such systems include those regulated by tetracycline ("tet systems"), interferon, estrogen, ecdysone, Lac operator, progesterone antagonist RU486, and rapamycin (FK506) with tet systems being particularly preferred (see, e.g., Gingrich and Roder, 1998, Annu. Rev. Neurosci. 21 : 377-405; incorporated herein by reference in its entirety). These drugs or hormones (or their analogs) act on modular transactivators composed of natural or mutant ligand-binding domains and intrinsic or extrinsic DNA binding and transcriptional activation domains. In certain embodiments, expression of the heterologous peptidecan be regulated by varying the concentration of the drag or hormone in medium in vitro or in the diet of the transgenic animal in vivo.

In a preferred embodiment, the control elements of the tetracycline-resistance operon of E. coli is used as an inducible or repressible transactivator or transcriptional regulation system ("tet system") for conditional expression of the transgene. A tetracycline-controlled transactivator can require either the presence or absence of the antibiotic tetracycline, or one of its derivatives, e.g. , doxycycline (dox), for binding to the tet operator of the tet system, and thus for the activation of the tet system promoter (Ptet).

In a specific embodiment, a tetracycline-repressed regulatable system (TrRS) is used (Agha-Mohammadi and Lotze, 2000, J Clin. Invest. 105(9): 1177-83; Shockett et /., 1995, Proc. Natl. Acad. Sci. USA 92: 6522-26; and Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. USA 89: 5547-51; incorporated herein by reference in their entireties).

In another embodiment, a reverse tetracycline-controlled transactivator, e.g., rtTA2 S-M2, is used. rtTA2 S-M2 transactivator has reduced basal activity in the absence doxycycline, increased stability in eukaryotic cells, and increased doxycycline sensitivity (Urlinger et al, 2000, Proc. Natl. Acad. Sci. USA 97(14): 7963-68; incorporated herein by reference in its entirety). In another embodiment, the tet-repressible system described by Wells et al. (1999, Transgenic Res. 8(5): 371-81; incorporated herein by reference in its entirety) is used. In one aspect of the embodiment, a single plasmid Tet-repressible system is used.. In another embodiment, the GAL4-UAS system (Ornitz et al, 1991, Proc. Natl Acad. Sci. USA 88:698-702; Rowitch et al. , 1999, J. Neuroscience 19(20):8954-8965;

5 Wang et al, 1999, Proc. Natl. Acad. Sci. USA 96:8483-8488; Lewandoski, 2001, Nature Reviews (Genetics) 2:743-755) or a GAL4-VP16 fusion protein system (Wang et al, 1999, Proc. Natl. Acad. Sci. USA 96:8483-8488) is used.

In other embodiments, conditional expression of a transgene is regulated by using a recombinase system that is used to turn on or off the gene's expression by recombination in

10 the appropriate region of the genome in which the potential drug target gene is inserted. The transgene is flanked by recombinase sites, e.g., FRT sites. Such a recombinase system can be used to turn on or off expression a transgene (for review of temporal genetic switches and "tissue scissors" using recombinases, see Hennighausen & Furth, 1999, Nature Biotechnol 17: 1062-63). Exclusive recombination in a selected cell type may be mediated

15 by use of a site-specific recombinase such as Cre, FLP-wild type (wt), FLP-L or FLPe. Recombination may be effected by any art-known method, e.g., the method of Doetschman et al (1987, Nature 330: 576-78; incorporated herein by reference in its entirety); the method of Thomas et al, (1986, Cell 44: 419-28; incorporated herein by reference in its entirety); the Cre-loxP recombination system (Sternberg and Hamilton, 1981, J Mol. Biol

20 150: 467-86; Lakso et al, 1992, Proc. Natl Acad. Sci. USA 89: 6232-36; which are both incorporated herein by reference in their entireties); the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al, 1991, Science 251: 1351-55); the Cre-loxP- tetracycline control switch (Gossen and Bujard, 1992, Proc. Natl Acad. Sci. USA 89: 5547- 51, incorporated herein by reference in its entirety); and ligand-regulated recombinase

25 system (Kellendonk et al, 1999, J Mol Biol 285: 175-82; incorporated herein by reference in its entirety). Preferably, the recombinase is highly active, e.g., the Cre-loxP or the FLPe system, and has enhanced thermostability (Rodriguez et al, 2000, Nature Genetics 25: 139- 40; incorporated herein by reference in its entirety).

In a specific embodiment, the ligand-regulated recombinase system of Kellendonk et

30 al (1999, J Mol Biol 285: 175-82; incorporated herein by reference in its entirety) can be used. In this system, the ligand-binding domain (LBD) of a receptor, e.g., the progesterone or estrogen receptor, is fused to the Cre recombinase to increase specificity of the recombinase.

In the case of an avian, a heterologous polypeptide or polypeptides encoded by the

35 transgenic nucleic acid may be secreted into the oviduct lumen of the mature animal and deposited as a constituent component of the egg white into eggs laid by the animal. It is also contemplated to be within the scope of the present invention for the heterologous polypeptides to be produced in the serum of a transgenic avian.

A leaky promoter such as the CMV promoter may be operably linked to a transgene, resulting in expression of the transgene in all tissues of the transgenic avian, resulting in production of, for example, immunoglobulin polypeptides in the serum. Alternatively, the transgene may be operably linked to an avian promoter that may express the transgene in a restricted range of tissues such as, for example, oviduct cells and macrophages so that the heterologous protein may be identified in the egg white or the serum of a transgenic avian. Transgenic avians produced by the sperm-mediated transfection methods of the present invention will have the ability to lay eggs that contain one or more desired heterologous protein(s) or variant thereof.

One embodiment of the present invention, therefore, is a transgenic avian produced by the sperm-mediated transfection methods of the present invention and having a heterologous polynucleotide sequence comprising a nucleic acid insert encoding a heterologous polypeptide and operably linked to an avian lysozyme gene expression control region, the gene expression control region comprising at least one 5' matrix attachment region, an intrinsically curved DNA region, at least one transcription enhancer, a negative regulatory element, at least one hormone responsive element, at least one avian CR1 repeat element, and a proximal lysozyme promoter and signal peptide-encoding region.

Another embodiment of the present invention provides a transgenic avian further comprising a transgene with a lysozyme 3' domain.

Accordingly, the invention provides transgenic avians produced by methods of the invention as described infra. In preferred embodiments, the transgenic avian contains a transgene comprising a heterologous peptide coding sequence operably linked to a promoter and, in certain embodiments, other regulatory elements. In more preferred embodiments, the transgenic avians of the invention produce heterologous proteins, preferably in a tissue specific manner, more preferably such that they are deposited in the serum and, most preferably, such that the heterologous protein is deposited into the egg, particularly in the egg white. In preferred embodiments, the transgenic avians produce eggs containing greater than 5 μg, 10 μg, 50 μg, 100 μg, 250 μg, 500 μg, or 750 μg, more preferably greater than 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 700 mg, 1 gram, 2 grams, 3 grams, 4 grams or 5 grams of the heterologous protein. In preferred embodiments, the transgenic avians produce an immunoglobulin molecule and deposit the immunoglobulin in the egg or serum of the avian, and preferably, the immunoglobulin isolated from the egg or serum specifically binds its cognate antigen. The antibody so produced may bind the antigen with the same, greater or lesser affinity than the antibody produced in a mammalian cell, such as a myeloma or CHO cell.

In specific embodiments, the transgenic avians of the invention were not produced or are not progeny of a transgenic ancestor produced using a eukaryotic viral vector, more particularly, not a retroviral vector (although, in certain embodiments, the vector may contain sequences derived from a eukaryotic viral vector, such as promoters, origins of replication, etc.). The transgenic avians of the invention include GO avians, founder transgenic avians, GI transgenic avians, avians containing the transgene in the sperm or ova, avians mosaic for the transgene and avians containing copies of the transgene in most or all of the cells. Contemplated by the invention are transgenic avians in which the transgene is episomal. In more preferred embodiments, the transgenic avians have the transgene integrated into one or more chromosomes. Chromosomal integration can be detected using a variety of methods well known in the art, such as, but not limited to, Southern blotting, PCR, etc.

6. EXAMPLES

The present invention is further illustrated by the following examples. Each example is provided by way of explanation of the invention, and is not intended to be a limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications, combination, additions, deletions and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment. It is intended that the present invention covers such modifications, combinations, additions, deletions and variations as come within the scope of the appended claims and their equivalents.

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. 6.1 Example 1: Vectors Having Sperm-Specific Reporter Genes

The specific activity of spermatogenesis-specific promoters, such as the protamine promoter necessary for post-meiotic-specific transcription of this gene may be used to selectively mark those sperm cells that have inherited the transgene of interest after meiotic segregation.

The construct contains two separate elements. In one example, the first element comprises an oviduct-specific promoter, such as that associated with a gene encoding ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin or ovomucin, The promoter is operatively linked to, and therefore drives the expression of a gene coding for a desired heterologous protein of interest, such as, but not limited to, a therapeutic protein like interferon, erythropoietin (EPO), or an immunoglobulin.

The second element, which can be located either upstream or downstream from the first element, contains the protamine promoter, or any fragment thereof that is sufficient to drive the expression of a marker gene encoding a vital and color marker, such as the Green Fluorescent Protein (GFP). Those sperm cells that incorporate the transgene into their genomic DNA are vitally labeled during the late stages of spermiogenesis by the expression of the GFP protein. Given that the construct contains both the above first and the second elements, positive sperm cells also contain the transgene of interest.

Large numbers of positive sperm cells expressing the GFP protein are isolated using Fluorescent Activated Cell Sorting (FACS). Sperm cells selected on the basis of the expression of the incorporated marker gene are then used to breed hens by artificial insemination protocols. Suitable avian insemination protocols have been described by Etches (1996) Reprod. in Poultry (CAB International, Wallingford, UK), incorporated herein by reference in its entirety. In those cases where the number of positive sperm obtained after FACS isolation is too low for the likelihood of successful artificial insemination, the females may be fertilized by the intramagnal insemination method of Engel (1991) Poult. Sci. 70:1965-1969 or Trefil (1996) Br. Poult. Sci. 37:661-664, incorporated herein by reference in their entireties. Alternatively, small numbers of positive sperm cells are isolated under a microscope using UV light and then microinjected into unfertilized eggs via the Intracytoplasmic Sperm Injection (ICSI) protocols of Perry (1999), incorporated herein by reference in its entirety.

6.2 Example 2: Lipofection Gene Transfer to Avian Oocytes

(a) Isolation of the ovum: Donor hens were inseminated using the protocol for avian artificial insemination described by Etches (1996), incorporated herein by reference in its entirety. Fertilized ova were collected from the magnum region of the oviduct of euthanized birds 1.5-3 hours after oviposition. Alternatively, a hen whose oviduct is fistulated allows the collection of eggs for enucleation as taught by Gilbert and Woodgush, (1963, J Reprod. Fertility 5: 451-453) and Pancer et al, (1989, Br. Poult. Sci. 30: 953-7). The thick albumen capsule surrounding the ovum was removed using spatulas and the ovum was placed in a well 48mm diameter and 23 mm in height containing Perry's salt solution (see Perry (1988), incorporated herein by reference in its entirety).

(b) Preparation of lipofection solutions: Two lipofection solutions were used. The first solution comprised 50 μg/ml of LIPOFECT AMINE™ (Gibco) pre-incubated for 1 hour with the restriction endonuclease Not I (500 Units Not I per ml of lipofection solution), and designated herein as "Lipofectamine/Not I solution". The second lipofection solution was composed of 50μg/ml of LIPOFECTAMINE™ pre-incubated for 1 hour with 500μg of peGFP linearized with Not I per ml of lipofection solution, herein described as "Lipofectamine/peGFP solution." Lipofectin-treated eggs were then incubated for 1 hour. (c) Gene transfer to avian oocytes by lipofection: The isolated ovum was then placed inside a glass conical chamber (Figure 1 A) so that the blastodisc was located in the center of a window that opens at the narrower end of the conical chamber. A 40 mm diameter and 8 mm high glass dish was used at the bottom of the cone to close the system. Perry salt solution was added to the bottom of the dish to prevent drying of the lower half of the ovum. The Perry's salt solution overlaying the blastodisc (accessed through the window opening of the cone) was then replaced by, for example, 100 μl, of a lipofection solution described below. The eggs were incubated for 1 hour. Alternatively, egg incubation can be done by adding the lipofection solutions to the well and inverting the position of the incubation chamber (Figure IB), or by using a cloning cylinder around the blastodisc (Figure IC).

(d) Transfer of the lipofected egg: In a preferred embodiment, the ovum is surgically transferred into the oviduct of the recipient hen shortly after lipofection according to a described surgical procedure. (Tanaka, 1994, supra). The recipient hens are anesthetized by wing vein injection with pentobarbital (0.7 ml of a 68 mg/ml solution) or using gas anesthetics such as Isoflurane shortly after laying. During this window, the infundibulum is receptive to receiving a donor ovum but that has not yet ovulated. Feathers are removed from the abdominal area, the area is scrubbed with betadine and rinsed with 70%) ethanol. The bird is placed in a supine position and a surgical drape is placed over the bird exposing the surgical area. An incision is made beginning at the junction of the sternal rib to the breastbone and running parallel to the breastbone. The length of the incision is approximately 6cm. After cutting through the smooth muscle layers and the peritoneum, the infundibulum is located. The infundibulum is externalized and opened using gloved hands. The donor ovum is gently placed in the open infundibulum. Gravity facilitates the movement of the ovum through the infundibulum and into the anterior magnum. The internalized ovum is placed into the body cavity and the incision closed using interlocking stitches both for the smooth muscle layer and the skin. The recipient hen is returned to her cage and allowed to recover with free access to both feed and water. The hens resume normal activities after a post-operative recovery time of less than 45 minutes. Once transferred, the embryo develops inside the recipient hen and travels through the oviduct where it is encapsulated by natural egg white proteins and a natural eggshell. Eggs laid by the recipient hens are collected the next day, set, and incubated in a Jamesway incubator. The eggs hatch 21 days later.

6.3 Example 3: Maintenance of Plasmid Linearization in the Remi Procedure

A plasmid that is to be integrated into the genomic nucleic acid of a sperm is linearized by cleavage with a selected restriction endonuclease. The linearized nucleic acid is then dephosphorylated at the exposed 5' ends of the newly formed cohesive regions by alkaline phosphatase treatment. Suitable protocols for the alkaline phosphatase dephosphorylation of nucleic acids are disclosed, for example, by Sambrook et al, (supra), incorporated herein by reference in its entirety.

While not wishing to be bound by any one theory, it is believed that dephosphorylated cohesive ends of the nucleic acid may hybridize to recircularize the cleaved plasmid. Dephosphorylation of the 5' termini, however, prevent a DNA ligase from covalently rejoining a 5' terminus to the adjacent 3' terminus, thereby preventing a stable circular plasmid molecule from reforming. The cohesive ends of the non-ligated circularized plasmid may dissociate within a sperm cell to give a linearized nucleic acid that may integrate into the sperm genomic DNA.

Alternatively, a circular plasmid having a heterologous nucleic acid that is to be integrated into the genomic nucleic acid of a sperm is digested with at least two different restriction endonucleases that generate a linearized plasmid having two non-cohesive ends, and wherein the desired transgenic element heterologous nucleic acid remains intact between the new termini of the cleaved plasmid. The restriction endonucleases are selected to give dissimilar cohesive ends that cannot hybridize together to recircularize the cleaved plasmid. The linearized nucleic acid is then delivered to the sperm with both of the restriction endonucleases used to cleave the plasmid. The restriction endonucleases may be delivered to the sperm sequentially or simultaneously and combined, or sequentially delivered, with the cleaved plasmid.

It can be advantageous, depending upon the positions of the endonuclease cleavage sites within the plasmid relative to the desired transgene, to use two different endonucleases that produce hybridizable cohesive ends. In this case, the 5' termini may also be dephosphorylated with alkaline phosphatase as described above, to prevent religation and stabilization of the cleaved plasmid.

6.4 Example 4: Methods for Determing the SV40 Ori Requirement in SMT

To determine the requirement for the SV40 origin of replication in sperm-mediated transgenesis, 5 μg each of the plasmids pl083 (with the CMV promoter controlling heavy chain transcription) and pl086 (where the CMV promoter controls light chain transcription) were digested with Dra III which excises the SV40 origin of replication from the pl083 plasmid while retaining the SV40 origin of replication of the pl086 plasmid. For comparison, 5 μg each of the plasmids pi 083 and pi 086 were digested with the restriction endonuclease Mlu I that linearizes both plasmids while retaining the S V40 origin of replication in each of the respective plasmids.

Digested plasmids were used to transfect sperm. In a polystyrene tube, Dra III- digested plasmids pl086 and pl083 (5 μg of each) were added to 100 μl of OPTIMEM™ medium (Life Technologies, Gaithersburg, MD) and 10 μg of LIPOFECTAMINE™ liposome (Life Technologies, Gaithersburg, MD). In a separate tube, 100 units of Dra III restriction enzyme were added to 100 μl of OPTIMEM™ medium followed by 10 μg of LIPOFECTAMINE™. The tubes were incubated at room temperature for 30 minutes, then added to freshly collected semen containing 10⁹ chicken sperm (approximately 300 μl of semen). The sperm, DNA-liposome, and restriction enzyme-liposome mixture was incubated at room temperature for 30 minutes.

Two White Leghorn hens were then artificially inseminated with 250 μl each of the transfection mixture. Eggs were collected for 7 days starting on the second day after fertilization, and set for hatch. Two weeks after hatch, serum samples were collected and assayed for human monoclonal antibodies by ELISA. The results are shown in Figure 10, wherein wing band number 3932 is the control. 6.5 Example 5: Gamma Irradiation of Chicken Sperm

Exogenous, linearized DNA can be integrated into the genome of a recipient sperm cell by cleaving the double-stranded genomic DNA by gamma irradiation of the sperm prior to lipofection thereof with the transgenic nucleic acid. Wooster et al found that rooster sperm irradiated with 12 Grays (Gy) of gamma irradiation resulted in about 43% residual fertility. (1977, Can. J. Genet. Cytol. 19, 437- 446). Therefore, rooster semen will be irradiated with the following doses of gamma radiation: 0, 1, 5, 10, 15, and 20 Gy. A liposomal complex will consist of 10 μg of linearized DNA containing a promoter (e.g., CMV, ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin, etc.) and transgene (e.g., IFN, erythropoeitin, human monoclonal antibody immunoglobulin heavy and light chains, and GM-CSF, etc.) and 10 μg of LIPOFECTAMINE™ (Life Technologies, Gaithersburg, MD) will then be transfected into the irradiated sperm. After one hour, the irradiated and transfected sperm will be introduced into the hen by traditional artificial insemination procedures. Resulting laid eggs will be set and hatched, and transgene integration will be confirmed by Southern analysis of blood DNA.

6.6 Examples 6: Ovum Transfer to a Laying Hen

At the time of laying, recipient hens are anesthetized by wing vein injection with pentobarbital (0.7 ml of a 68 mg/ml solution) or by a gaseous anesthetic such as Isoflurane. Pentobarbital is the preferred anesthetic. At this time, the infundibulum is receptive to receiving a donor ovum but has not yet ovulated. Feathers are removed from the abdominal area, and the area is scrubbed with betadine, and rinsed with 70%> ethanol. The bird is placed in a supine position and a surgical drape is placed over the bird with the surgical area exposed. An incision is made beginning at the junction of the sternal rib to the breastbone and running parallel to the breastbone. The length of the incision is approximately two inches. After cutting through the smooth muscle layers and the peritoneum, the infundibulum is located. The infundibulum is externalized and opened using gloved hands and the donor ovum is gently applied to the open infundibulum. The ovum is allowed to move into the infundibulum and into the anterior magnum by gravity feed. The internalized ovum is placed into the body cavity and the incision closed using interlocking stitches both for the smooth muscle layer and the skin. The recipient hen is returned to her cage and allowed to recover with free access to both feed and water. Recovery time for the bird to be up, moving and feeding is usually within 45 minutes of the operation's end. Eggs laid by the recipient hens are collected the next day, set, and incubated. They will hatch 21 days later.

6.7 Example 7: Generation of Transgenic Chickens by Sperm-Mediated Transfection of Heterologous Nucleic Acid

Plasmid pRC/CMV-EGFP, 10 μg, was added to 100 μl of OPTIMEM™ medium (Life Technologies, Gaithersburg, MD) and 10 μg of LIPOFECTAMINE™ (Life Technologies, Gaithersburg, MD) liposomes, in a polystyrene tube. In a separate tube, 100 units of Dra III restriction enzyme was added to 100 μg of OPTIMEM™ medium followed by 10 μg of LIPOFECTAMINE™. As negative controls, plasmids ρl086 and pl083 were used for pRC/CMV-EGFP in the transfection mixture. Tubes were incubated at room temperature for 30 minutes, then added to 10⁹ freshly collected chicken sperm (approximately 300 μl of sperm). The sperm, DNA-liposome, and restriction enzyme- liposome mixture was incubated at room temperature for 30 minutes. Two White Leghorn hens were inseminated with the transfection mixture, each hen receiving approximately 250 μl of the transfection mixture. Eggs were collected for 7 days starting on the second day after fertilization, and set for hatch.

Four days after hatching, blood drops from chicks were collected from leg veins with heparinized capillary tubes and placed on microscope slides. Blood smears were viewed with FITC illumination with an inverted microscope (Olympus 1X70, 100 watt mercury lamp, HQ-FITC Band Pass Emission filter cube, excitation 480/40 nm, emission 535/50 nm, and 20X phase contrast objective). Auto-fluorescence was assessed using a TRITC filter (Olympus Modular B-MAX Filter cube, excitation 535/50 nm, emission 610/75 nm). Two chicks that resulted from sperm transfected with pRC/CMV-EGFP had white blood cells showing green fluorescence. No fluorescence was seen when viewed with the TRITC filter, indicating that the green fluorescence was not due to auto-fluorescence. None of the control chicks, derived from sperm transferred with control plasmids, had green fluorescence in their blood.

6.8 Example 8: Sperm-Mediated Transfection of Japanese Quail Ova

Prophetic Example

Japanese Quail hens will be artificially inseminated with sperm transfected with vectors capable of expressing α-IFN, erythropoietin or a monoclonal antibody. ELIS As will be used to detect and measure the amount of an expressed transgene product in the animal's serum and egg. As little as 15pg of α-interferon (α-IFN) or erythropoietin can be detected by this procedure.

To prepare the quail flock for artificial insemination, the females will be separated from the males. Once the isolated females are no longer laying fertile eggs and the males are consistently producing sufficient semen, the birds will be used for artificial insemination

(A.I) procedures.

Sperm mediated transgenesis (SMT) of the quail will be performed with two plasmid vectors, pRC/CMV-IFNMM-SV40 and pRC/CMV-EPOMM-SV40. Transgenesis resulting in the integration of, and expression from, a heterologous nucleic acid encoding α- IFN has been used successfully in chickens with both viral-based and sperm-mediated transfer (SMT)-based systems. The second vector will carry the gene encoding for erythropoietin. This protein requires more extensive post-translational modification, i.e. four glycosylations, than does α-IFN. Both of the plasmid vectors will produce their respective expressed polypeptides in serum and in ovo. Assaying for α-IFN or EPO production in serum will begin at two weeks of age and egg production will occur shortly thereafter. SMT will be performed with vectors having immunoglobulin heavy and light chain under the expression control of a lysozyme promoter.

About 50 chicks will be obtained from the SMT-A.I.'s. Based on results from our chicken SMT experiments, at least 2 to 4 transgenic quail for every 50 birds will be produced from the SMT-A.I. ' s.

6.9 Example 9: Preparation of Female and Male Japanese Quails for Sperm-Mediated Transfection by Artificial Insemination

The birds used will be selected for their optimal age for fertility, according to the average life history of the quail as shown in Table 1.

Table 1 : Japanese Quail life history

Females: Female Japanese Quail are separated from males and eggs inspected for fertilization over a 10 day period. Females will begin producing infertile eggs about 7 to 10 days after removal from the males.

Males: Males birds will be separated from females and conditioned for semen collection. The conditioning will be continued for 10 days. About 60% of males at their sexual peak will produce good semen, and consistently high volume semen producing birds will be progressively selected.

(a) Semen Quality and Lipofection Optimization: The extracted quail sperm will be added to a diluent that is at a higher pH than is typically used with chicken sperm. Quail sperm, compared to chicken sperm, require a higher pH to maintain motility once collected from the animal, as reported by Holm. L. & Wishart G.J. in Animal Reprod. Sci. 54: 45-54 (1998) and incorporated herein by reference in its entirety. A semen diluent having a pH of between about 8 and about 9 maintains motility better than does a pH of 7.

Artificial insemination (A.I): Each hen will be artificial inseminated with a 25 μl dose containing 2.5 x 10⁷ sperm per hen. Hens will be divided into Group 1 : 4 females inseminated with semen only; Group 2: 4 females inseminated with semen treated with LIPOFECTAMINE™; Group 3: 4 females sperm-mediated transfected with pCMV-IFN- SV40; Group 4: 4 females sperm-mediated transfected with pCMV-EPO-SV40. Since the average fertility of hens after artificial insemination is about 4 days, the hens will be inseminated twice a week to ensure delivery of a fresh supply of transfected semen. Transgenic positive birds will be mated to produce G_x chicks. The first three eggs of each bird will be screened for IFN, EPO or immunoglobulin polypeptide expression and the remaining eggs will be incubated to hatching.

(b) Hatchling care: Newly hatched chicks will be grown at 105 ° F-l 10°F for the first 4-5 days. The temperature will then be reduced by 5°F after the first and second week in 2° F increments. By the third week the house temperature will be sufficient. A 16/8 lighting schedule will also be used.

6.10 Example 10: Quail Semen Collection The male bird is grasped so its breastbone rests in the palm of the right hand. The tail is positioned so the first two fingers of right hand lay on either side of the vent just below the legs. Holding the male in an almost vertical position, the left hand gently squeezes four times at the base of the cloaca to remove the foamy secretions of the glandula proctodealis (foam gland). The vent is wiped to remove traces of the foamy substance and to prevent contamination of the semen. The left hand maintains firm pressure against the base of the cloaca and gently pulls back on dorsal proctodeal wall to achieve erection. The first two fingers of the right hand gently massage the abdomen and apply moderate pressure just below the vent to force semen from the vas defer ens into the copulatory organ. The semen will appear shortly thereafter. The viscous, pale yellow to white semen is collected with a 20 μl pipette and immediately diluted with 150 mM NaCl and 20mM N-tris[Hydroxymethyl]methyl-2-aminoethane-sulfonic acid (TES), at pH 8.0.

6.11 Example 11: Lipofection of Quail Sperm

Quail semen will be diluted, immediately after harvesting, to a concentration of 10⁸ sperm/ml in 150 mM NaCl and 20mM N-tris[Hydroxymethyl]methyl-2-aminoethane- sulfonic acid (TES), pH 8.0 buffer. Semen extender that is optimized for chicken sperm may not be used since it rapidly immobilizes quail semen within five minutes of contact. The lipofection procedures used with quail sperm will be similar to those adopted for chicken lipofection, including REMI sperm mediated tranfections (SMT). With the chicken SMT procedure, artificial insemination is with approximately 6 x 10⁸ sperm. Due to the limited amount of semen produced by male quail lxl 0^s quail sperm will be used per hen. The lowest number of sperm that will still gives maximum insemination will be adopted. Typically, the DNA (l.Oμg), restriction enzyme, LIPOFECTAMINE™ (l.Oμg) and sperm (10⁸) will be incubated together at a ratio of/ respectively for 30 minutes. All reactions will be carried out in OptiMEM™ medium (Gibco-BRL, Gaithersburg, MD).

6.12 Integration of Adeno- Associated Virus (Aav) Inverted Terminal Repeats-Flanked Genes Introduced by Sperm-Mediated Transgenesis

The chromosomal integration of plasmid DNA into the genome of an avian cell will be mediated by flanking the gene of interest and sequences related to its expression, with AAV inverted terminal repeat (ITR) sequences. A method for gene delivery and integration of heterologous nucleic acid sequences into the genomic DNA of a mammalian cell is described by Solis et al in U.S. Patent Serial No. 5,843,742 incorporated herein by reference in its entirety. A nucleic acid segment will also be included with the gene of interest that will result in the expression of the AAV Rep protein within the same cell. For example, a plasmid nucleic acid vector containing an expression cassette consisting of a CMV immediate early promoter driving the expression of human erythropoetin, will be flanked by AAV ITR sequences. This plasmid will be introduced by sperm-mediate transgenesis into targeted host cells together with a second nucleic acid vector plasmid. This second plasmid will include an expression cassette comprising the CMV immediate early promoter driving expression of the nucleic acid sequence encoding the AAV Rep 78 protein. Alternatively, a single nucleic acid vector comprising the expression cassette comprising the CMV immediate early promoter driving expression of the nucleic acid sequence encoding the AAV Rep 78 protein and the cassette expressing the gene of interest, such as erythropoetin, will be introduced together into an avian male gem cell.

6.13 Example 13: DNA Construct Modification to Improve Germline Transmission of Trangenes Following genetic modification in vertebrates, a low percentage of offsprings derived from the founder animals are transgenic given the low number of germline cells that carry the transgene. As a result, costly and cumbersome breeding of the founder animals is required to expand the number of transgenic animals derived from the original founder animals. A number of articles (e.g., Peschon, 1989, Ann. NY Acad Sci. 564: 186-197;

Peschon, 1987, PNAS 84: 5316-5319; Zambrowicz, 1993, PNAS 90: 5071; Braun, 1989, Gene Dev. 3:793-802; Rhim, 1995, Biol. Reprod. 52:20-32) as well as patent application(s) (O'Gorman et al, PCT Publication No. WO 99/10488) have identified and used the elements of the protamine promoter necessary for post-meiotic-specific transcription of this gene. Other spermiogenesis-specific promoters have also been described and used in the context of genetic manipulation (Sage, 1999, Mech. Dev. 80: 29-39; Vidal, 1998, Mol Reprod. Dev. 51: 274-280). In this example, we take advantage of the specific activity of these promoters to selectively mark those sperm cells that have inherited the transgene of interest after meiotic segregation. In the example described here, the construct would contain two independent elements. In a preferred example, the first element would comprise an oviduct-specific promoter, such as ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin. The promoter would drive expression of a gene coding for a protein of interest, such as a therapeutic protein like Interferon, erythroprotin (EPO). Alternatively, constitutive promoters such as CMV or RSV may also be used.

The second element, located up or downstream from the first, would contain the protamine promoter, or a segment of this promoter that is sufficient to drive the expression of a marker gene. In a preferred example, the protamine promoter would drive the expression of a marker, preferably a vital and color marker, such as the Green Fluorescent Protein (GFP). In such example, those sperm cells that have inherited the transgene would be vitally labeled during the late stages of spermiogenesis with the expression of the GFP protein. Given that the construct used contains both the first and the second elements described above, positive sperm cells would also contain the transgene of interest. Large numbers of positive sperm cells expressing the GFP proteins could be isolated using Fluorescent Activated Cell Sorting (FACS). These sperm cells could subsequently be used to breed hens by described artificial insemination protocols. (Etches, 1996, Mol. Reprod. Dev. 45:2918). In cases where the number of positive sperm after FACS isolation is low and insufficient for Al, the females could be bred through intramagnal insemination. (Engel, 1991, Poultry Sci. 70: 1965; Trefil, 1996, Br. Poult. Sci. 37: 661-664).

Alternatively, small numbers of positive sperm cells could be isolated under a microscope using UV light and injected into unfertilized eggs via described Intracytoplasmic Sperm Injection (ICSI) protocols. (Perry, 1999, Science 284: 1180-83).

6.14 Example 14: Use of Chicken Centromeric and Telomeric Sequences to

Create a Chicken Artificial Chromosome (ChAC)

The Shemesh et al. procedure (2000, Molecular Reproduction and Development 56: 306-308) for introducing linearized plasmid DNA into chicken sperm appears to rely on vector sequences which include an SV40 origin of replication. It is possible that the exogenous DNA therefore replicates as an episome and would most likely be lost in subsequent cell divisions due to improper segregation at mitosis. To insure proper segregation at mitosis, chicken centromere and telomere sequences could be included in the transgenic construct. Chicken centromere and telomere sequences could be obtained on a BAC (bacterial artificial chromosome) library clone from Texas A&M University or Martin Groenen at Wageningen Agricultural University, The Netherlands. The SV40 origin of replication and the promoter (i.e. CMV, ovalbumin, lysozyme, ovomucoid, ovotransferrin, conalbumin, and ovomucin, etc.) and transgene (i.e. IFN, EPO, human monoclonal antibody heavy and light chains, GM-CSF, etc.) combination could be cloned into the BAC clone containing the chicken centromere. This BAC would therefore contain an origin of replication, a centromere, telomere, and the promoter/transgene combination which could be transfected into sperm with the Shemesh procedure. Due to the chicken centromere and telomeres, the construct would replicate and segregate as a chicken artificial chromosome (ChAC).

6.15 Example 15: Construction of Lysozyme Promoter Plasmids

The chicken lysozyme gene expression control region was isolated by PCR amplification. Ligation and reamplification of the fragments thereby obtained yielded a contiguous nucleic acid construct comprising the chicken lysozyme gene expression control region operably linked to a nucleic acid sequence optimized for codon usage in the chicken (SEQ ID NO: 5) and encoding a human interferon α2b polypeptide optimized for expression in an avian cell.

White Leghorn Chicken (Gallus gallus) genomic DNA was PCR amplified using the primers 5ρLMAR2 (SEQ ID NO: 1) and LE-6.1kbrevl (SEQ ID NO: 2) in a first reaction, and Lys-6.1 (SEQ ID NO: 3) and LysElrev (SEQ ID NO: 4) as primers in a second reaction. PCR cycling steps were: denaturation at 94°C for 1 minute; annealing at 60°C for 1 minute; extension at 72°C for 6 minutes, for 30 cycles using TAQ PLUS PRECISION DNA polymerase (STRATAGENE®, LaJolla, CA). The PCR products from these two reactions were gel purified, and then united in a third PCR reaction using only 5pLMAR2 (SEQ ID NO: 1) and LysElrev (SEQ ID NO: 4) as primers and a 10-minute extension period. The resulting DNA product was phosphorylated, gel-purified, and cloned into the EcoR V restriction site of the vector PBLUESCRIPT® KS, resulting in the plasmid pl2.0-lys. pl2.0-lys was used as a template in a PCR reaction with primers 5pLMAR2 (SEQ ID NO: l) and LYSBSU (5'-CCCCCCCCTAAGGCAGCCAGGGGCAGGAAGCAAA-3') (SEQ ID NO: 5) and a 10 minute extension time. The resulting DNA was phosphorylated, gel-purified, and cloned into the EcoR V restriction site of PBLUESCRIPT® KS, forming plasmid pl2.01ys-B. pl2.01ys-B was restriction digested with Not I and Bsu36 1, gel-purified, and cloned into Not I and Bsu36 I digested pCMV-LysSPIFNMM, resulting in pl2.0-lys-LSPIFNMM. pl2.0-lys-LSPIFNMM was digested with Sal I and the SalltoNotl primer (5'- TCGAGCGGCCGC-3') (SΕQ ID NO: 13) was annealed to the digested plasmid, followed by Not I digestion. The resulting 12.5 kb Not I fragment, comprising the lysozyme promoter region linked to IFNMAGMAX-encoding region and an SV40 polyadenylation signal sequence, was gel-purified and ligated to Not I cleaved and dephosphorylated PBLTJESCRIPT® KS, thereby forming the plasmid pAVIJCR-Al 15.93.1.2, which was then sequenced.

6.16 Example 16: Construction of Plasmids Which Contain the 3' Lysozyme Domain

The plasmid pAVIJCR-Al 15.93.1.2 (containing the - 12.0 kb lysozyme promoter controlling expression of human interferon α2b) was purified with a QIAGEN® Plasmid Maxi Kit (QIAGEN®, Valencia, CA), and 100 μg of the plasmid were restriction digested with Notl restriction enzyme. The digested DNA was phenol/CHCl₃ extracted and ethanol precipitated. Recovered DNA was resuspended in ImM Tris-HCl (pH 8.0) and 0.1 mM EDTA, then placed overnight at 4°C. DNA was quantified by spectrophotometry and diluted to the appropriate concentration. The DNA samples were bound to the SV40 T antigen NLS peptide by incubation for 15 minutes.

The plasmid pAVIJCR-Al 15.93.1.2 was restriction digested with Fseϊ and blunt- ended with T4 DNA polymerase. The linearized, blunt-ended pAVIJCR-Al 15.93.1.2 plasmid was then digested with^TzoI restriction enzyme, followed by treatment with alkaline phosphatase. The resulting 15.4 kb DNA band containing the lysozyme 5' matrix attachment region (MAR) and -12.0 kb lysozyme promoter driving expression of a human interferon was gel purified by electroelution.

The plasmid plllilys was restriction digested with Mlu\, then blunt-ended with the Klenow fragment of DNA polymerase. The linearized, blunt-ended plllilys plasmid was digested with^ol restriction enzyme and the resulting 6 kb band containing the 3' lysozyme domain from exon 3 to the 3' end of the 3' MAR was gel purified by electroelution. The 15.4 kb band from pAVIJCR-Al 15.93.1.2 and the 6 kb band from plllilys were ligated with T4 DNA ligase and transformed into STBL4 cells (Invitrogen Life Technologies, Carlsbad, CA) by electroporation. The resulting 21.3 kb plasmids from two different bacterial colonies were named pAVIJCR-A212.89.2.1 and pAVIJCR-A212.89.2.3 respectively. 6.17 Example 17: Construction of an ALN-based Vector Having β-lactamase Encoding Sequences

The lacZ gene of pΝLB, a replication-deficient avian leukosis virus (ALV)-based vector (Cosset et al, J. Virol. 65: 3388-94 (1991)), was replaced with an expression cassette

5 consisting of a cytomegaloviras (CMV) promoter and the reporter gene β-lactamase (β-La or BL).

To efficiently replace the lacZ gene of pΝLB with a transgene, an intermediate adaptor plasmid was first created, pΝLB-Adapter. pΝLB-Adapter was created by inserting the chewed back ApaUApal fragment of pΝLB (Cosset et al, 1991, J. Virol 65:3388-94)

10 (in pΝLB, the 5 ' Apaϊ sites reside 289 bp upstream of lacZ and the 3 ' Apal sites reside 3 ' of the 3' LTR and Gag segments) into the c ewed-back Kpnl/Sacl sites of PBLUESCRIPT®KS(- ). The filled-inMwI/ b l fragment of pCMV-BL (Moore et al, Anal. Biochem. 247: 203-9 (1997)) was inserted into the chewed-back KpnVNdel sites of pΝLB-Adapter, replacing lacZ with the CMV promoter and the BL gene (in pΝLB, Kpnl resides 67 bp upstream of

15 lacZ and Nde resides 100 bp upstream of the lacZ stop codon), thereby creating pΝLB- Adapter-CMV-BL. To create pΝLB-CMV-BL, the Hindlϊl/Blpl insert of pΝLB (containing lacZ) was replaced with the Hindϊl/Blpϊ insert of pΝLB-Adapter-CMV-BL. This two step cloning was necessary because direct ligation of blunt-ended fragments into the HinάHllBlpl sites of pΝLB yielded mostly rearranged subclones, for unknown reasons.

20

6.18 Example 18: Production of Transduction Particles Having an ALV- based Vector Having β-lactamase Encoding Sequences

Sentas and Isoldes were cultured in F10 (GIBCO®), 5%> newborn calf serum (GIBCO®), 1% chicken serum (GIBCO®), 50 μg/ml phleomycin (Cayla Laboratories) and

25 50 μg/ml hygromycin (SIGMA®). Transduction particles were produced as described in Cosset et al, 1991, herein incorporated by reference, with the following exceptions. Two days after transfection of the retroviral vector pΝLB-CMV-BL (from Example 10, above) into 9 x 10⁵ Sentas, virus was harvested in fresh media for 6-16 hours and filtered. All of the media was used to transduce 3 x 10⁶ Isoldes in three 100 mm plates with polybrene

30 added to a final concentration of 4 μg/ml. The following day the media was replaced with media containing 50 μg/ml phleomycin, 50 μg/ml hygromycin and 200 μg/ml G418 (SIGMA®). After 10-12 days, single G418^r colonies were isolated and transferred to 24- well plates. After 7-10 days, titers from each colony was determined by transduction of Sentas followed by G418 selection. Typically 2 out of 60 colonies gave titers at 1-3 x 10⁵. 5 Those colonies were expanded and the virus concentrated to 2-7 x 10⁷ as described in Allioli et al, 1994, Dev. Biol. 165:30-7, herein incorporated by reference. The integrity of the CMV-BL expression cassette was confirmed by assaying for β-lactamase in the media of cells transduced with NLB-CMV-BL transduction particles.

6.19 Example 19: pNLB-CMV-IFN Vector Having an IFN Encoding

Sequence

The DNA sequence for human interferon α2b based on hen oviduct optimized codon usage was created using the BACKTRANSLATE program of the Wisconsin Package, version 9.1 (Genetics Computer Group. Inc., Madison, Wl) with a codon usage table compiled from the chicken (Gallus gallus) ovalbumin, lysozyme, ovomucoid, and ovotransferrin proteins. The template and primer oligonucleotides (SEQ ID NOS: 14-31) shown in Figures 8A-B were amplified by PCR with Pfu polymerase (STRATAGENE®, La Jolla, CA) using 20 cycles of 94°C for 1 min., 50°C for 30 sec, and 72°C for 1 min. and 10 sec. PCR products were purified from a 12%> polyacrylamide-TBE gel by the "crash and soak" method (Maniatis et al. 1982), then combined as templates in an amplification reaction using only IFN-1 (SEQ ID NO: 21) and IFN-8 (SEQ ID NO: 31) as primers. The resulting PCR product was digested with Hind III and Xba I and gel purified from a 2% agarose-TAE gel, then ligated into Hind HI and Xba I digested, alkaline phosphatase-treated, PBLUESCRIPT® KS (STRATAGENE®), resulting in the plasmid pBluKSP-IFNMagMax. Both strands were sequenced by cycle sequencing on an ABI PRISM 377 DNA Sequencer (Perkin-Elmer, Foster City, CA) using universal T7 or T3 primers. Mutations in pBluKSP- IFN derived from the original oligonucleotide templates were corrected by site-directed mutagenesis with the Transformer Site-Directed Mutagenesis Kit (Clontech, Palo Alto, CA). The interferon coding sequence was then removed from the corrected pBluKSP-IFN with Hind III and Xba 1, purified from a 0.8%o agarose-TAE Gel, and ligated to Hind III and Xba I digested, alkaline phosphatase-treated pCMV-BetaLa-3B-dH. The resulting plasmid was pCMV-IFN which contained IFN coding sequence controlled by the cytomegaloviras immediate early promoter/enhancer and SV40 polyA site. To clone the IFN coding sequence controlled by the CMV promoter/enhancer into the NLB retroviral plasmid, pCMV-IFN was first digested with CM andXbal, then both ends were filled in with Klenow fragment of DNA polymerase (New England BioLabs, Beverly, MA). pNLB-adapter was digested with Nde I and Kpn I, and both ends were made blunt by T4 DNA polymerase (New England BioLabs). Appropriate DNA fragments were purified on a 0.8% agarose-TAE gel, then ligated and transformed into DH5α cells. The resulting plasmid was pNLB-adapter-CMV-IFN.

This plasmid was then digested with Mlu I and partially digested with Blp I and the appropriate fragment was gel purified. pNLB-CMV-EGFP was digested with Mlu I and Blp 5 I, then alkaline-phosphatase treated and gel purified. The Mlu llBlp I partial fragment of pNLB-adapter-CMV-IFN was ligated to the large fragment derived from the Mlu VBlp I digest of pNLB-CMV-EGFP, creating pNLB-CMV-IFN.

6.20 Example 20: Production of pNLB-CMV-IFN Transduction Particles

10 Senta packaging cells (Cosset et al, 1991) were plated at a density of 3 x 10⁵ cells/35 mm tissue culture dish in F-10 medium (Life Technologies) supplemented with 50% calf serum (Atlanta Biologicals), 1%> chicken serum (Life Technologies), 50 μg/ml hygromycin (SIGMA®), and 50 μg/ml phleomycin (CAYLA, Toulouse, France). These cells were transfected 24h after plating with 2 μg of CsCl-purified pNLB-CMV-IFN DNA

15 and 6 μl of Lipofectin liposomes (Life Technologies) in a final volume of 500 μl Optimem (Life Technologies). The plates were gently rocked for four hours at 37° C in a 5%> CO₂ incubator. For each well, the media was removed, washed once with 1 ml of Optimem and re-fed with 2 mis of F-10 medium supplemented with 50% calf serum, 1% chicken serum, 50 μg/ml hygromycin, and 50 μg/ml phleomycin. The next day, medium from transfected

20 Sentas was recovered and filtered through a 0.45 micron filter.

This medium was then used to transduce Isolde cells. 0.3 ml of the filtered medium recovered from Senta cells was added to 9.6 ml of F-10 (Life Technologies) supplemented as described above, in addition to polybrene (SIGMA®) at a final concentration of 4 μg/ml. This mixture was added to 10⁶ Isolde packaging cells (Cosset et al, 1991) plated on a

25 100mm dish the previous day, then replaced with fresh F-10 medium (as described for Senta growth) 4 hours later.

The next day, the medium was replaced with fresh medium which also contained 200 μg/ml neomycin (G418, SIGMA®). Every other day, the medium was replaced with fresh F-10 medium supplemented with 50% calf serum, 1%> chicken serum, 50 μg/ml

30 hygromycin, 50 μg/ml phleomycin, and 200 μg/ml neomycin. Eleven to twelve days later, single colonies were visible by eye, and these were picked and placed into 24 well dishes. When some of the 24 well dishes became confluent, medium was harvested and titered to determine the cell lines with the highest production of retrovirus.

Titering was performed by plating 7.5 x 10⁴ Senta cells per well in 24 well plates on

35 the day prior to viral harvest and transduction. The next day 1 ml of fresh F-10 medium supplemented with 50%> calf serum, l%o chicken serum, 50 μg/ml hygromycin, and 50 μg/ml phleomycin was added to each well of the isolated Isolde colonies. Virus was harvested for 8-10 hours. The relative density of each well of Isoldes was noted. After 8-10 hours, 2 and 20μl of media from each well of Isoldes was added directly to the media of duplicate wills of the Sentas. Harvested medium was also tested for the presence of interferon by IFN ELIS A and for interferon bioreactivity. The next day the media was replaced with F-10 medium supplemented with 50% calf serum, 1% chicken serum, 50 μg/ml hygromycin, 50 μg/ml phleomycin, and 200 μg/ml neomycin. When obvious neomycin-resistant colonies were evident in the wells of transduced Sentas, the number of colonies was counted for each well.

The Isolde colony producing the highest titer was determined by taking into account the number of colonies and correcting for the density of the Isolde cells when the viral particles were harvested (i.e., if two Isolde colonies gave rise to media with the same titer, but one was at a 5% density and the other was at a 50%> density at the time of viral harvest, the one at the 5%> density was chosen for further work, as was the case in the present example).

The Isolde cell line producing the highest titer of IFN-encoding transducing particles was scaled up to six T-75 tissue culture flasks. When flasks were confluent, cells were washed with F-10 medium (unsupplemented) and transducing particles were then harvested for 16 hours in 14 ml/flask of F-10 containing 1% calf serum (Atlanta Biologicals) and 0.2%) chicken serum (Life Technolocyies). Medium was harvested, filtered through a 0.45 micron syringe filter, then centrifuged at 195,000xg in a Beckman 60Ti rotor for 35 min. Liquid was removed except for 1 ml, and this was incubated with the pellet at 37°C with gentle shaking for one hour. Aliquots were frozen at -70°C. Transducing particles were then titered on Senta cells to determine concentrations used to inject avian sperms.

6.21 Example 21: Construction of Lysozyme Promoter Plasmids

The chicken lysozyme gene expression control region isolated by PCR amplification is fully disclosed in U.S. Patent Application Serial No. 09/922,549, filed August 3, 2001 and incorporated herein by reference in its entirety. Ligation and reamplification of the fragments thereby obtained yielded a functionally contiguous nucleic acid construct comprising the chicken lysozyme gene expression control region operably linked to a nucleic acid sequence encoding a human interferon α2b polypeptide and optimized for codon usage in the chicken. Briefly, chicken (Gallus gallus (White Leghorn)) genomic DNA was PCR amplified using the primers 5pLMAR2 and LE-6.1kbrevl in a first reaction, and Lys-6.1 and LysElrev as primers in a second reaction. PCR cycling steps were: denaturation at 94 °C for 1 minute; annealing at 60 °C for 1 minute; extension at 72 °C for 6 minutes, for 30 cycles using TAQ PLUS PRECISION™ DNA polymerase (STRATAGENE®, La Jolla, CA). The PCR products from these two reactions were gel

5 purified, and then united in a third PCR reaction using only 5pLMAR2 and LysElrev as primers and a 10 minute extension period. The resulting DNA product was phosphorylated, gel-purified, and cloned into the EcoR V restriction site of the vector PBLUESCRIPT® KS, resulting in the plasmid pl2.0-lys. pl2.0-lys was used as a template in a PCR reaction with primers 5pLMAR2 and

10 LYSBSU and a 10 minute extension time. The resulting DNA was phosphorylated, gel- purified, and cloned into the EcoR V restriction site of PBLUESCRIPT® KS, forming plasmid pl2.01ys-B. pl2.01ys-B was restriction digested with Not I and Bsu361, gel-purified, and cloned into Not I and Bsu36 1 digested pCMV-LysSPIFNMM, resulting in pl2.0-lys-LSPIFNMM.

15 pl2.0-lys-LSPIFNMM was digested with Sal I and the SalltoNotl primer was annealed to the digested plasmid, followed by Not I digestion. The resulting 12.5 kb Not I fragment, comprising the lysozyme promoter region linked to IFNMAGMAX-encoding region and an SV40 polyadenylation signal sequence, was gel-purified and ligated to Not I cleaved and dephosphorylated PBLUESCRIPT® KS, thereby forming the plasmid pAVIJCR-Al 15.93.1.2.

20

6.22 Example 22: Complete Lysozyme Promoter and IFNMAGMAX Sequences

The complete sequences of the lysozyme gene promoter and the codon-optimized human interferon α2b nucleic acid are fully disclosed in U.S. Patent Application No.

25 09/922,549, filed 03 August 2001 and incorporated herein by reference in its entirety. The complete nucleotide sequence of the approximately 12.5 kb chicken lysozyme promoter region/IFNMAGMAX construct spans the 5' matrix attachment region (5' MAR), through the lysozyme signal peptide, to the sequence encoding the gene IFNMAGMAX and the subsequent polyadenylation signal sequence. The IFNMAGMAX nucleic acid sequence

30 had been synthesized as described in Example 21 above. The expressed IFN α2b sequence within plasmid pAVIJCR-A115.93.1.2 functioned as a reporter gene for lysozyme promoter activity. This plasmid construct may also be used for production of interferon α2b in the egg white of transgenic chickens.

35 6.23 Example 23: Synthesis of the MDOT promoter construct

Amplification of the ovomucoid and ovotransferrin promoter sequences

Oligonucleotide primers 1 (SEQ ID NO: 32) and 2 (SEQ ID NO: 33), as shown in Figure 9 were used to amplify the ovomucoid sequences. Oligonucleotide primers 3 (SEQ ID NO: 34) and 4 (SEQ ID NO: 35) were used to amplify the ovotransferrin sequence by PCR. The primers were designed such that the PCR-amplified ovomucoid sequences contained an Xho I restriction cleavage site at the 5' end and a Cla I site at the 3 ' end. Similarly, the PCR-amplified ovotransferrin product had a Cla I restriction site at the 5' end and a Hind III site at the 3 ' end. The overlapping Cla I site was used to splice the two-PCR products to create the MDOT promoter constract. The nucleic acid sequence SEQ ID NO: 11 of the MDOT promoter constract is shown in Figure 11. The final product was cloned in a bluescript vector between the Xho I and Hind III sites. From the bluescript vector the promoter region was released by Kpn II Hind III restriction digestion and cloned into the prc- CMV-IFN vector to replace the CMV promoter to create MDOT-IFN (clone #10). This plasmid was tested in vitro .

6.24 Example 24: Testicular Injection

5 weeks old White Leghorn male chickens were anesthetized using Isoflourane. Small incision was made between the last two ribs to expose the testes. A 5-10 μl virus suspension of pLNHX-CMV-EGFP/VSVg (9 x 10⁶ per ml) was injected into either both testes or only one of the testes.

At 20 weeks of age, semen samples were collected. Only one bird had sperm in his semen. Genomic DNA was isolated from the semen and used to amplify the transgene (CMV-EGFP) by PCR reaction using different DMSO concentrations. The samples were separated on agarose gel, transferred onto nitrocellulose membrane and hybridized with EGFP probe. As shown in Figure 11, EGFP positive bands are detected at two different DMSO concentrations suggesting that (1) specific PCR conditions are required for the amplification of the transgene and (2) the sperm samples have incorporated the transgene in their genome.

EQUIVALENTS

Reference now will be made in detail to the various embodiments of the invention, one or more examples of which are illustrated in the accompanying drawings. Each example is provided by way of explanation of the invention, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications, combinations, additions, deletions and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment. It is intended that the present invention covers such modifications, combinations, additions, deletions and variations as fall within the scope of the appended claims and their equivalents.

Claims

What is Claimed Is:

1. A method of generating a transgenic avian zygote by sperm-mediated transfection, said method comprising:

(a) obtaining a suspension of avian male germ cells selected from the group consisting of spermatozoa and spermatozoal precursor cells;

(b) introducing a nucleic acid comprising a transgene comprising a nucleotide sequence encoding a heterologous polypeptide to the avian male germ cells by lipofection, electroporation or restriction enzyme mediated integration;

(c) delivering the avian male germ cells having the nucleic acid to an avian oocyte , thereby generating a transgenic avian zygote having the nucleic acid incorporated therein.

2. The method of Claim 1, wherein the avian male germ cells and the avian oocyte are obtained from a chicken.

3. The method of Claim 1, wherein the avian male germ cells and the avian oocytes are obtained from a quail.

4. The method of Claim 1, wherein the nucleotide sequence encoding said heterologous polypeptide is operably linked to a transcriptional regulatory element that can direct gene expression in one or more cells of said transgenic avian.

5. The method of Claim 4, wherein the transcriptional regulatory element is selected from the group consisting of the promoter regions of the avian genes encoding ovalbumin, lysozyme, ovomucoid, ovomucin, conalbumin and ovotransferrin.

6. The method of Claim 5, wherein the selected nucleic acid further comprises a chicken lysozyme gene expression controlling region comprising the nucleotide sequence of SEQ ID NO: 7.

7. The method of Claim 4, wherein the transcriptional regulatory element is a tissue specific promoter.

8. The method of Claim 7, wherein the tissue specific promoter is specific for the magnum.

9. The method of Claim 1 , wherein the transgene comprises at least one cytomegaloviras promoter.

10. The method of Claim 9, wherein the transcriptional regulatory element comprises at least two regions derived from the promoter of an avian gene, said regions being from a different promoter.

11. The method of Claim 10, wherein the transcriptional regulatory element has the nucleotide sequence of SEQ ID NO: 11.

12. The method of Claim 1, wherein the transgene comprises at least one matrix attachment region (MAR).

13. The method of Claim 12, wherein the transgene comprises a 5' MAR and a 3' MAR which flank said nucleotide sequence.

14. The method of Claim 1, wherein the heterologous polypeptide is selected from the group consisting of a cytokine, a hormone, an enzyme, a structural polypeptide, and an immuoglobulin polypeptide.

15. The method of Claim 14, wherein the cytokine is selected from the group consisting of interferon, interleukin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, stem cell factor, erythropoietin, thrombopoietin, and stem cell factor.

16. The method of Claim 15, wherein the cytokine is an interferon.

17. The method of Claim 1 , wherein the transgene comprises an internal ribosome entry site (IRES).

18. The method of Claim 17, wherein the transgene comprises at least two nucleotide sequences each encoding a heterologous polypeptide.

19. The method of Claim 18, wherein the at least two nucleotide sequences encode at least two heterologous peptides that form a multimeric protein.

20. The method of Claim 19, wherein the multimeric protein specifically binds a selected ligand.

21. The method of Claim 20, wherein the multimeric protein is an antibody.

22. The method of Claim 1 , wherein the heterologous polypeptide comprises a peptide region suitable for the isolation of the heterologous polypeptide.

23. The method of Claim 1 , wherein the nucleic acid is a eukaryotic viral vector.

24. The method of Claim 23, wherein the eukaryotic viral vector is derived from any of the group consisting of avian leukosis viras, adenoviras, transferrin-polylysine enhanced adenoviral vectors, human immunodeficiency viras vectors, lentiviral vectors, and Moloney murine leukemia virus-derived vectors.

25. The method of Claim 1 , wherein the nucleic acid is a plasmid vector.

26. The method of Claim 1 , wherein the nucleic acid is a bacterial artificial chromosome (BAC).

27. The method of Claim 1 , wherein the nucleic acid is not a eukaryotic viral vector.

28. The method of Claim 4, wherein the transcriptional regulatory element is a regulatable promoter.

29. The method of Claim 6, wherein the selected nucleic acid further comprises a region encoding the 3' region of the chicken lysozyme gene and having the nucleotide sequence of SEQ ID NO: 9.

30. The method of Claim 1 , wherein the nucleotide sequence encoding said heterologous polypeptide comprises an origin of replication.

31. The method of Claim 30, wherein the origin of replication is the SV40 origin of replication.

32. The method of Claim 1, wherein the nucleic acid is selected from the group consisting of a linear nucleic acid, a plasmid, a viral nucleic acid, and an artificial chromosome.

5 33. The method of Claim 32, wherein the artificial chromosome further comprises a centromere and optionally a telomere.

34. The method of Claim 32, wherein the linear nucleic acid has at least one cohesive end characterized by the cohesive end generated by a restriction endonuclease.

10

35. The method of Claim 32, wherein the linear nucleic acid has at least one blunt end.

36. The method of Claim 34, wherein the at least one cohesive end is generated by chemical synthesis.

15

37. The method of Claim 34, wherein the at least one cohesive end is generated by an enzyme other than a restriction endonuclease.

38. The method of Claim 34, wherein the at least one cohesive end is generated by a 20 combination of chemical and enzymatic methods.

39. The method of Claim 1 , wherein the nucleic acid is introduced to the avian male germ cells by restriction enzyme mediated integration.

25 40. The method of Claim 39, further comprising the step of delivering to the avian male germ cells a restriction endonuclease capable of cleaving the genomic nucleic acid of the avian male germ cells.

41. The method of Claim 40, wherein the nucleic acid is delivered sequentially with the 30 restriction endonuclease to the avian male germ cells.

42. The method of Claim 1 , wherein the nucleic acid is delivered to the avian male germ cells by adeno-associated virus-derived vector.

35

43. The method of Claim 42, wherein the nucleic acid is bounded by inverted terminal repeat sequences.

44. The method of Claim 42, wherein the nucleic acid is bounded by inverted terminal 5 repeat sequences derived from an adeno-associated virus-derived vector.

45. The method of Claim 42, wherein the adeno-associated virus-derived vector further comprises a transcription cassette capable of expressing an adeno-associated viras Rep protein.

10

46. The method of Claim 45, wherein the Rep protein is Rep 78.

47. The method of Claim 45, wherein the nucleic acid bounded by inverted terminal repeat sequences is inserted in a first nucleic acid vector and the transcription cassette

15 capable of expressing an adeno-associated virus Rep protein is inserted in a second nucleic acid vector.

48. The method of Claim 1 , further comprising the step of irradiating the avian male germ cells, thereby cleaving the nuclic acid, wherein the radiation is selected from the group

20 consisting of ultraviolet light, gamma rays, X-rays, and ultrasound.

49. The method of Claim 1, wherein the avian oocyte is an isolated oocyte, and wherein the avian male germ cells having the nucleic acid are delivered to the isolated oocyte by a method selected from the group consisting of microinjection, intracytoplasmic sperm

25 injection (ICSI), and artificial insemination.

50. The method of Claim 49, wherein the avian male germ cells having the nucleic acid therein are delivered to the nucleus of the oocyte.

30 51. The method of Claim 1 , wherein the nucleic acid forms an episome in the avian male germ cells.

52. The method of Claim 1, wherein the nucleic acid in the avian oocyte is an episome.

35

53. The method of Claim 1, further comprising isolating an avian oocyte from the female of an avian by:

(a) removing an ovum from a bird after ovulation and before fertilization; and

(b) removing an albumen layer from the ovum.

5

54. The method of Claim 1, further comprising the steps of:

(a) fistulating an avian female;

(b) delivering the transgenic avian zygote to the infundibulum of the avian female such that said transgenic avian zygote is subsequently laid by said

10 avian female as a shelled egg; and

(c) incubating the shelled egg until said shelled egg hatches, thereby producing a transgenic avian containing the transgene.

15 55. The method of Claim 54, wherein the heterologous polypeptide is expressed in one or more cells of said transgenic avian .

56. The method of Claim 55, wherein the heterologous polypeptide is expressed in the serum of said transgenic avian.

20

57. The method of Claim 55, wherein the heterologous polypeptide is expressed in the magnum of said transgenic avian.

58. The method of Claim 54, further comprising the step of allowing the transgenic 25 avian to develop to sexual maturity.

59. The method of Claim 58, wherein the heterologous polypeptide is delivered to the white of a developing avian egg produced by the transgenic avian.

30 60. The method of Claim 55 or 59 further comprising isolating said heterologous polypeptide from said transgenic avian or an egg produced by the transgenic avian.

61. A transgenic avian that produces at least one heterologous polypeptide in egg white, wherein the transgenic avian or founder ancestor of said transgenic avian was not produced 35 using a eukaryotic viral vector.

62. A transgenic avian produced by the method of Claim 54.

63. The transgenic avian of Claim 61 or 62, wherein the avian is a chicken.

5 64. The transgenic avian of Claim 63, wherein the heterologous polypeptide is selected from the group consisting of a cytokine, a hormone, an enzyme, a structural protein, and an immunglobulin polypeptide.

65. The transgenic avian of Claim 63, wherein the cytokine is an interferon. 10

66. The transgenic avian of Claim 61 or 62, wherein the transgenic avian produces a heterologous multimeric protein.

67. The transgenic avian of Claim 66, wherein the heterologous multimeric protein 15 specifically binds a selected ligand.

68. The transgenic avian of Claim 66, wherein the heterologous multimeric protein is an antibody.

20 69. An avian egg produced by the transgenic avian of Claim 61 or 62.

70. An avian egg produced by the transgenic avian of any of Claims 63-68.

71. A heterologous protein heterologous protein produced by the transgenic avian of 25 Claim 61 or 62, wherein the heterologous protein comprises a heterologous polypeptide selected from the group consisting of a cytokine, a hormone, an enzyme, a stractural protein, and an immunoglobulin polypeptide.

72. The heterologous polypeptide of Claim 71, wherein the cytokine is an interferon. 30

73. The heterologous protein of Claim 71 , wherein the heterologous protein is a multimeric protein.

74. The heterologous protein of Claim 71 , wherein the heterologous protein is an 35 antibody.