Methods and materials for producing transgenic artiodactyls

Abstract

Swine animal models comprising a genomic disruption of an endogenous gene chosen from the group consisting of a Low-Density Lipoprotein Receptor gene LDLR, Duchene's Muscular Dystrophy (DMD) gene, and hairless gene (HR). Methods of preparing transfected cells useful for making a transgenic animal comprising exposing a first group of cells to a transfection agent and reseeding the group with additional cells that have not been exposed to the agent. The transgenic animals are useful for medical and scientific animal models of human diseases and conditions, as well as sources for cells, tissues, and biomaterials.

1 . A transgenic swine comprising a genomic disruption of an endogenous gene chosen from the group consisting of a Low-Density Lipoprotein Receptor gene (LDLR), Duchene's Muscular Dystrophy (DMD) gene, and hairless gene (HR). 
     
     
         2 . The swine of  claim 1 , with said genomic disruption preventing expression of a functional protein. 
     
     
         3 . The swine of  claim 1  being homozygous or heterozygous for said disrupted gene. 
     
     
         4 . The swine of  claim 1  being free of a marker gene. 
     
     
         5 . The swine of  claim 1  wherein the disrupted endogenous gene is the LDLR. 
     
     
         6 . The swine of  claim 5  wherein the swine exhibits a phenotype chosen from the group consisting of hypercholesterolemia, atherosclerosis, and atherosclerotic lesions. 
     
     
         7 . The swine of  claim 5  wherein the disrupted LDLR gene is disrupted at exon 4. 
     
     
         8 . The swine of  claim 5  wherein all of the LDLR genes in the swine are disrupted. 
     
     
         9 . The swine of  claim 1  wherein the disrupted endogenous gene is the Duchene's Muscular Dystrophy (DMD) gene. 
     
     
         10 . The swine of  claim 9  wherein the swine exhibits a muscular dystrophy phenotype. 
     
     
         11 . The swine of  claim 9  wherein the disrupted DMD gene is disrupted at exon 7. 
     
     
         12 . The swine of  claim 9  wherein all of the DMD genes are disrupted. 
     
     
         13 . The swine of  claim 9  wherein the DMD gene disruption prevents expression of a functional Dp427 dystrophin isoform. 
     
     
         14 . The swine of  claim 1  wherein the disrupted endogenous gene is the HR. 
     
     
         15 . The swine of  claim 14  wherein the swine exhibits a phenotype chosen from the group consisting of hairlessness and reduced hair. 
     
     
         16 . The swine of  claim 5  wherein the disrupted HR gene is disrupted at HR exon 2. 
     
     
         17 . The swine of  claim 5  wherein all of the HR genes are disrupted. 
     
     
         18 . The swine of  claim 1  wherein the disruption is inducible upon administration of an induction agent. 
     
     
         19 . The swine of  claim 1  wherein the swine is chosen from the group consisting of pig, miniature pig, and Ossabaw pig. 
     
     
         20 . Tissue isolated from the transgenic pig of  claim 1 . 
     
     
         21 . A transfected somatic swine cell comprising a disrupted gene chosen from the group consisting of a Low-Density Lipoprotein Receptor gene (LDLR), Duchene's Muscular Dystrophy (DMD) gene, and a hairless gene (HR). 
     
     
         22 . The cell of  claim 1  wherein the cell is chosen from the group consisting of embryonic blastomere, fetal fibroblast, adult ear fibroblast, and granulosa cell. 
     
     
         23 . The cell of  claim 1  wherein the disrupted endogenous gene is the LDLR. 
     
     
         24 . The cell of  claim 1  wherein the disrupted endogenous gene is the DMD gene. 
     
     
         25 . The swine of  claim 1  wherein the disrupted endogenous gene is the HR. 
     
     
         26 . A transgenic swine prepared by nuclear transfer of the cell of  claim 21 . 
     
     
         27 . The swine of  claim 26  wherein the nuclear transfer is chosen from the group consisting of pronuclear microinjection, sperm mediated gene transfer, and somatic cell nuclear transfer. 
     
     
         28 . A method of introducing an exogenous nucleic acid into a swine cell in vitro comprising:
 exposing a first group of swine cells to a transfection agent that comprises an exogenous nucleic acid during a first culture time period and   subsequently adding a second group of swine cells to the first group for a second culture time period, wherein the second group of cells have not been exposed to the transfection agent.   
     
     
         29 . The method of  claim 28 , wherein the first group of cells are chosen from the group consisting of primary fetal swine cells and swine fibroblasts. 
     
     
         30 . The method of  claim 28  wherein a ratio of the second group of cells to the first group of cells is between 1:1 and 20:1. 
     
     
         31 . The method of  claim 28  wherein the exogenous nucleic acid disrupts a target gene chosen from the group consisting of a Low-Density Lipoprotein Receptor gene (LDLR), Duchene's Muscular Dystrophy (DMD) gene, and a hairless gene (HR).