Transgenic avians and protein production

Abstract

This invention provides vectors and methods for the stable introduction of exogenous nucleic acid sequences into the genome of avians in order to express the exogenous sequences to alter the phenotype of the avians or to produce desired proteins. In particular, transgenic avians are produced which express exogenous sequences in their oviducts and which deposit exogenous proteins into their eggs. Avian eggs that contain exogenous proteins are encompassed by this invention. The instant invention further provides novel forms of interferon and erythropoietin which are efficiently expressed in the oviduct of transgenic avians and deposited into avian eggs.

1 . A transgenic avian comprising a transgene which includes a retroviral vector derived from a lentivirus wherein the vector contains a heterologous coding sequence operably linked to a promoter such that the transgenic avian expresses the heterologous coding sequence in an oviduct cell of the transgenic avian. 
     
     
         2 . The transgenic avian of  claim 1  wherein the transgene is present in the germ-line. 
     
     
         3 . The transgenic avian of  claim 1  wherein the avian is a G1 transgenic avian. 
     
     
         4 . The transgenic avian of  claim 1  wherein the avian is a G2 transgenic avian. 
     
     
         5 . The transgenic avian of  claim 1  wherein the avian is a chicken. 
     
     
         6 . The transgenic avian of  claim 1  wherein the heterologous coding sequence encodes a pharmaceutical protein. 
     
     
         7 . The transgenic avian of  claim 1  wherein the heterologous coding sequence encodes a cytokine. 
     
     
         8 . The transgenic avian of  claim 1  wherein the heterologous coding sequence encodes a cytokine selected from the group consisting of interferon, G-CSF and erythropoietin. 
     
     
         9 . The transgenic avian of  claim 1  wherein the heterologous coding sequence includes a signal peptide coding sequence. 
     
     
         10 . The transgenic avian of  claim 9  wherein the signal peptide directs secretion of the heterologous protein into egg white. 
     
     
         11 . The transgenic avian of  claim 9  wherein the signal peptide is a lysozyme signal peptide. 
     
     
         12 . The transgenic avian of  claim 1  wherein the heterologous coding sequence and the promoter are both positioned between 5′ and 3′ LTRs of the vector. 
     
     
         13 . The transgenic avian of  claim 1  wherein the transgene comprises one or more insulator elements. 
     
     
         14 . The transgenic avian of  claim 1  wherein the retroviral vector is replication deficient. 
     
     
         15 . The transgenic avian of  claim 1  wherein the transgene comprises an IRES. 
     
     
         16 . The transgenic avian of  claim 1  wherein the heterologous coding sequence is expressed in tubular gland cells. 
     
     
         17 . The transgenic avian of  claim 1  wherein protein encoded by the heterologous coding sequence is present in egg white. 
     
     
         18 . The transgenic avian of  claim 1  wherein protein encoded by the heterologous coding sequence is present in a hard shell egg produced by the transgenic avian. 
     
     
         19 . The transgenic avian of  claim 1  wherein the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, a MDOT promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemia virus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter, an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter, an ovomucoid promoter, an ovomucin promoter, an ovotransferrin promoter, and segments thereof. 
     
     
         20 . An egg produced by the transgenic avian of  claim 1 . 
     
     
         21 . A transgenic avian comprising a transgene which includes a retroviral vector derived from a lentivirus containing a heterologous coding sequence operably linked to a promoter such that the transgenic avian expresses the heterologous coding sequence in an oviduct cell of the transgenic avian. 
     
     
         22 . The transgenic avian of  claim 21  wherein the transgene is present in the germ-line. 
     
     
         23 . The transgenic avian of  claim 21  wherein the avian is a G1 transgenic avian. 
     
     
         24 . The transgenic avian of  claim 21  wherein the avian is a chicken. 
     
     
         25 . The transgenic avian of  claim 21  wherein the heterologous coding sequence encodes a pharmaceutical protein. 
     
     
         26 . The transgenic avian of  claim 21  wherein the heterologous coding sequence encodes a cytokine. 
     
     
         27 . The transgenic avian of  claim 21  wherein the heterologous coding sequence is expressed in tubular gland cells. 
     
     
         28 . The transgenic avian of  claim 21  wherein protein encoded by the heterologous coding sequence is present in egg white. 
     
     
         29 . The transgenic avian of  claim 21  wherein protein encoded by the heterologous coding sequence is present in a hard shell egg produced by the transgenic avian. 
     
     
         30 . The transgenic avian of  claim 21  wherein the retroviral vector is replication deficient. 
     
     
         31 . A method of making a transgenic avian comprising: introducing a transgene which includes a retroviral vector derived from a lentivirus containing a heterologous coding sequence operably linked to a promoter into embryonic blastodermal cells and obtaining a mature transgenic avian from the blastodermal cells comprising the transgene wherein the coding sequence is expressed in an oviduct cell of the transgenic avian. 
     
     
         32 . The method of  claim 31  wherein the wherein the embryonic blastodermal cells are present in an embryo.