Abstract:
Transgenes encoding exogenous proteins are stably integrated into embryonic stem cells and are present in the somatic tissue of transgenic or chimeric birds. The transgenes encode exogenous proteins and are expressed in any of endodermal, ectodermal, mesodermal, or extra embryonic tissue. Tissue specificity is provided by selecting the content of the transgene accordingly. Transgenic birds whose genome is comprised of trangene derived exogenous DNA express exogenous proteins with tissue specificity, and specifically express exogenous proteins in the tubular gland cells of the oviduct to concentrate exogenous proteins in egg white.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a divisional of U.S. application Ser. No. 10/524,089 filed Feb. 9, 2005, which claims priority to the National phase of WO 2004/015123 filed Aug. 11, 2003, the disclosure of which is fully incorporated herein by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    This invention relates to the field of protein expression in transgenic and chimeric animals, and enabling technologies such as genetic engineering, transgenics, and the long-term culture of embryonic stem cells. Embryonic stem cells are engineered with specially designed genetic constructs to introduce genetic modification into birds, including by the insertion of transgenes that yield tissue specific expression of exogenous proteins. Transgenic birds of the invention express the transgene-derived exogenous protein in the oviduct and the protein is deposited in large quantities in the egg. 
       BACKGROUND OF THE INVENTION 
       [0003]    Genetically engineered animals offer the potential for tremendous advances in the production of valuable pharmaceutical products from the cells of such animals. However, the production of genetically modified animals involves significant technical hurdles that have only been overcome for a few species. The ability to incorporate genetic modifications into the permanent DNA of a species requires several distinct technologies that must be developed for each genetically engineered species. One approach to alter the genetic and physical characteristics of an animal is to use embryonic stem cells that contribute to the phenotype of an animal when injected into an embryonic form of the animal. Embryonic stem cells have the ability to contribute to the tissue of an animal born from the recipient embryo and to contribute to the genome of a transgenic organism created by breeding chimeras. 
         [0004]    Significant expenditure of time and resources has been committed to the study and development of embryonic stem cell lines, the manipulation of the genome of the cells, and cell culture techniques that permit such engineered cells to be maintained in culture. Although many attempts have been made, the ability to sustain the pluripotency of engineered embryonic stem cells in culture has been achieved for only a few species, notably mice. For other species, the promise of genetic engineering in transgenics for protein production has been frustrated by the lack of sustainable long-term embryonic stem cell cultures. 
         [0005]    If sustainable cultures of embryonic stem cells were readily available and susceptible to genetic engineering while maintaining pluripotency, a broad application of new technologies would be available. Because embryonic stem cells contribute to the permanent DNA of an animal, the physiological characteristics of the animal from which an embryonic stem cell was derived can be transferred to a recipient embryo by incorporating these cells into the recipient animal in an embryonic state. This offers two principal advantages: first, the phenotype of an animal from which embryonic stem cells are derived can be selectively transferred to a recipient embryo. Second, as noted above, when the embryonic stem cell cultures are particularly stable, the genome of the cells can be modified genetically to introduce genetic modifications into a recipient embryo in which the cells are introduced. 
         [0006]    In certain cases, the embryonic stem cells can be engineered with a transgene that encodes an exogenous protein. The transgene is a genetic construct that contains DNA that acts as the blueprint for the production of a valuable protein and contains sufficient coding and regulatory elements to enable the expression of the protein in the tissue of the animal that is created from the insertion of the stem cells into a recipient embryo. In many cases, the expression of a protein is particularly valuable because the protein can be collected and isolated from the transgenic animal. However, the collection of a valuable protein from the tissues of an animal typically requires that the expression be limited to certain specific tissues that facilitate collection of the expressed protein. For example, in cows, the expression of a protein in the milk would enable the ready collection of the protein by simply collecting the milk of the cow and separating the exogenous protein. In chickens, the robust production of proteins in the white of the egg also provides an attractive vehicle for the expression of exogenous proteins. If the expression of valuable proteins could be achieved in this manner, the animal could be used as a vehicle for production of proteins that is superior to other production methods. Thus, one particularly attractive field of research and attractive area for commercial development is genetically engineered animals that express selected exogenous proteins in specific tissue that facilitate isolation and collection of the protein. The ability to produce exogenous proteins in specifically selected cells of an animal is also particularly valuable because the absence of tissue specificity simply results in the protein being expressed in all of the tissues of an animal. Under such circumstances, it is unlikely that a meaningful quantity of the protein could be separated from the animal, and furthermore the ubiquitous expression of an exogenous protein is usually very damaging to the overall health and well being of the animal. 
         [0007]    If an embryonic stem cell culture is sufficiently stable to allow a transgene to become integrated into the genome of the embryonic stem cell, a transgene encoding tissue specific expression of a protein can be passed to a new chimeric or transgenic organism by several different techniques depending on the specific construct used as the transgene. Whole genomes can be transferred by cell hybridization, intact chromosomes by microcells, subchromosomal segments by chromosome mediated gene transfer, and DNA fragments in the kilobase range by DNA mediated gene transfer (Klobutcher, L. A. and F. H. Ruddle, Annu. Rev. Biochem., 50: 533-554, 1981). Intact chromosomes may be transferred to an embryonic stem cell by microcell-mediated chromosome transfer (MMCT) (Fournier, R. E. and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A., 74: 319-323, 1977). The specific design of the transgene also must consider the content of the DNA sequences encoding the exogenous protein, the specific tissue in which expression is targeted, the host organism in which expression occurs, and the nature of the protein to be expressed. Because proteins ordinarily expressed in vivo vary dramatically in their size, biochemical characteristics, and functionality, the transgene designed for tissue specific expression must satisfy several parameters to enable successful integration into the genome of an embryonic stem cell and to insure successful expression in the selected tissue of the host organism. 
         [0008]    As noted above, the performance of genetic modifications in embryonic stem cells to produce transgenic animals has been demonstrated in only a very few species. For mice, the separate use of homologous recombination followed by chromosome transfer to embryonic stem (ES) cells for the production of chimeric and transgenic offspring is well known. Powerful techniques of site-specific homologous recombination or gene targeting have been developed (see Thomas, K. R. and M. R. Capecchi, Cell 51: 503-512, 1987; review by Waldman, A. S., Crit. Rev. Oncol. Hematol. 12: 49-64, 1992). Insertion of cloned DNA (Jakobovits, A., Curr. Biol. 4: 761-763, 1994), and manipulation and selection of chromosome fragments by the Cre-loxP system techniques (see Smith, A. J. et al., Nat. Genet. 9: 376-385, 1995; Ramirez-Solis, R. et al., Nature 378: 720-724, 1995; U.S. Pat. Nos. 4,959,317; 6,130,364; 6,091,001; 5,985,614) are available for the manipulation and transfer of genes into murine ES cells to produce stable genetic chimeras. Many such techniques that have proved useful in mammalian systems would be available to be applied to non-mammalian embryonic stem cells if the necessary cell cultures were available and if transgenes could be designed that yielded tissue specific expression in specific tissues that facilitate isolation and collection of the exogenous protein. 
         [0009]    The transgenes that enable tissue specific expression are complex and the genetic manipulations that are necessary to incorporate the transgenes into an embryonic cell line require extensive manipulation of the embryonic stem cells and can threaten the pluripotency of the stem cells unless the culture conditions are optimized for transgenesis. Thus, embryonic stem cell lines suitable for use in transgenesis must be both stable in culture and must maintain pluripotency when the ES cell is transfected with a genetic construct that is large and complex enough to contain all of the elements necessary for protein expression. Moreover, the genetic construct must be expressed in the ES cell to allow selection of successfully transformed cells, and the ES cell must maintain potency and the transgene must remain viable during the injection into recipient embryos and the formation of resulting animals. In the resulting animal, the transgene must be effectively expressed in specific individual tissue types in which the transgene is designed to be expressed, and, should not be expressed in other tissues such that the viability of the animal is compromised. For example, transgenes encoding DNA derived from the lymphoid elements of the immune system might be specifically targeted to be expressed in B lymphocytes of a chimeric or transgenic animal. Specifically for the expression of valuable proteins, the transgene may be designed to express protein in the oviduct such that the resulting protein is deposited in egg white. In an avian species, tissue-specific expression of exogenous proteins in the cell types of the oviduct would yield a valuable biological system for protein production. 
         [0010]    For the production of exogenous proteins, avian biological systems offer many advantages including efficient farm cultivation, rapid growth, and economical production. Globally, chickens and turkeys are a major source of protein in the human diet. Further, the avian egg offers an ideal biological design, both for massive synthesis of a few proteins and ease of isolation and collection of protein product. However, application of the full range of mammalian transgenic techniques to avian species has been unsuccessful. Most notably, the transmission to a mature, living animal of a genetic modification encoding an exogenous protein introduced into an avian embryonic stem cell and expressed with tissue specificity has not been demonstrated. 
         [0011]    In many cases, the techniques necessary to introduce genetic modifications into embryonic stem cells, the screening of modified embryonic stem cells to select specific cell modifications in which the genetic constructs have been introduced, and the ability to manipulate the ES cells for injection into embryos to produce transgenic chickens, requires at least several weeks for all of the steps to be performed. For the embryonic stem cells to be useful in transgenesis, the pluripotential state must be maintained for the entire time period up until injection into an embryo and the ES cell must be incorporated into a recipient embryo to a degree necessary to express meaningful quantities of the exogenous protein in the resulting animal. 
       SUMMARY OF INVENTION 
       [0012]    The present invention includes transgenic and chimeric birds exhibiting non-specific and tissue specific expression of exogenous proteins, transgene constructs that enable exogenous protein expression, isolated compositions of exogenous proteins, and related methods. The invention uses long term avian ES cell cultures and special techniques to produce chimeric and transgenic birds derived from prolonged embryonic stem cell cultures, wherein the genome of the ES cells have a stably integrated transgene expressing an exogenous protein such that progeny of the ES cells contain the transgene. In some embodiments, these genetic constructs modify the DNA of the ES cell to facilitate tissue specific expression of an exogenous protein. When combined with a host avian embryo, by the procedures described below, those modified ES cells produce chimeric birds that incorporate the transgene into specific, selected somatic tissue of the resulting animals. These chimeric or transgenic birds exhibit an ES-cell derived phenotype and express the foreign protein across all tissues or in a selected tissue. Preferably, a specific expression pattern focuses expression in a tissue or tissue type to the substantial exclusion of other tissues and facilitates concentration and collection of the protein. 
         [0013]    This invention also includes compositions comprising long-term cultures of chicken embryonic stem cells that have been genetically modified to incorporate large amounts of foreign DNA, and that contribute to the somatic tissues and the germline of recipient embryos. The invention also includes transgenic chickens expressing exogenous protein in the oviduct tissue such that exogenous protein is concentrated in the egg white. In one preferred embodiment, the exogenous protein is a monoclonal antibody encoded by the transgene construct incorporated into the genome of the embryonic stem cell and progeny. The monoclonal antibody sequence is contained within a transgene that is specifically constructed for expression in the oviduct and which contains appropriate promoters and regulatory sequences to facilitate tissue specific expression. In the embodiment of a transgenic or chimeric bird expressing exogenous proteins, the invention includes compositions specific to the animal and the protein, such as egg white, albumen containing exogenous proteins. For all of these embodiments, methods of using the constructs, animals, and exogenous proteins of the invention are also included. 
     
    
     
       DESCRIPTION OF THE FIGURES 
         [0014]      FIG. 1  shows the characteristic morphology of chicken ES cells where the small cells grow in a single layer with little cytoplasm and a prominent nucleolus. 
           [0015]      FIG. 2  shows in vitro properties of chicken ES cells, specifically the reaction with the antibodies SSEA-1 and EMA-1, and expression of alkaline phosphatase. 
           [0016]      FIG. 3  is a karyotype of chicken ES cells that have been in culture for 189 days. The cells are diploid, carry  38  pairs of autosomal chromosomes and one pair of Z chromosomes. 
           [0017]      FIG. 4  is two Barred Rock chicks and two chimeras formed by injecting Barred Rock ES cells into a White Leghorn recipient embryo. The chimeras and the Barred Rocks are indistinguishable indicating that the contributions of the ES cells to the melanocyte lineage is extensive. 
           [0018]      FIG. 5  are chimeras made by injecting Barred Rock ES cells into White Leghorn recipients. The pair of chimeras in the left panel exhibit minor contributions to the melanocyte lineage whereas the pair in the left panel show more extensive contributions. 
           [0019]      FIG. 6  is a diagram of the pCX/GFP/Puro plasmid construct used for transfection of ES cells. 
           [0020]      FIG. 7  shows chicken ES cells that have been transfected with the pCX/GFP/Puro construct and grown in the presence of puromycin. The upper panel is photographed to reveal fluorescence; the lower panel is the same field viewed by phase contrast microscopy. 
           [0021]      FIG. 8  is a FACS analysis of non-transfected chicken ES cells (upper panel) and chicken ES cells that have been transfected with the pCX/GFP/Puro construct and grown in the presence of puromycin. The analysis shows that substantially all of the transfected cells are expressing the transgene. 
           [0022]      FIG. 9  is a Southern analysis of ES cells that have been transfected with the pCX/GFP/Puro construct. The difference in the location of the probe in preparations of DNA digested with BamH1, EcoR1 and a combination of the two endonucelases indicates that the transgene in incorporated into the genome at different sites in the cell lines TB01 and TB09. 
           [0023]      FIGS. 10A-10D  are a transgene construct providing tissue specific expression in the tubular gland cells of the oviduct of a chicken and physiological evidence confirming the tissue specific expression.  FIG. 10A  is a diagram of a transgene designated Ov7.5 MAbdns (upper panel); (B) A diagrammatic representation of a tissue-specific expression transgene containing DNA sequences derived from the host ovalbumin promoter combined with coding DNA for a monoclonal antibody.  FIG. 10B  is a section of the magnum of a chimeric chicken containing the tissue-specific expression transgene showing the expression of monoclonal antibody selectively in tubular gland cells to the exclusion of other cell types.  FIGS. 10C and 10D  are RT-PCR analyses showing expression of both the light and heavy chain of a monoclonal antibody in the oviduct, to the exclusion of brain, gut, pancreas and muscle tissue of the chimeras. 
           [0024]      FIG. 11  is the genomic PCR analysis of BAC-A transfected cES cells. 
           [0025]      FIG. 12  is a Southern analysis of chimeras made with cES cells transfected with CX/GFP/Puro. 
           [0026]      FIG. 13  is a photograph of a chimeric chicken made with cES cells transfected with CX/GFP/Puro under fluorescent light (left panel) and white light (right panel). Green fluorescence can be observed in the eyes and beak, confirming that the transfected ES cells contributed to these tissues. 
           [0027]      FIG. 14  is a photograph of a chimeric chicken made with cES cells transfected with CX/GFP/Puro under fluorescent light. The green fluorescence in the buccal cavity, legs, and feet, confirming that the transfected ES cells contributed to these tissues. 
           [0028]      FIG. 15  is a photograph of a chimeric chicken made with cES cells transfected with CX/GFP/Puro under fluorescent light (right panel) and white light (left panel). Green fluorescence in the bones and in cells in the feather pulp of the emerging primary wing feathers can be observed, confirming that the transfected ES cells contributed to these tissues. 
           [0029]      FIG. 16  is a photograph of a chimeric chicken made with cES cells transfected with CX/GFP/Puro under fluorescent light (left panel) and white light (right panel). Green fluorescencean be observed in the intestinal tissue and in breast muscle, confirming that the transfected ES cells contributed to these tissues. 
           [0030]      FIG. 17  is a photograph of a chimeric chicken made with cES cells transfected with CX/GFP/Puro under fluorescent light (left panel) and white light (right panel). Green fluorescence can be observed in the leg muscles, confirming that the transfected ES cells contributed to these tissues. 
           [0031]      FIG. 18  is a photograph of a chimeric chicken made with cES cells transfected with CX/GFP/Puro under fluorescent light (left panel) and white light (right panel). Green fluorescence can be observed in the pancreas, confirming that the ES cells contributed to this tissue. 
           [0032]      FIG. 19  shows the correlation between estimates of chimerism derived by scoring the level of fluorescence in skin and comb tissues and derived by scoring the extent of black pigmentation of the chick at hatch. 
           [0033]      FIG. 20  is a photograph of the allantois of a chimeric embryo made with cES cells transfected with CX/GFP/Puro under fluorescent light (left panel) and white light (right panel). Green fluorescence can be observed in the allantois, demonstrating that ES cells contributed to this tissue. 
           [0034]      FIG. 21  is a summary of FACS data from chimeras made with non-transfected cells (control), chimeras made with ES cells that were transfected with CX/GFP/Puro and estimated to contain &lt;5% donor-derived cells by feather pigmentation, chimeras made with ES cells that were transfected with CX/GFP/Puro and estimated to contain &gt;5% and &lt;10% donor-derived cells by feather pigmentation, chimeras made with ES cells that were transfected with CX/GFP/Puro and estimated to contain &gt;10% and &lt;30% donor-derived cells by feather pigmentation, chimeras made with ES cells that were transfected with CX/GFP/Puro and estimated to contain &gt;30 and &lt;75% donor-derived cells by feather pigmentation, and chimeras made with ES cells that were transfected with CX/GFP/Puro and estimated to contain &gt;75% donor-derived cells by feather pigmentation. Cells were prepared as described in the text from liver, brain, and muscle. The mean number of fluorescent cells detected above the autofluorescent threshold is shown in the left panel; the mean number of fluorescent cells detected above the M2 threshold, which is an order of magnitude higher than the autofluorescent threshold is shown in the right panel. 
           [0035]      FIG. 22  is an example of a FACS analysis of cells prepared from the brain of a chimera made with non-transfected ES cells (upper panel) and chimeras that were made with cells transfected with CX/GFP/Puro (lower panel). Substantially all of the cells from the chimera made with transfected cells display a level of fluorescence that is above that of the cells from the control bird indicating that the contribution of the donor ES cells to the brain tissue of the chimera is extensive. 
           [0036]      FIG. 23  is an example of a FACS analysis of cells prepared from leg muscles of a chimera that was made with cells transfected with CX/GFP/Puro. The right leg of this chimera was green to the naked eye indicating substantial contribution to this tissue. A preparation of cells from the fluorescent leg muscle was shown to contain primarily fluorescing cells by FACS analysis (lower panel). The left leg of the chimera was normal in color and a preparation of these cells was shown to contain non-fluorescing cells by FACS analysis. These data show that chimerism in tissues within the same animal may receive different contributions from donor ES cells. 
       
    
    
     DETAILED DESCRIPTION OF INVENTION 
       [0037]    Pursuant to this invention, chicken ES cell lines are derived from stage X embryos that have a large nucleus and contain a prominent nucleolus ( FIG. 1 ). These cells are confirmed to be chicken embryonic stem (cES) cells by morphology after long-term culturing and to yield chimeras when injected into recipient embryos. Moreover, the ES cells enable a high degree of contribution to somatic tissues as determined by extensive feather chimerism. Still further, these embryonic stem cells are demonstrated to be transfected with transgenes carrying DNA encoding an exogenous protein. The ES cells stably integrate the transgene and express the transgene to enable selection of transformed cells. These transformed cells are capable of forming chimeras wherein an exogenous protein encoded by the transgene is present in the selected tissues of the chimera. Cells derived from the chimera express the exogenous protein encoded by the transgene. In a particularly preferred embodiment, an exogenous proteins encoded by the transgene is expressed in a specific tissue or tissue types according to the protein encoded by the transgene. Embryonic stem cell progeny are derivatives of ES cells that differentiate into non-ES cell phenotypes after introduction of the ES cells into recipient embryos and the formation of a chimera. A transgenic chicken is the progeny of a chimera which has been produced from chicken ES cells carrying a transgene which is stably integrated into the genome when cells derived from the transgenic ES cells have incorporated into the germline. 
         [0038]    Broad expression of a transgene in somatic tissue is demonstrated by expression in extraembryonic and somatic tissues. Analysis of the protein content of eggs of transgenic animals demonstrates selective expression in the egg white of transgene-encoded exogenous proteins obtained from the transgenes and using the techniques of the present invention. Tissue specific expression is demonstrated by expression that is substantially confined to one organ, tissue, or cell type. 
       Example 1 
     Derivation of Chicken Embryonic Stem Cells (cES Cells) 
       [0039]    Chicken ES cells were derived from one of two crosses: Barred Rock X Barred Rock or Barred Rock X Rhode Island Red. These breeds were selected to obtain a feather marker when testing the developmental potential of cES cells. The cES cells are injected into White Leghorn embryos, which are homozygous dominant at the dominant, white locus. Chimeric chickens resulting from injection of these ES cells display black feathers from the cES cells and white feathers from the recipient embryo. 
         [0040]    Initial establishment of the cES cell culture was initiated according to the protocol described in U.S. Pat. No. 5,565,479. At stage X, the embryo is a small round disk, consisting of approximately 40,000-60,000 cells, situated on the surface of the yolk. To retrieve the embryo a paper ring is put on the yolk membrane, exposing the embryo in the middle. The yolk membrane is cut around the ring, which is then lifted off the yolk. The embryo, attached to the ventral side of the ring, is placed under the microscope and the area pellucida isolated from the area opaca using a fine loop. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 cES cell lines derived on either STO feeder cells or a polyester insert in CES-80 
               
               
                 medium. The cultures were initiated from both single and pooled embryos. 
               
             
          
           
               
                   
                 Donor 
                 Substrate used to 
                   
               
               
                 Cell line 
                 embryo 
                 derive cES cells 
                 Endpoint of cell line 
               
               
                   
               
               
                 009 
                 pooled 
                 STO 
                 cultured for 3 months, injected &amp; cryopreserved 
               
               
                 029 
                 pooled 
                 insert 
                 cultured for over 3 months, injected &amp; cryopreserved 
               
               
                  31 
                 pooled 
                 STO 
                 injected at 4 days 
               
               
                  36 
                 pooled 
                 STO 
                 injected at 13 days 
               
               
                  50 
                 pooled 
                 STO 
                 grown for over 8 months, injected &amp; cryopreserved 
               
               
                  63b 
                 pooled 
                 insert 
                 grown for 3 months and cryopreserved 
               
               
                  67I 
                 single 
                 insert 
                 injected at 45 days of culture 
               
               
                 307 
                 pooled 
                 STO 
                 injected at 15 days and fixed for staining 
               
               
                 314 
                 pooled 
                 STO 
                 cultured for over 3 months, injected &amp; cryopreserved 
               
               
                 317 
                 pooled 
                 STO 
                 injected at 12 days and fixed for staining 
               
               
                 324A 
                 single 
                 insert 
                 cultured for over 6 months and injected 
               
               
                 328 
                 single 
                 insert 
                 cultured for over 6 months, injected &amp; cryopreserved 
               
               
                 329 
                 single 
                 insert 
                 cultured for 5 months, injected &amp; cryopreserved 
               
               
                 330 
                 single 
                 insert 
                 cultured for 3 months and cryopreserved 
               
               
                 331 
                 single 
                 24 w insert 
                 cultured for over 3 months and terminated 
               
               
                 332 
                 single 
                 96 w STO 
                 cultured for 3 months and cryopreserved 
               
               
                 333 
                 single 
                 12 w insert 
                 cultured for over 3 months and terminated 
               
               
                 334 
                 single 
                 12 w insert 
                 cultured for over 3 months and terminated 
               
               
                 335 
                 single 
                 96 w insert 
                 cultured for over 3 months and terminated 
               
               
                   
               
             
          
         
       
     
         [0041]    Embryos are dispersed mechanically into a single cell suspension and seeded on a confluent feeder layer of mitotically inactivated STO cells at a concentration of 3×10 4  cells/cm 2 . The cES culture medium consists of DMEM (20% fresh medium and 80% conditioned medium) supplemented with 10% FCS, 1% pen/strep; 2 mM glutamine, 1 mM pyruvate, 1× nucleosides, 1× non-essential amino acids and 0.1 mM β-mercaptoethanol. Before use, the DMEM medium is conditioned on Buffalo Rat Liver (BRL) cells. Briefly, after BRL cells are grown to confluency, DMEM containing 5% serum is added and conditioned for three days. The medium is removed and a new batch of medium conditioned for three days and repeated. The three batches are combined and used to make the cES medium. Chicken ES cells become visible 3-7 days after seeding of the blastodermal cells. These cES cells were morphologically similar to mES cells; the cells were small with a large nucleus and a pronounced nucleolus (See  FIG. 1 ). 
         [0042]    The growth characteristics of cES cells are different from mES cells, which grow in tight round colonies with smooth edges and individual cells that are difficult to distinguish. Chicken ES cells grow in single layer colonies with clearly visible individual cells. Tight colonies are often the first sign of differentiation in a cES culture. 
         [0043]    To test for markers of pluripotency of the cells that were derived in culture, the cells were fixed and stained with SSEA-11 (Solter, D. and B. B. Knowles, Proc. Natl. Acad. Sci, U.S.A. 75: 5565-5569, 1978), EMA-1, which recognize epitopes on primordial germ cells in mice and chickens (Hahnel, A. C. and E. M. Eddy, Gamete Research 15: 25-34, 1986) and alkaline phosphatase (AP) which is also expressed by pluripotential cells. The results of these tests, which are shown in  FIG. 2 , demonstrate that chicken ES cells express alkaline phosphatase and the antigens recognized by SSEA-1 and EMA-1. 
         [0044]    Although cES cells are visible after using the above protocol, such cultures cannot be maintained longer than a few weeks. Several modifications in culture conditions were initiated, as discussed below, which led to the derivation of 19 cell lines (Table 1) of which 14 were tested for their developmental potential by injection into recipient embryos. Eleven of the 14 cell lines contributed to recipient embryos as determined by feather pigmentation (See Table 2 below). This protocol yields sustained cultures of pluripotent cells expressing an embryonic stem cell phenotype. At any point, the cells can be cryopreserved and when injected into compromised recipient embryos have the potential to substantially contribute to somatic tissues (See Examples 3 and 5 below). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Passage number and time in culture of embryonic stem cell lines derived from single 
               
               
                 or pooled embryos. Frequency and extent of somatic chimerism after injection of these cES 
               
               
                 cells into stage X recipients. 
               
             
          
           
               
                   
                   
                   
                 time in 
                 # of 
                   
                   
                   
                 Extent of 
               
               
                 Cell 
                 Donor 
                 Passage 
                 culture 
                 embryos 
                   
                 # 
                 % 
                 chimerism 1   
               
               
                 line 
                 embryo 
                 number 
                 (days) 
                 injected 
                 # chimeras 
                 analyzed 
                 chimeras 
                 (%) 
               
               
                   
               
             
          
           
               
                  31 
                 pooled 
                 0 
                 4 
                 15 
                 2 
                 7 
                 28.5 
                 1-5  
               
               
                 317 
                 pooled 
                 4 
                 12 
                 29 
                 2 
                 10 
                 20 
                 25-65  
               
               
                  36 
                 Pooled 
                 1 
                 13 
                 24 
                 1 
                 5 
                 20 
                 15 
               
               
                 307 
                 pooled 
                 4 
                 15 
                 21 
                 1 
                 6 
                 17 
                  5 
               
               
                 330 
                 single 
                 6 
                 33 
                 11 
                 3 
                 8 
                 25 
                 5-50 
               
               
                  63b 
                 pooled 
                 11 
                 72 
                 36 
                 4 
                 21 
                 19 
                 1-10 
               
               
                  67I 
                 single 
                 3 
                 45 
                 28 
                 0 
                 15 
                 0 
                 — 
               
               
                 324A 
                 single 
                 10 
                 65 
                 25 
                 0 
                 15 
                 0 
                 — 
               
               
                 009 
                 pooled 
                 20 
                 61 
                 27 
                 0 
                 9 
                 0 
                 — 
               
               
                 329 
                 single 
                 3 
                 15 
                 31 
                 8 
                 17 
                 47 
                 3-75 
               
               
                 329 
                   
                 6 
                 25 
                 30 
                 9 
                 19 
                 47 
                 3-95 
               
               
                 329 
                   
                 6 
                 28 
                 26 
                 1 
                 12 
                 8 
                 23 
               
               
                 329 
                   
                 11 
                 49 
                 10 
                 1 
                 4 
                 25 
                 60 
               
               
                 029 
                 pooled 
                 4 
                 33 
                 40 
                 9 
                 27 
                 33 
                 5-80 
               
               
                 029 
                   
                 9 
                 37 
                 40 
                 4 
                 15 
                 27 
                 4-15 
               
               
                 328 
                 Single 
                 6 
                 56 
                 19 
                 4 
                 11 
                 36 
                 10-80  
               
               
                 328 
                   
                 12 
                 98 
                 33 
                 7 
                 22 
                 32 
                 5-50 
               
               
                 314 
                 Pooled 
                 17 
                 52 
                 30 
                 2 
                 5 
                 40 
                 5-65 
               
               
                 314 
                   
                 15-17 
                 53 
                 29 
                 1 
                 4 
                 25 
                 30 
               
               
                 314 
                   
                 17 
                 55 
                 37 
                 3 
                 15 
                 30 
                 3-80 
               
               
                 314 
                   
                 16 
                 65 
                 27 
                 2 
                 11 
                 18 
                 5-40 
               
               
                 314 
                   
                 14 
                 61 
                 25 
                 0 
                 13 
                 0 
                 — 
               
               
                 314 
                   
                 16 
                 65 
                 32 
                 3 
                 14 
                 21 
                 10-60  
               
               
                 314 
                   
                 20 
                 61 
                 30 
                 4 
                 5 
                 80 
                 4-50 
               
               
                 314 
                   
                 21 
                 67 
                 30 
                 2 
                 11 
                 18 
                 5-15 
               
               
                 314 
                   
                 21 
                 71 
                 8 
                 0 
                 2 
                 0 
                 — 
               
               
                  50 
                 pooled 
                 7 
                 53 
                 35 
                 7 
                 23 
                 30 
                 4-65 
               
               
                  50 
                   
                 14 
                 106 
                 36 
                 3 
                 21 
                 14 
                 10-30  
               
               
                   
               
               
                   1 Extent of chimerism was determined by the proportion of black feathers. 
               
             
          
         
       
     
         [0045]    As with the mouse, avian embryonic stem cells, which are sometimes referred to as embryonic germ cells, are derived on a variety of feeder layers including STO, STO-snl and others that are readily available. Leukemia Inhibitory Factor (LIF) produced by these feeders, and the addition of fetal bovine serum contributes to the maintenance of ES cells in an undifferentiated state. In a preferred embodiment of this invention, chicken ES cell cultures are initiated on a STO feeder layer. STO cells are grown to confluency, treated with 10 μg/ml mitomycin for 3-4 hours, washed, trypsinized and seeded on gelatin coated dishes at 4×10 4  cells/cm 2 . cES cells appear to grow more rapidly when the feeder of STO cells are sparser. Reducing the STO feeder cell concentration to between 10 3  and 10 5 , and preferably below 10 4  cells/cm 2 , facilitates the derivation and propagation of cES cells. However, when chicken embryonic fibroblasts and mouse primary fibroblasts are used as feeders, no cES cells were derived. Also, when previously established cES cells were plated on these feeders, all of them differentiated within 1 week. 
         [0046]    Growing cES cells on synthetic inserts, such as polymer membranes (Costar, Transwell type) in the absence of feeders avoids contamination of the recipient embryo with feeder cells when the ES cells are injected. As Table 3 and 4 show, culturing on inserts, instead of STO feeders, facilitates the derivation of cES cells, and inserts can be used for initial derivation. However, after initially growing rapidly on inserts, the mitotic activity of the ES cells declines after 4-6 weeks of culture. To extend the culture the cells have to be transferred to a feeder of STO cells. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Establishment of cES cells from single embryos on either 
               
               
                 inserts or a feeder of STO cells (10 4  cells/cm 2 ). 
               
             
          
           
               
                   
                   
                 # of cultures 
                 # of cell lines 
               
               
                   
                 Substrate 
                 started 
                 obtained 
               
               
                   
                   
               
               
                   
                 STO feeder 
                 56 
                 3 (5%) 
               
               
                   
                 insert 
                 45 
                  7 (16%) 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Establishment of cES cells from pooled embryos on either a STO 
               
               
                 feeder or a synthetic insert. 
               
             
          
           
               
                   
                   
                 # of cultures 
                 # of cell lines 
               
               
                   
                 Substrate 
                 started 
                 obtained 
               
               
                   
                   
               
               
                   
                 STO feeder 
                 73 
                 7 (9.5%) 
               
               
                   
                 insert 
                 17 
                 2 (12%)  
               
               
                   
                   
               
             
          
         
       
     
         [0047]    The data in Tables 3 and 4 show that chicken embryonic feeder cells and mouse primary fetal fibroblasts do not support the derivation or the culture of cES cells. A feeder of STO cells supports derivation and growth but only when present in a limited concentration of between 10 3  and 10 5  STO cells but preferably in the present embodiment at a concentration of less than or appropriately 10 4  cells/cm 2 . A dense STO feeder layer impairs the growth of cES cells, while the specified concentration of STO cells provides factor(s) necessary for ES cell proliferation. When the cells are sustained over an extended culture period and continue to express an embryonic stem cell phenotype, and differentiate into non-embryonic stem cell phenotypes in vivo, they are referred to as “ES cell progeny.” 
         [0048]    The cES cell culture medium consists of 80% conditioned medium and preferably contains certain BRL conditioned medium with factors necessary for the derivation and growth of cES cells. At a concentration of 50%, growth of the cES cells is not as reliable as in 80% conditioned medium. When the percentage of conditioned medium is reduced to less than 50%, the growth of the cES cells is affected, as evidenced by an increase in differentiated cells and, at a concentration of 30% or less, the cES cells differentiate within 1 week. This conditioned medium found necessary for the derivation and maintenance of cES cells does not maintain mES but causes their differentiation. 
         [0049]    Fetal bovine serum is a preferred component of the ES cell medium according to the present invention and contains factors that keep cES cells in an undifferentiated state. However, serum is also known to contain factors that induce differentiation. Commercially available serum lots are routinely tested by users for their potential to keep ES cells in an undifferentiated state. Serum used for cES cell cultures are known to differ from serum used for mouse ES cell cultures. For example, serum used for the culture of mouse ES cells that is low in cytotoxin and hemoglobin concentration, which is known to maintain mouse ES cells in an undifferentiated state, did not support the sustained growth of chicken ES cells. 
         [0050]    Therefore, serum to be used on chicken ES cells should not be tested on mouse ES cells to determine suitability as a media component, but instead should be evaluated on chicken ES cells. To do so, chicken ES cell cultures are divided into two and used to test each new batch of serum. The new batch tested must clearly support the growth of chicken ES cells when empirically tested. 
         [0051]    Chicken chromosomal spreads require special evaluation techniques different than mice because the complex karyotype consisting of 10 macrochromosomes, 66 micro-chromosomes and a pair of sex chromosomes (ZZ in males and ZW in females). The long-term cES cells of the present invention shown in  FIG. 3  were analyzed after 189 days in culture and being cyopreserved twice. Referring to  FIG. 3 , they exhibited a normal karyotype with 10 macro chromosomes; 2 Z-chromosomes and 66 microchromosomes. 
         [0052]    Chicken ES cells are cryopreserved in 10% DMSO in medium. After thawing and injecting several cell lines into recipient embryos, somatic chimeras are obtained, indicating that the cES cells maintain their developmental potential during the cryopreservation process. 
       Example 2 
     Injection of Chicken Embryonic Stem Cells into Recipient Embryos 
       [0053]    To permit access to the embryo in a freshly laid egg the shell must be breeched, inevitably leading to a reduction in the hatch rate at the end of the 21-day incubation period. The convention was to cut a small hole (less than 10 mm diameter) in the side of the egg, through which the embryo was manipulated, and re-seal with tape, a glass cover slip, shell membrane or a piece of shell. Though relatively simple to perform, this “windowing” method caused embryonic mortality between 70 and 100%. Improved access to the embryo and increased survival and hatchability can be achieved if the embryo is transferred to surrogate eggshells for incubation to hatching using two different shells and a method (adapted from Callebaut) (Callebaut, Poult. Sci 60: 723-725, 1981) and (Rowlett, K. and K. Simkiss, J. Exp. Biol. 143: 529-536, 1989), which are specifically incorporated herein by reference with this technique, the mean hatch rate is approximately 41% (range 23-70%) with 191 chicks hatched from 469 cES-cell injected embryos. 
         [0054]    Incubation of embryos following injection of donor ES cells into recipient embryos can be divided into two parts comprising System A and System B as described below: 
         [0055]    System A covers the first three days of post-oviposition development. Fertile eggs containing the recipient embryos are matched with eggs 3 to 5 grams heavier. A 32 mm diameter window is cut at the pointed pole, the contents removed and the recipient embryo on the yolk, together with the surrounding albumen, is carefully transferred into the surrogate shell. 
         [0056]    Cells are taken up in a sterile, finely tapered glass pipette connected to a mouth aspirator fitted with a 2 micron filter. The opening of the pipette can be from 50 to 120 microns in diameter and possesses a 30° spiked bevel. The embryo is visualized under low magnification and with blue light. Chicken ES cells are trypsinized into a single cell suspension and between approximately 2,000 and 26,000 cells and preferably about 20,000 cells, are injected into an embryo. The cells are gently expelled into the space either below or above the embryo, i.e. into the sub-embryonic cavity or between the apical surface of the area pellucida and the perivitelline layer (yolk membrane) [Rob: preferably into the sub-germinal cavity]. Extra albumen collected from fresh fertile eggs is added and the shell sealed with Saran Wrap plastic film. 
         [0057]    System B covers the period from day three to hatching. At day three of incubation the embryo has reached around stage  17  (H&amp;H). Water has been transported from the albumen to the sub-embryonic cavity, causing the yolk to enlarge and become very fragile. The contents of the System A shell are very carefully transferred to a second surrogate shell (usually a turkey egg) 30 to 35 grams heavier than the original egg. Penicillin and streptomycin are added to prevent bacterial contamination and the 38 to 42 mm window in the blunt pole is sealed with plastic film. This larger shell allows for an artificial airspace. At day  18  to  19  of incubation the embryo cultures are transferred to tabletop hatchers for close observation. As lung ventilation becomes established, holes are periodically made in the plastic film to allow ambient air into the airspace. Approximately 6-12 hours before hatching the film is replaced with a small petri dish, which the chick can easily push aside during hatching. 
         [0058]    For incubation, conventional temperature (37.5 to 38° C.) and relative humidity (50 to 60%) are maintained for the embryos in surrogate shells, but periodic egg rocking, which is normally hourly and through 90 degrees, has to be modified to ensure good survival. In System A rocking is through 90° every 4 to 5 minutes; in System B it is through 40 to 600 every 40 to 45 minutes. In both systems the speed of rocking is maintained at 15 to 200 per minute. 
         [0059]    The contribution of cES cells to chimeras is improved if the recipient embryo is prepared by (1) irradiated by exposure to 660 rads of gamma irradiation (2) altered by mechanically removing approximately 1000 cells from the center of the embryo, or by combining (1) and (2) above before the injection of the cES cells. Referring to Table 5, contribution of cES cells to the somatic tissues increased substantially when recipient embryos were compromised, either by removing cells from the center of the recipient embryo or by exposure to irradiation. When the recipient embryos are compromised by a combination of irradiation and mechanical removal of the cells, the contribution of the ES cells is increased further, even though the cES cells had been in culture for longer periods of time. Some of the resulting chimeric chicks are indistinguishable from pure Barred Rock chicks ( FIG. 4 ). As the data in Table 5 show, chimerism rates as well as the extent of chimerism per embryo increases after compromising the recipient embryo. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Frequency of somatic chimerism after injection of cES cells into recipient embryos 
               
               
                 that were compromised by different methods. 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 Extent 
               
               
                   
                 # 
                   
                   
                 # Embryos 
                 Frequency 
                 feather 
               
               
                 Treatment to compromise 
                 Cell 
                 Time cells 
                 # 
                 &amp; chicks 
                 of 
                 chimerism 
               
               
                 the recipient embryo 
                 lines 
                 in culture 
                 Chimeras 
                 evaluated 
                 chimerism % 
                 (%) 
               
               
                   
               
             
          
           
               
                 None 
                 14 
                 4-106 
                 days 
                 83 
                 347 
                 24 
                 26 
               
               
                 Mechanical removal of cells 
                 1 
                 6 
                 months 
                 34 
                 63 
                 54 
                 20 
               
               
                 Irradiation 
                 1 
                 6-7 
                 months 
                 56 
                 95 
                 59 
                 29 
               
               
                 Irradiation &amp; Mechanical 
                 1 
                 7-8 
                 months 
                 52 
                 59 
                 88 
                 49 
               
               
                 removal of cells 
               
               
                   
               
             
          
         
       
     
         [0060]    Recipient embryos substantially younger than stage X may also be used to produce chimeras using ES cell as the donor. Early stage recipient embryos are retrieved by injecting the hens with oxytocin to induce premature oviposition and fertile eggs are retrieved at stages VII to IX. 
         [0061]    Alternatively, the retrieval of embryos from the magnum region of the oviduct provides access to stage I to VI embryos, consisting of approximately 4-250 cells, and enables the development of chimeras from all embryonic stages as potential recipient embryos. 
       Example 3 
     Somatic Chimeras from Chicken Embryonic Stem Cells (cES) 
       [0062]    To demonstrate that pluripotency of the cES cells of the invention, cES cells are injected into White Leghorn recipient embryos. In the first round of experiments, a total of 14 cell lines in 28 experiments are injected into stage X recipient embryos (See Table 2). The cES cells have been propagated in culture between 4 and 106 days and some lines had been cryopreserved. Chicken ES cells are lightly trypsinized, resulting in small clumps of cES cells, and resuspended in DMEM supplemented with 25 mM HEPES+10% fetal calf serum. Three to five pi of the cell suspension, containing between 2000-5000 cells, are injected into the subgerminal cavity of the recipient embryos. All embryos that developed feathers are analyzed and twenty four percent of embryos (83/347) are chimeric as determined by feather color. Feather chimeras are obtained from 11/14 cell lines. The extent of chimerism varied from 1%-95% with a mean extent of 25.9% (SD=20.4). 
         [0063]    Table 2 illustrates the variance in the somatic chimerism between experiments performed within and between cell lines. Examples of the contribution of ES cells to chimeras is shown in  FIGS. 4 and 5 . In  FIG. 4 , two chicks are chimeras and two are Barred Rocks; it is apparent that there are no phenotypic differences between these chicks indicating that the contribution of ES cells to the chimera is extensive, particularly in the ectodermally derived lineages. In  FIG. 5 , the chimeras on the left have relatively low levels of contribution from ES cells whereas those on the right have intermediate contributions. 
       Example 4 
     Transfection of cES Cells by Lipofection and Electroporation 
       [0064]    Referring to Table 6, an appropriate amount of DNA compatible with the size of the well being transfected is diluted in media absent of serum or antibiotics. The appropriate volume of Superfect (Stratagene) is added and mixed with the DNA, and the reaction is allowed to occur for 5-10 minutes. The media is removed and the wells to be transfected are washed with a Ca/Mg free salt solution. The appropriate volume of media, which can contain serum and antibiotics, is added to the DNA/superfect mixture. The plates are incubated for 2-3 hours at 37 C. When the incubation is completed, the Superfect is removed by washing the cells 1-2× and fresh culture media is added. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Conditions for transfection of chicken ES cells using Superfect. 
               
             
          
           
               
                   
                 Volume of 
                   
                   
                 Time to 
                 Volume of 
                   
               
               
                   
                 media used 
                 Total 
                   
                 form 
                 media 
               
               
                   
                 to dilute 
                 amount of 
                 ul 
                 complex 
                 added to 
                 Incubation 
               
               
                 Plate Size 
                 DNA 
                 DNA 
                 Superfect 
                 (min) 
                 complex 
                 time 
               
               
                   
               
             
          
           
               
                 96 
                 well 
                 30 ul 
                 1 
                  5 ul 
                 5-10 
                 150 
                 2-3 hrs 
               
               
                 48 
                 well 
                 50 ul 
                 1.5 
                  9 ul 
                 5-10 
                 250 ul 
                 2-3 hrs 
               
               
                 24 
                 well 
                 60 ul 
                 2 
                 10 ul 
                 5-10 
                 350 ul 
                 2-3 hrs 
               
               
                 12 
                 well 
                 75 ul 
                 3 
                 15 ul 
                 5-10 
                 400 ul 
                 2-3 hrs 
               
               
                 6 
                 well 
                 100 ul  
                 4 
                 20 ul 
                 5-10 
                 600 ul 
                 2-3 hrs 
               
               
                 60 
                 mm 
                 150 ul  
                 10 
                 50 ul 
                 5-10 
                 1000 ul  
                 2-3 hrs 
               
               
                 100 
                 mm 
                 300 ul  
                 20 
                 120 ul  
                 5-10 
                 3000 ul  
                 2-3 hrs 
               
               
                   
               
             
          
         
       
     
         [0065]    A petri-pulser is used to electroporate cES cells that are attached to the plate in a 35 mm diameter well. The media is removed and the well is washed with a salt solution without Ca ++  and Mg ++ . One ml of electroporation solution is added to the well. DNA is added and the media is gently mixed. The petri-pulser is lowered onto the bottom of the well and an electrical current is delivered. (Voltage preferably varies from 100-500 V/cm and the pulse length can be from 12-16 msec). The petri-pulser is removed and the electroporated well is allowed to stand for 10 minutes at room temperature. After 10 minutes, 2 mls of media is added and the dish is returned to the incubator. 
         [0066]    To transfect cells in suspension, media is removed and cells are washed with a Ca/Mg free salt solution. Tryspin with EDTA is added to obtain a single cell suspension. Cells are washed, centrifuged and resuspended in a correctional electroporation buffer solution such as PBS. The ES cell suspension is placed into a sterile cuvette, and DNA added (minimum concentration of 1 mg/ml) to the cell suspension and mixed by pipetting up and down. The cells are electroporated and allowed to sit at RT for 10 minutes. Cells are removed from cuvette and distributed to previously prepared wells/dishes. Cells are placed in an incubator and evaluated or transient transfection 24-48 hours after electroporation. Selection of antibiotic resistant cells may also be started by including an antibiotic such as puromycin in the culture medium. 
         [0067]    In a preferred embodiment, the concentration of puromycin required for selecting transfected cells is calculated as a titration kill curve. Titration kill curves for chicken embryonic stem cells are established by exposing cells in culture to puromycin concentrations varying from 0.0 to 1.0 μg/ml for 10 days (Table 7) and neomycin concentrations varying from 0.0 to 200 μg/ml (Table 8). The medium is changed every 2 days and fresh puromycin or neomycin is added. When exposed to a concentration of 0.3 μg/ml puromycin, ES cells were absent from all wells after 3 changes of medium with fresh puromycin over a six day period (see Table 7). Puromycin concentrations of 0.3-1.0 μg/ml are used for selection of the transfected cultures. Neomycin concentrations over 40 μg/ml eliminated all cES cells within 7 days (Table 8). 
         [0068]    After 10 days of selection, cES cells colonies are visible and can be picked for further expansion. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Morphology of cES cells after exposure of various concentrations of 
               
               
                 puromycin and different lengths of time (days after addition of puromycin). 
               
             
          
           
               
                 Puromycin conc. 
                 Time under selection (days) 
               
             
          
           
               
                 (μg/ml) 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
               
               
                   
               
             
          
           
               
                 0.0 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
               
               
                 0.1 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
               
               
                 0.2 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
               
               
                 0.25 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 diff 
                 diff 
                 diff/ 
                 diff/ 
                 diff/ 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 gone 
                 gone 
                 gone 
               
               
                 0.3 
                 ES 
                 ES 
                 diff 
                 diff/ 
                 diff/ 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
               
               
                   
                   
                   
                   
                 gone 
                 gone 
               
               
                 0.4 
                 ES 
                 diff 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
               
               
                 0.5 
                 diff 
                 gone 
                 Gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
               
               
                 0.6 
                 diff 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
               
               
                 0.7 
                 diff 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
               
               
                 0.8 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
               
               
                   
               
               
                 ES: ES cells are present. 
               
               
                 diff: ES cells are differentiated. 
               
               
                 gone: no morphologically recognizable cells are present 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Morphology of cES cells after exposure of various concentrations of neomycin and 
               
               
                 different lengths of time (days after addition of neomycin). 
               
             
          
           
               
                 Neomycin 
                   
               
               
                 conc. 
                 Time under selection (days) 
               
             
          
           
               
                 (μg/ml) 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
               
               
                   
               
             
          
           
               
                 0.0 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
               
               
                 10 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
               
               
                 20 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
               
               
                 30 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES/Diff 
                 ES/diff 
                 Diff 
                 Diff/ 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 gone 
               
               
                 40 
                 ES 
                 ES 
                 ES 
                 ES 
                 ES/Diff 
                 Diff/ 
                 dead 
                 gone 
                 gone 
                 gone 
               
               
                   
                   
                   
                   
                   
                   
                 dead 
               
               
                 50 
                 ES 
                 ES 
                 ES 
                 ES/Diff 
                 ES/Diff 
                 Diff/ 
                 Dead/ 
                 gone 
                 gone 
                 gone 
               
               
                   
                   
                   
                   
                   
                   
                 dead 
                 gone 
               
               
                 60 
                 ES 
                 ES 
                 ES 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
               
               
                 100 
                 ES/Diff 
                 Diff 
                 dead 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
               
               
                 150 
                   
                 dead 
                 dead 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
               
               
                 200 
                   
                 dead 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
                 gone 
               
               
                   
               
             
          
         
       
     
         [0069]    Selection of transfected chicken ES cells and their identification in chimeras requires that the transgene confer a selective advantage to the cells in culture (e.g. resistance to puromycin in the medium) and that it produce an identifiable gene product in the cells in the chimera which are derived from the ES cells. This can be accomplished using pCX/GFP/Puro which provides resistance to puromycin in cES cells and produces a green fluorescent protein (GFP) in most, if not all, donor-derived cells in chimeras. 
         [0070]    Referring to  FIG. 6 , PCX/GFP/Puro ( FIG. 6 ) was produced in three cloning steps involving two intermediates before make the final pCX/GFP/Puro plasmid. In step 1, the PGK-driven Puromycin resistant gene cassette (1.5 Kb) was released from pKO SelectPuro (Stratagene) by Asc I digestion. The fragment was then blunted and Kpn I linkers were added. The resulting fragment (GFP/Puro) was inserted into the corresponding Kpn I site of pMIEM (courtesy of Jim Petitte (NCSU), a GFP expression version derived from LacZ expression pMIWZ, see  Cell Diff and Dev.  29: 181-186 (1990) to produce the first intermediate (pGFP/Puro). The PGK-Puro cassette was in same transcription orientation as GFP (determined by BamH I and Sty I digestion). In step 2, the GFP/Puro expression cassette (2.5 Kb) was released from pGFP/Puro by BamH I and EcoR I double digestion. The resulting fragment was inserted into the BamH I and EcoR I sites of pUC18 (Invitrogen). It contains 5′ unique sites, Hind III, Pst I and Sal I. The resulting plasmid pUC18/GFP/Puro was verified by a BamH I, EcoR I, and Not I triple digestion. In the third step, the Cx promoter including 384 bp CMV-IE enhancer, 1.3 kb chicken beta-actin promoter and portion of 1 st  intron was released from pCX-EGFP (Masahito, I. et al., FEBS Letters 375: 125-128, 1995) by Sal I and EcoR I digestion. A 3′ EcoR I (null)-Xmn I-BamH I linker was attached to the fragment and it was inserted into the Sal I and BamH I sites of pUC18/GFP/Puro. The plasmid pCX/GFP/Puro ( FIG. 6 ) was verified by a BamH I and Pst I double digestion. pCX/GFP/Puro DNA can be linearized by Sca I digestion for transfection into cES cells. 
         [0071]    Transfection and selection of ES cells using the procedures described above produced a population of cells that would grow in the presence of 0.5 μg of puromycin. These cells exhibited green fluorescence when examined by conventional fluorescence microscopy (See  FIG. 7 ). When preparations of the ES cells are examined by fluorescence activated cell sorting, it is evident that essentially all of the cells carry and express the transgene (See  FIG. 8 ). Southern analysis of DNA from the transfected ES cell lines TB01 and TB09 that was digested with BamHI, EcoRI or both restriction endonucleases revealed the transgene in DNA fragments of various sizes, providing evidence that the transgene is integrated into the genome (See  FIG. 9 ). 
         [0072]    The CX/GFP/Puro construct demonstrates that transgenes of at least 4.5 kb can be inserted into chimeric chickens. Using the cES cells described herein, chicken ES cells can be transfected with different or larger constructs. Depending on the design of the transgene, DNA encoding an exogenous protein may be widely detected in somatic cells of the resulting chimeric or transgenic animal, i.e., the endoderm, mesoderm, ectoderm, and extra embryonic tissue, or may be designed to express significant levels of an exogenous protein only in a selected tissue. The protein is encoded by DNA contained in a tissue specific transgene construct. The transgene construct may be comprised of genetic elements derived from the genome of the host organism and selected on the basis of known expression, or patterns of expression, of a protein in a selected tissue. For expression in a particular tissue, a gene encoding a protein that is normally expressed, and usually highly expressed in the selected tissue, is selected and regulatory elements from the gene are chosen to drive expression of the exogenous protein. When combined with DNA coding sequences for the exogenous protein, other regulatory elements, selectable markers, etc. the transgene yields preferential expression of the exogenous protein in the selected tissue. Preferential expression in a specific tissue type may be defined as 3 to 4 orders of magnitude greater expression in the selected tissue in comparison to non-selected tissues. 
         [0073]    For tissue specific protein expression in a transgenic bird, the tissue specific expression is preferably directed to a region of the oviduct including the magnum, isthmus, shell gland, or infundibulum. The selection of the individual tissue for protein expression depends on the type of protein to be expressed. The magnum contains the tubular gland cells that express the predominant proteins of the egg white, while the isthmus contains cells that express the shell membrane. Soluble protein expression is preferably directed to the tubular gland cells of the magnum of the oviduct by selecting regulatory sequences, usually comprising a promoter, from genes expressing egg white proteins, preferably ovalbumin, but including ovotransferrin, ovomucoid, lysozyme, ovoglobulin G2, ovoglobulin G3, ovoinhibitor, cystatin, ovoglycoprotein, ovoflavoprotein, ovomacroglobulin, and avidin. Selective expression in the tubular gland cells, to the exclusion of expression in the epithelial cells is demonstrated below and termed preferential expression to distinguish selectivity between the two cell types. 
         [0074]    In the following example, a transgene containing an endogenous egg white regulatory sequence comprising a promoter together with an exogenous immunoglobulin locus is constructed to yield tissue specific antibody expression in the tubular gland cells of the oviduct. The antibody molecules so expressed are then deposited in the egg white of a transgenic chicken. In this embodiment, antibodies encoded by any rearranged immunoglobulin gene are expressed specifically in tissue comprising the tubular gland cells of the magnum region of the oviduct and can be isolated from the whites of eggs. The rearranged immunoglobulin gene encoding a monoclonal antibody is preferentially expressed in the oviduct to the substantial exclusion of expression in other tissues, although expression in other tissues may exist above detectable levels. 
         [0075]    In this embodiment, the monoclonal antibody cassette under the control of the ovalbumin regulatory sequences is comprised of at least 3.4 kb and preferably at least approximately 7.5 kb of the 5′ regulatory sequence and may include 15 kb or more of the 3′ regulatory sequence. Preferably, the construct includes regions of the ovalbumin gene flanking both the 5′ and 3′ ends of the exogenous antibody coding region although a large enough segment of the endogenous promoter sequence of the 5′ flanking region may avoid the need for a 3′ flanking region. The coding regions of both the heavy and light chains of the antibody are provided in the transgene and include the variable, diversity, joining and constant regions of the selected isotype. In a preferred embodiment, the antibody is encoded by an immunoglobulin gene that is characteristically human and contains at least a human heavy chain. Also, the isotype is preferably IgG, and most preferably IgG1. 
         [0076]    A preferred transgene construct for an ovalbumin derived monoclonal antibody construct is provided in  FIG. 10A . This transgene construct is designated Ov7.5 and has approximately 7.5 kb of an egg white regulatory sequence comprising a promoter, in this specific embodiment the ovalbumin promoter, flanking the MAb coding at the 5′ end sequence and 15 kb of the promoter sequence  3 ′ of the coding region. The coding regions comprise the variable regions for both the light chain and heavy chain, J-C intron sequences, the kappa light chain constant region, an IRES sequence and the gamma 1 isotype heavy chain constant regions. The 3′ end of the construct comprises a GFP gene and a selectable marker, in this case a puromycin resistance gene driven by the CX promoter described herein. The lengths of the ovalbumin promoter sequence both 3′ and 5′ of the monoclonal antibody coding region are examples only and analogous constructs may include 25-100 kb or more of the 5′ sequence as well as varying lengths in the 3′ sequence. Those of ordinary skill in the art will appreciate that the GFP marker is present only for detection in physiological specimens and can be removed without departing from the utility of the transgene. The puromycin resistance marker can be substituted with any marker that provides the ability to select embryonic stem cells that are successfully transformed with the transgene. Several types of analogous selectable marker are well known in the art and can be used essentially interchangeably with the puromycin resistance gene of this embodiment. 
         [0077]    As noted above, this monoclonal antibody is only one example of several types of monoclonal antibody products that may be expressed using the transgene constructs of the invention. Moreover, monoclonal antibodies as a class of proteins are only one example of many classes of protein products that may be expressed in tissue-specific fashion pursuant to the methods and techniques described herein. 
         [0078]    Referring to  FIG. 10B , a section of the magnum of two-week old chimeras in which expression of the Ov 7.5 transgene was induced by estrogen injection shows tissue specific expression of the anti-dansyl monoclonal antibody producing cells derived from the transformed embryonic stem cell express GFP, which shows as green in the top left panel of  FIG. 10B , confirm contribution by the embryonic stem cell transformed with the Ov 7.5 transgene. Referring to the bottom left panel of  FIG. 10B , the monoclonal antibody stains red in the tubular gland cells, while the epithelial cells, which are also derived from the donor embryonic stem cell, stain green and do not stain red. This difference in staining demonstrates that expression of the construct is tissue specific and selected by the content of the transgene for the specific tissue type. While the example below demonstrates tissue specific expression in the tubular gland cells of the oviduct, the demonstration of expression across all cell and tissue types demonstrates that each or any tissue type could be chosen for tissue- or cell-specific expression by corresponding selection of the components of the transgene construct, and e.g. the promoter or other regulatory elements. 
         [0079]    In the top right panel of  FIG. 10B , all of the cell types are shown by DAPI staining. In the bottom right panel, the stains are overlaid to demonstrate that only donor-derived tubular gland cells express the monoclonal antibody, while recipient-derived cells and donor-derived epithelial cells do not express the monoclonal antibody.  FIGS. 10C and 10D  are RT-PCR analyses showing that the heavy chain and light chain, respectively, of the anti-dansyl monoclonal antibody are selectively expressed in only the oviduct tissue of 3 or 5 chimeras and not in the brain, gut, pancreas or muscle of these chimeras above levels detected by RT-PCR. 
         [0080]    To demonstrate that a human IgG isotype monoclonal antibody could be selectively expressed and the protein deposited is egg white, a total of 18 chimeric females were produced by injecting cES cells carrying the Ov7.5 construct. Six chimeras from this group were used for testing transgene expression by early estrogen induction. The remaining 12 chimeric females were reared to sexual maturity for egg collection. Nine chimeric females commenced laying at 17-22 weeks of age and one of these chimeras laid sporadically. Three of the chimeric females had not laid eggs at 35 weeks of age and at autopsy it was evident that their gonad was masculized by the presence of male ES cell derived tissue. 
         [0081]    Eggs from the nine egg laying chimeras were collected and representative egg white samples were prepared by ammonium sulfate precipitation and analyzed by ELISA. The microtiter plates were coated with a goat anti-human IgG antibody and the presence of human IgG MAb in the egg white samples was revealed by either a labeled goat anti-human IgG (γ chain specific) antibody for heavy chain and/or a labeled goat anti-human kappa (K chain specific) antibody for light chain. The standard curves were established with purified human Igγ1, κ proteins. The sensitivity of the ELISA was 0.8 ng/ml. Egg white samples from non-transgenic White Leghorn hens were used as negative controls. No human IgG MAb deposition was detectable in eggs from non-transgenic White Leghorn hens (4 eggs) or from 6 chimeric hens (8 eggs from hen #OV11-17, 8 eggs from hen #OV11-53, 6 eggs from hen #OV11-73, 6 eggs from hen #OV11-88, 4 eggs from hen #OV12-97 and 5 eggs from hen #OV13-13). 
         [0082]    Human IgG MAb deposition were detected in eggs from 3 different chimeric hens (˜1.4-6.3 ng/ml for eggs from hen #OV11-13, ˜2.0-2.9 ng/ml for eggs from hen #OV11-37 and ˜2.9-10.8 ng/ml for eggs from hen #OV11-43 as determined by ELISA for IgH). The concentration of human IgG MAb in representative eggs is summarized in Table 9. The concentration of human IgG MAb in eggs determined by ELISA for IgL was consistently lower than that determined by ELISA for IgH. In general, the concentration determined by IgL was 60% of the concentration determined by IgH (compare columns 3 and 5 in Table 9). The difference was also present in spiked samples made with purified human Igγ1, κ proteins. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Deposition of human IgG MAb in eggs from chimeras 
               
             
          
           
               
                   
                   
                 Human IgG 
                 Corrected 
                 Human IgG 
               
               
                   
                   
                 level by IgH 
                 IgG value in 
                 level by IgL 
               
               
                   
                   
                 ELISA 
                 egg white 
                 ELISA 
               
               
                 Egg ID 
                 Date laid 
                 (ng/ml)* 
                 (ng/ml)** 
                 (ng/ml)* 
               
               
                   
               
             
          
           
               
                 OV11-43-9 
                 Nov. 12, 2002 
                 7.6 
                 10.8 
                 4.6 
               
               
                 OV11-43-10 
                 Nov. 13, 2002 
                 5.2 
                 7.4 
                 3.4 
               
               
                 OV11-43-11 
                 Nov. 14, 2002 
                 6.8 
                 9.7 
                 4.2 
               
               
                 OV11-43-13 
                 Nov. 17, 2002 
                 5.4 
                 7.7 
                 3.4 
               
               
                 OV11-43-14 
                 Nov. 18, 2002 
                 4.6 
                 6.6 
                 3.2 
               
               
                 OV11-43-25 
                 Nov. 30, 2002 
                 3.0 
                 4.3 
                 1.8 
               
               
                 OV11-43-26 
                 Dec. 01, 2002 
                 2.0 
                 2.9 
                 1.2 
               
               
                 OV11-43-33 
                 Dec. 08, 2002 
                 7.0 
                 10.0 
                 4.2 
               
               
                 OV11-13-1 
                 Nov. 24, 2002 
                 3.6 
                 5.1 
                 2.4 
               
               
                 OV11-13-2 
                 Nov. 26, 2002 
                 4.4 
                 6.3 
                 2.6 
               
               
                 OV11-13-3 
                 Dec. 2, 2002 
                 3.8 
                 5.4 
                 2.4 
               
               
                 OV11-13-4 
                 Dec. 10, 2002 
                 2.6 
                 3.7 
                 1.4 
               
               
                 OV11-13-5 
                 Dec. 11, 2002 
                 3.6 
                 5.1 
                 2.2 
               
               
                 OV11-13-11 
                 Dec. 17, 2002 
                 3.2 
                 4.6 
                 2.2 
               
               
                 OV11-13-37 
                 Jan. 27, 2003 
                 1.0 
                 1.4 
                 ~0.6 
               
               
                 OV11-37-1 
                 Dec. 08, 2002 
                 2.0 
                 2.9 
                 1.2 
               
               
                 OV11-37-2 
                 Dec. 11, 2002 
                 2.0 
                 2.9 
                 1.2 
               
               
                 OV11-37-3 
                 Jan. 06, 2003 
                 1.4 
                 2.0 
                 ~0.6 
               
               
                   
               
               
                 *Some egg white samples were prepared and assayed multiple times and the values were averaged. 
               
               
                 **The recovery for sample preparation was estimated at 50-70% using negative control egg white samples spiked with known amount of purified human Igγ1, κ proteins. The IgG value given in Column 4 was corrected based on a recovery of 70% for sample preparation to provide a conservative estimate of the concentration of human Ig in egg white. 
               
             
          
         
       
     
         [0083]    The concentration of human IgG MAB proteins in blood samples from hens #OV11-13, #OV11-37 (which deposited the human Ig in their eggs) and a White Leghorn was less than the sensitivity of the assay (0.8 ng/ml). These data are consistent with the absence of ectopic expression of human Ig in chimeric chickens that was observed using RT-PCR to evaluate the presence of the human Ig transcripts in gut, brain, pancreas and muscle in estrogen induced chimeric chicks (Figures Ti  10 C and D). The Ov7.5 construct, therefore, appears to deliver tissue specific, hormonally induced and developmentally regulated gene expression in transgenic chimeric hens. Furthermore, the protein appears to be exported from the tubular gland cells in the magnum and deposited in egg white. 
         [0084]    The ovalbumin-derived tissue specific protein expression transgenes were constructed as follows: 
         [0085]    A chicken genomic BAC library (Crooijmans, R. P. et al., Mamm. Genome 11: 360-363, 2000), (Texas A &amp; M BAC Center) is screened to isolate a region of 46 Kb in the ovalbumin locus. Two different vectors were constructed having different fragments of the ovalbumin promoter located 5′ of the MAb coding region: (1) Ov7.5 MAb-dns: A 42 Kb expression vector contains 9.2 Kb 5′sequences from the ovalbumin gene (including 7.5 Kb promoter) and 15.5 Kb 3′ flanking sequences ( FIG. 10A ). This 42 kb expression vector contains 9.2 kb of 5′ sequence from the ovalbumin gene (including 7.5 kb promoter) and 15.5 kb 3′ flanking sequences. A bicistronic monoclonal antibody cassette encodes the light chain, an IRES and the heavy chain of an anti-dansyl antibody. (2) Ov15 MAb-dns: A 49 Kb expression vector contains 16.8 Kb 5′sequences from the ovalbumin gene (including 15 Kb promoter) and 15.5 Kb 3′ flanking sequences (not shown). The 49 kb expression vector contains 16.8 kb of 5′ sequence from the ovalalbumin gene (including 15 kb promoter) and 15.5 kb3′ flanking sequences. The monoclonal antibody cassette is identical in both constructs. 
         [0086]    As noted above, the gene to be expressed in both vectors is a mouse-human hybrid anti-dansyl monoclonal antibody (MAbdns). The CxEGFP/CxPuro cassette is cloned in the most 3′ end to allow selection with puromycin for stable transfection in cES cells and easy identification of transfected cells in chimeras. Both constructs are linearized and purified before transfection into cES cells. Transfections of cES cells are performed with Ov7.5 MAbdns and Ov15 MAb using either SuperFect (Stratagene) or petri-pulser electroporation. After selection with puromycin, 6 resistant clones are picked for molecular analysis. The presence of the transgene is confirmed by PCR with primers located in the MAbdns cassette, in the GFP gene and in the Puro gene. Transfected ES cells are used to create transgenic or chimeric birds as described in detail below. 
         [0087]    In yet another example, a very large transgene encoding part of the unrearranged human heavy chain immunoglobulin locus has been transfected into chicken ES cells. A 139 kilobase bacterial artificial chromosome (BAC) clone was co-transfected with the pCX-EGFP-CX-puro selectable marker into cES cells by co-lipofection of circular BAC DNA and linear selectable marker DNA. The BAC clone contains a human genomic DNA insert from an unrearranged immunoglobulin heavy chain locus and contains the most 3′ variable region (V H 6-1), all the diversity (D) segments, all the joining (J) segments, the Cmu and Cgamma constant regions, the J-intronic enhancer, and all the intervening DNA between these elements. It also includes the human gene KIAA0125, a gene that encodes a non-translated RNA of unknown function that is found between V H 6-1 and the D segment region. pCX-EGFP-CX-puro is a plasmid that contains the Enhanced Green Fluorescent Protein (EGFP) gene driven by the CX promoter (consisting of a cytomegalovirus enhancer and the chicken P-actin promoter) and a puromycin resistance gene driven by the same promoter. The cES cells transfected with this plasmid are green fluorescent and resistant to the antibiotic puromycin. The presence of the unrearranged human heavy chain locus in the transfected ES cells that were growing in the presence of puromycin was examined by PCR analysis of transgenes spread throughout the 139 kb construct. The primer sequences used in the PCR analysis were: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 V H 6-1: 
                   
                   
               
               
                   
                 V6-1F 
                 AGTGTCAGGGAGATGCCGTATTCA 
               
               
                   
                   
               
               
                   
                 V6-1R 
                 ACTTCCCCTCACTGTGTCTCTTG 
               
               
                   
                   
               
               
                   
                 D1-26: 
               
               
                   
                 D1-26F 
                 GGGCGCCTGGGTGGATTCTGA 
               
               
                   
                   
               
               
                   
                 D1-26R 
                 GTGGCCCCTAAACCTGAGTCTGCT 
               
               
                   
                   
               
               
                   
                 D1-20: 
               
               
                   
                 D1-20F 
                 CCCGAGCACCGTCCCCATTGA 
               
               
                   
                   
               
               
                   
                 D1-20R 
                 GTGCCGGTGATCCCTGTCTTTCTG 
               
               
                   
                   
               
               
                   
                 Cμ: 
               
               
                   
                 Mu1F 
                 GCGGGAGTCGGCCACCATCACG 
               
               
                   
                   
               
               
                   
                 Mu1R 
                 AGCACAGCCGCCGCCCCAGTAG 
               
               
                   
                   
               
               
                   
                 Cδ: 
               
               
                   
                 Delta 1F 
                 TGGGGAGAGGAGAGCACAGT 
               
               
                   
                   
               
               
                   
                 Delta 1R 
                 GGCGGGCGTAGGGGTCAGC 
               
             
          
         
       
     
         [0088]    cES cells are co-transfected with the selectable marker pCX-EGFP-CX-puro and the BAC CTD-2005N2, resulting in a cES cell line designated BAC-A. Genomic DNA is prepared and PCR performed using 5 different primer sets corresponding to markers along the length of the BAC clone. These markers are: V H 6-1 (24 kb from the 5′ end [relative to the human heavy chain locus] of the human genomic insert), D1-26 (83 kb from the end), D1-20 (73 kb from the end), Cμ (˜108 kb from the end), and Cδ (˜120 kb from the end). Only V H 6-1, Cμ, and D1-26 are shown but all gave similar results. As a control for amplification from the cES cell sample, a chicken β-actin PCR is also run. The samples are:
       1. BAC-A cells;   2. Mouse STO cells used as a feeder layer for the cES cells (negative control);   3. Barred Rock embryo DNA (the same strain a the parental cES cell line, negative control);   4. Human genomic DNA (positive control);   5. cES cell medium (negative control).       
 
         [0094]    As shown in  FIG. 11 , all segments of the transgene are present in the transfected and selected ES cells. 
       Example 5 
     Identification of Transfected Donor-Derived Cells in Chimeras 
       [0095]    Transfected donor derived cells are detected in chimeras by a number of methods. For example, the transgene is detected by Southern analysis of tissue taken from chimeras that are identified by the presence of black pigmentation in their feathers. Also, DNA is harvested from embryos or from comb tissue and digested with BamH1, EcoR1 or a combination of these endonucleases. When DNA from tissues in the chimeras are examined by Southern analysis, bands that were identical to those seen in TB01, which was the donor cell used to make the chimeras, were evident after digestion with either BamH1, EcoR1 or a combination of these two restriction endonucleases ( FIG. 12 ), thus providing evidence that the chimera contains progeny of the ES cells that were introduced into the recipient embryo to form the chimera. 
         [0096]    The presence of the transgene in tissues is detected by illuminating the chimeras with fluorescent lights, which shows that the transgene is expressed in the eye and the beak ( FIG. 13 ), the buccal cavity, legs and feet ( FIG. 14 ), and in the bones of the wing and in cells in the feather pulp ( FIG. 15 ). Examination of the internal organs revealed that donor-derived ES cells had contributed to the intestinal tissues and breast muscle ( FIG. 16 ) and leg muscle ( FIG. 17 ). In another bird, extensive contributions to the pancreas were observed ( FIG. 18 ). These data provide compelling evidence that the progeny of ES cells contribute to the ectoderm which gives rise to the beak, feathers and skin, to the mesoderm which gives rise to muscle and bone and to the endoderm, which gives rise to the pancreas. 
         [0097]    Illumination of the bird with fluorescent light is used to score the birds on a scale of 1 to 4 where 4 indicates that all of the visible skin, eyes and comb are fluorescent. The fluorescent score was determined by screening several areas of the skin with a fluorescent lamp and scoring the chicks on a scale of 0 to 4. A similar score is used to rate the extent of chimerism estimated by feather pigmentation. Feather chimerism was estimated as % of black down compared to down at hatch. Approximately 25% of the down of a Barred Rock chick is white. The maximum score that is possible in a chimera derived completely from Barred Rock derived ES cells is, therefore, 75%. The correlation between the two which is described by the equation y=0.41×+0.25 where y is the fluorescence score and x is the extent of ES-cell derived feather pigmentation is shown in  FIG. 19 . 
         [0098]    Examination of the chorioallantois indicated that cES cells also contributed to the extraembryonic tissues (See  FIG. 20 ). These data indicate that the pluripotentiality of chicken ES cells may be greater than that of murine ES cells, which do not contribute to the trophectoderm. 
         [0099]    In addition to examination of live tissues in chimeras, cells were prepared from a variety of tissues and the proportion of ES-cell derived fluorescing cells was determined. Tissue (muscle, liver or brain) was removed from chickens post-mortem, then rinsed with PBS to remove blood or other fluids. The outer membrane of the tissue was dissected away and the remaining sample was minced. Each tissue sample was transferred to a microcentrifuge tube containing 1 ml of 1 mg/ml collagenase (Type IV, Sigma) in either DMEM (muscle and liver) or Liebovitz L15 medium (brain) without serum. These tubes were incubated in a 37° C. water bath for 30-45 minutes (the tubes were inverted every 10-15 minutes to shake the suspension) and then the tissues were dispersed into single cell suspensions using a 100-200 ul pipette equipped with a disposable plastic tip and purified according to the tissue type. All centrifugation steps were executed in a microcentrifuge. 
         [0100]    For the muscle cell suspension, the suspension was centrifuged for 5 min at 4000 rpm, the supernatant removed and replaced with 500 ul of DMEM containing 10% heat inactivated horse serum. The pellet was resuspended, and the tube centrifuged for a further 60 seconds at 2600 rpm, the supernatant was then removed and filtered through 40 micron nylon mesh (sterile disposable, Falcon brand). A sample of the cell suspension was inspected by microscopy at this stage to ensure correct cell morphology and sufficient cell density for flow cytometry (sub optimal densities were corrected by dilution, or by pelleting and resuspension). 
         [0101]    For the liver cell suspension, the suspension was filtered through 40-micron nylon mesh, transferred to a clean microcentrifuge tube and pelleted as for muscle. The top layer of the pellet was carefully removed to a clean tube with complete DMEM (500 ul), taking care not to disturb the red blood cells which form a distinct and visible layer at the bottom of the pellet. The cell suspension was inspected at this stage and the cell density adjusted. If red blood cells were present in the cell preparation they were removed with a lysis step (incubating the cells in 1 ml lysis buffer containing 130 mM Ammonium Chloride, 17 mM Tris, 10 mM Sodium Bicarbonate for 5 minutes at room temperature). 
         [0102]    For the brain cell suspension, the suspension was filtered and pelleted as described above. The pellet was resuspended in 250 ul complete Liebowitz (L-15) medium containing 10% horse serum and 6 g/l glucose, Percoll was added to produce a 50% solution (approx. 260-280 ul). The suspension was centrifuged at 3.5 k for 5 minutes, following which the top layer of cells was carefully removed into a clean microcentrifuge tube and diluted with at least 1 ml of L-15 medium. The cells were pelleted by centrifuging at 4000 rpm for 5 minutes and then were resuspended in an appropriate volume of L-15. 
         [0103]    A flow cytometric analysis of GFP expression was conducted in liver, brain and muscle cells from chimeras. Single cell suspensions were transferred into polystyrene tubes and loaded into a flow cytometer, for which the operating parameters had been set to detect the particular cell type of the sample, and which was equipped to detect emitted light at a green wavelength in response to an excitory laser beam. At each analysis, at least one group of samples from a non-transgenic chimera was analyzed in order to set the baseline for fluorescence measurement (since the flow cytometer detects some autofluorescence from each cell). These are referred to as control samples in  FIG. 21 . The data produced by the flow cytometer included the number of cells detected (within the parameters specified) and summarized the fluorescence of the cells in that population. Examples of the data from analyses of brain and muscle tissue is shown in  FIGS. 22 and 23 , respectively. Data was collected for the number of cells which exhibited a fluorescence intensity greater than the autofluorescent level set by the non-transgenic sample for each tissue type (designated M1). A susbset of these data was collected for cells which exhibited a fluorescence intensity at least ten times greater than the autofluorescent level (designated M2). 
         [0104]    Brain, liver and muscle (breast and leg) samples were removed from twenty six chickens, of which 18 were chimeras that had been produced by injecting transgenic chicken ES cells carrying the GFP transgene into non-transgenic White Leghorn recipient embryos, as described above. The remaining 8 chickens were chimeras that were produced by injecting non-transfected cES cells into non-transgenic White Leghorn recipient embryos. Male and female chickens were present in both the groups. Green fluorescence was detected in brain-derived, liver-derived and muscle-derived cells from the transgenic chimeras. The fluorescence intensity and the ratio of fluorescent to non-fluorescent cells varied between birds and between tissue type. The tabulated results are shown in  FIG. 21 . In birds which had been awarded a low score for donor-derived feather pigmentation, or for green skin when screened with a UV lamp prior to post-mortem analysis, the number of fluorescent cells in the tissue samples was generally low or zero. Birds which scored well for these criteria (e.g. greater than 75% donor-derived feathers) were found to have fluorescent cells in all three tissue sample types, and this was the only group in which highly fluorescent (M2) cells were present in all three tissue types. Of the three tissue types, the number of fluorescent cells derived from the liver was the lowest. Brain and muscle-derived fluorescent cells were present in greater numbers and in a greater number of samples derived from the transgenic chimeras. The data is shown graphically in  FIG. 19 . 
         [0105]    The data shows that transgenic cES cells, when injected into recipient stage X embryos, can thrive in the host and differentiate into liver, muscle and brain cells. Liver, muscle and brain are tissues that are derived from endoderm, mesoderm and ectoderm, respectively; thus cES cells may differentiate into the three major somatic cell lineages, from which all other somatic cells are derived. Further, this experiment shows that transgenic cES-derived cells persist beyond the embryonic stages and can be seen in juvenile chickens, and that the transgene continues to express in these diverse cell types. 
         [0106]    The transgene is also present in lymphocytes derived from ES cells. The hemopeotic lineage, which comprises the lymphoid and myeloid lineages, is of particular interest in transgenic chimeras derived from ES cells because B cells in the lymphoid lineage produce antibodies. Lymphocytes are prepared either from blood samples taken from chimeric chickens at any time from hatching to adulthood or from the bursa of Fabricius of chimeric embryos. Bursae are removed from chicks at day  20  of embryonic development (E20) and macerated by forcing through steel mesh in 10 ml of Hanks&#39; Buffered Salt Solution (HBSS) with the plunger of a 20 ml syringe. The resulting tissue fragments and cells are collected into a tube and incubated at room temperature for 5 minutes to allow the large fragments to settle. The cell supernatant is harvested and the cells are counted and then collected by centrifugation at 1500 g for 10 minutes at 4° C. and resuspended at a maximum of 1×10 8  cells per ml of FIBSS in a 15 ml conical tube. Blood is collected (0.5 ml) from chimeric chickens&#39; wing veins using a heparinized syringe and deposited into a vacuum tube containing EDTA to prevent clotting. Blood samples are mixed 1:1 with HBSS to give a final volume of 1 ml in a 15 ml conical tube. From this point on blood samples and bursa samples are treated the same way. One ml of cell suspension is underlayed with 0.75 ml Fico/Lite-LM (Atlanta Biologicals catalog number 1406) by dispensing the Fico/Lite at the bottom of the tube, underneath the cell suspension. The tubes are then centrifuged at 1500 g for 15 minutes at 4° C., no brake. The interface between the Fico/Lite and the HBSS is carefully harvested to collect the mononuclear cells that have concentrated there in a discrete layer of material. This material is transferred to a new tube triturated to break up the compacted cells and then mixed with 3 ml of HBSS/2% heat inactivated fetal bovine serum. The cells are collected by centrifugation at 1500 rpm in a Sorvall benchtop centrifuge, 10 minutes at 4° C., then washed two more times in HBSS/2% FBS. A small aliquot (25 μl) is mounted on a microscope slide for preliminary assessment of the extent of donor-derived GFP fluorescence under the microscope. The remainder of the cells are then ready for antibody staining or fixation. 
         [0107]    To store cells for longer than a few hours before analysis or if they are to be permanently mounted on slides for microscopy, they are first fixed in paraformaldehyde. An aliquot of cells (50 μl, 0.5-1×10 6  cells) is fixed by adding 1 ml 4% paraformaldehyde and incubating at room temperature for 15 minutes. The cells are then washed three times by centrifugation at 500 g for 6 minutes in a microfuge to collect the cells, followed by resuspending the cells in PBS/2% heat inactivated fetal bovine serum. 
         [0108]    In antibody staining, an aliquot of 0.5×10 6  cells in fresh PBS/2% FBS/0.1% sodium azide is placed in a tube or a well of a 96-well plate, on ice. Monoclonal antibodies conjugated to R-phycoerythrin (Southern Biotechnology Associates) are added to the cells and incubated for 30 minutes on ice, covered to protect the fluorophore. Antibodies that recognize the chicken B lymphocyte marker Bu-1 (used at a working dilution of 0.2 g/10 6  cells) or the chicken T cell marker CD3 (used at 0.5 g/10 6  cells) are used. After incubation the cells are washed three times by centrifugation at 500 g for 6 minutes, resuspending in 0.5 ml PBS/2% FBS/0.1% azide each time. After the final wash the cells are stored in 0.5% paraformaldehyde up to one week before analysis by flow cytometry. The paraformaldehyde is replaced with PBS/2% FBS on the day the cells are to be analyzed by flow cytometry; buffers without phenol red are used for flow cytometry. FACS analysis is performed for both GFP fluorescence and R-phycoerythrin fluorescence simultaneously, to detect the total proportion of cells that are donor derived (GFP-positive), the proportion of cells (both donor and recipient derived) that are stained by the antibodies (R-phycoerythrin-positive), and the proportion of donor derived cells that are also stained with the antibodies (GFP, R-phycoerythrin double positive). 
         [0109]    Chimeras made with three different ES cell lines were analyzed for donor contribution to the lymphoid lineage, representing three different insertion sites for the CX-GFP marker (Table 9). A total of 27 chimeras have been analyzed, ranging in age from pre-hatch to adult. The proportion of donor-derived GFP-positive cells in the lymphocyte fraction ranged from 0 to 10% as judged by FACS analysis (animals with 0% are not shown in the table). Antibody staining of peripheral blood lymphocytes resulted in 5-17% of the cells staining with anti-Bu-1 antibodies and 75-85% of the cells staining with anti-CD3 antibodies. Staining of bursal lymphocytes with anti-Bu-1 antibodies resulted in over 90% of the cells stained. Double positive cells with GFP fluorescence and staining for either Bu-1 or CD3 and GFP were observed at low frequencies in several of the samples. 
         [0110]    These data show that ES cells containing a gene encoding an exogenous protein contributed to the hemopoietic lineage in hatched chicks and mature animals. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Contribution of cES cells to the lymphocyte lineage in somatic chimeras. 
               
             
          
           
               
                   
                   
                   
                   
                   
                 GFP+ and 
                 GFP+ and 
                   
                   
                 Green 
                   
               
               
                 Chimera 
                 Age 
                 GFP 
                 Bu-1+ 
                 CD3+ 
                 Bu-1+ 
                 CD3+ 
                 Sex 
                 Feather 
                 score 
                 Cell line 
               
               
                   
               
             
          
           
               
                 OV-36 
                 10 
                 10 
                 ND 
                 ND 
                 ND 
                 ND 
                 F 
                 15 
                 ND 
                 OVF 
               
               
                 OV-21 
                 10 
                 7 
                 5 
                 85 
                 0 
                 0 
                 M 
                 0 
                 ND 
                 OVF 
               
               
                 10881 
                 67 
                 5 
                 17 
                 75 
                 0.03 
                 1 
                 M 
                 60 
                 3 
                 TB01 
               
               
                 10891 
                 49 
                 3 
                 12 
                 74 
                 0 
                 0.1 
                 F 
                 5 
                 1 
                 TB01 
               
               
                 10821 
                 111 
                 1 
                 ND 
                 ND 
                 ND 
                 ND 
                 M 
                 75 
                 3 
                 TB01 
               
               
                 10877 
                 98 
                 1 
                 11 
                 ND 
                 0.04 
                 ND 
                 F 
                 75 
                 4 
                 TB01 
               
               
                 IG1-25 
                 E20 
                 0.2 
                 95 
                 ND 
                 0.03 
                 ND 
                 F 
                 70 
                 ND 
                 BAC A 
               
               
                 10845 
                 146 
                 0.18 
                 8.5 
                 ND 
                 0 
                 ND 
                 M 
                 60 
                 3 
                 TB01 
               
               
                   
               
             
          
         
       
     
         [0111]    The eight chimeras listed in the table were found to contain cES cell-derived lymphocytes. Peripheral blood lymphocytes (or bursal lymphocytes in the case of chimera IG1-25) were prepared with Fico/Lite-LM as described in the text. Lymphocytes were then analyzed for expression of green fluorescent protein (GFP), the Bu-1 B cell associated alloantigen (Bu-1), and the CD3 member of the T cell receptor-associated CD3 complex (CD3). The numbers in the columns labeled with GFP, Bu-1 and CD3 indicate the percentage of cells in each sample that were positive for those markers by FACS analysis (except for OV-36 and 10821 in which the percentage of GFP fluorescing cells was determined by counting cells under the microscope). The age of the chimeras when samples were taken for analysis is indicated in days, and the sex of each chimera is indicated. The column labeled “Feather” indicates the percentage estimate of black, cES cell derived feather pigmentation, with 75 being the highest amount of black possible (as pure Barred Rock chickens themselves have a plumage that is approximately 75% black). “Green score” is a subjective evaluation of overall green fluorescence in the whole animal visualized by shining a handheld UV lamp on the live animal. The scale is from 0 (no green) to 4 (the most green). This score is used as a further indication of the overall extent of cES cell contribution in each animal. “Cell line” indicates the names of the different cES cell lines used to generate the chimeras. 
         [0112]    There will be various modifications, improvements, and applications of the disclosed invention that will be apparent to those of skill in the art, and the present application encompasses such embodiments to the extent allowed by law. Although the present invention has been described in the context of certain preferred embodiments, the full scope of the invention is not so limited, but is in accord with the scope of the following claims. All references, patents, or other publications are specifically incorporated by reference herein.