Method and materials for producing a genetically modified animal

Abstract

Transgenic artiodactyls are described as well as methods of making and using such artiodactyls.

1 . A method of introducing an exogenous gene to a cell or an embryo comprising
 providing the exogenous gene on a first transposon and a selectable marker on a second transposon to a collection of cells or embryos,   testing the collection to identify a cell or embryo of the collection that has a chromosome that incorporates the selectable marker and expresses the selectable marker, and   creating a transgenic animal from the cell or embryo, with the transgenic animal being a founder animal expressing the exogenous gene.   
     
     
         2 . The method of  claim 1  wherein the selectable marker is flanked by recombinase recognition sites. 
     
     
         3 . The method of  claim 2  wherein the recombinase recognition sites are selected from the group consisting of FRT and loxP. 
     
     
         4 . The method of  claim 2  wherein the selectable marker is removed before using the cell or embryo for creating the transgenic animal and the founder animal is free of selectable markers. 
     
     
         5 . The method of  claim 1  wherein the selectable marker is selected from the group consisting of a puromycin-resistance gene, adenosine deaminase, aminoglycoside phosphotransferase), dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidine kinase, xanthin-guanine phosphoribosyltransferase, fluorescent polypeptides, green fluorescent protein, and yellow fluorescent protein. 
     
     
         6 . The method of  claim 1  wherein the ratio of the first transposon to the second transposon is not 1:1. 
     
     
         7 . The method of  claim 1  wherein the ratio of the first transposon to the second transposon is at least 3:1. 
     
     
         8 . The method of  claim 1  wherein the ratio of the first transposon to the second transposon is within a range from 3:1 to 10:1. 
     
     
         9 . The method of  claim 1  wherein the founder animal expresses the selectable marker, wherein the exogenous gene and the selectable maker are on different chromosomes. 
     
     
         10 . The method of  claim 1  wherein the transposon is selected from the group consisting of Sleeping Beauty, Tol2, PiggyBac, Frog Prince, Minos, and Hsmar1. 
     
     
         11 . The method of  claim 1  wherein the cell is an artiodactyl cell. 
     
     
         12 . A transgenic animal that expresses an exogenous gene and is free of exogenous selectable marker genes. 
     
     
         13 . The animal of  claim 12  wherein the animal is free of exogenous selectable marker genes selected from the group consisting of a puromycin-resistance gene, adenosine deaminase, aminoglycoside phosphotransferase), dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidine kinase, xanthin-guanine phosphoribosyltransferase, fluorescent polypeptides, green fluorescent protein, and yellow fluorescent protein. 
     
     
         14 . The animal of  claim 12  being an artiodactyl. 
     
     
         15 . A method of introducing an exogenous gene to a cell or an embryo comprising
 providing, to a collection of cells or embryos, the exogenous gene on a transposon and a selectable marker flanked by recombinase recognition sites on the same or a separate transposon,   testing the collection to identify a cell or embryo of the collection that has a chromosome that incorporates the selectable marker and expresses the selectable marker, and   creating a transgenic animal from the cell or embryo, with the transgenic animal being a founder animal expressing the exogenous gene.   
     
     
         16 . The method of  claim 15  wherein the recombinase recognition sites are selected from the group consisting of FRT and loxP. 
     
     
         17 . The method of  claim 15  wherein the selectable marker is removed before using the cell or embryo for creating the transgenic animal and the founder animal is free of selectable marker genes. 
     
     
         18 . The method of  claim 15  wherein the selectable marker is selected from the group consisting of a puromycin-resistance gene, adenosine deaminase, aminoglycoside phosphotransferase), dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidine kinase, xanthin-guanine phosphoribosyltransferase, fluorescent polypeptides, green fluorescent protein, and yellow fluorescent protein. 
     
     
         19 . The method of  claim 15  wherein the selectable marker and the exogenous gene are on separate transposons. 
     
     
         20 . The method of  claim 15  wherein the cell is an artiodactyl cell. 
     
     
         21 . A genetically modified cell comprising at least five copies of an exogenous gene, the copies being integrated at chromosomal locations that are independent of each other. 
     
     
         22 . The cell of  claim 21  having a number of the copies in a range from 5 to 15. 
     
     
         23 . The cell of  claim 21  being an artiodactyl cell. 
     
     
         24 . A method of making a transgenic animal comprising providing a cell with at least five copies of an exogenous gene, the copies being integrated at chromosomal locations that are independent of each other. 
     
     
         25 . The method of  claim 24  wherein the cell is a one-cell embryo. 
     
     
         26 . The method of  claim 24  comprising cloning the cell to make the animal. 
     
     
         27 . The method of  claim 24  comprising gestating the embryo in a surrogate mother. 
     
     
         28 . The method of  claim 24  having a number of the copies in a range from 5 to 15. 
     
     
         29 . The method of  claim 24  wherein the cell is an artiodactyl cell. 
     
     
         30 . A transgenic animal comprising at least five copies of an exogenous gene at independent chromosomal locations, with the exogenous gene being expressed in the animal. 
     
     
         31 . The animal of  claim 30  having a number of the copies in a range from 5 to 15. 
     
     
         32 . The animal of  claim 30  being an artiodactyl.