Porcine cmp-n-acetylneuraminic acid hydroxylase gene

Abstract

The present invention provides porcine CMP-N-Acetylneuraminic-Acid Hydroxylase (CMP-Neu5Ac hydroxylase) protein, cDNA, and genomic DNA regulatory sequences. Furthermore, the present invention includes porcine animals, tissues, and organs, as well as cells and cell lines derived from such animals, tissues, and organs, which lack expression of functional CMP-Neu5Ac hydroxylase. Such animals, tissues, organs, and cells can be used in research and in medical therapy, including in xenotransplantation, and in industrial livestock farming operations.

1 - 66 . (canceled) 
     
     
         67 . A targeting vector for homologous recombination in a somatic cell comprising a marker sequence and a sequence homologous to a porcine genomic CMP-N-acetylneuraminic-acid (CMP-Neu5Ac) hydroxylase sequence, wherein said homologous sequence is sufficient to provide targeted insertion of the selectable marker sequence into a target CMP-Neu5Ac gene in a host. 
     
     
         68 . The targeting vector of  claim 67  wherein said vector comprises:
 i. a first nucleotide sequence comprising at least 17 contiguous nucleotides of the porcine genomic CMP-Neu5Ac hydroxylase sequence;   ii. a second nucleotide sequence comprising at least 17 contiguous nucleotides of the porcine genomic CMP-Neu5Ac hydroxylase sequence; and   iii. a marker sequence;   wherein said first and second nucleotide sequences do not overlap.   
     
     
         69 . The targeting vector of  claim 68  wherein the selectable marker gene is green fluorescent protein. 
     
     
         70 . The targeting vector of  claim 68  wherein the first nucleotide sequence represents a 5′ recombination arm. 
     
     
         71 . The targeting vector of  claim 68  wherein the second nucleotide sequence represents a 3′ recombination arm. 
     
     
         72 . The targeting vector of  claim 68  wherein the first or second nucleotide sequence is homologous to Intron 5 of porcine genomic CMP-Neu5Ac hydroxylase sequence. 
     
     
         73 . The targeting vector of  claim 68  wherein the first or second nucleotide sequence is homologous to Intron 6 of the porcine genomic CMP-Neu5Ac hydroxylase sequence. 
     
     
         74 . The targeting vector of  claim 68  wherein the first or second nucleotide sequence comprises at least 50 contiguous nucleotides of the porcine genomic CMP-Neu5Ac hydroxylase sequence. 
     
     
         75 . The targeting vector of  claim 68  wherein the first or second nucleotide sequence comprises at least 100 contiguous nucleotides of the porcine genomic CMP-Neu5Ac hydroxylase sequence. 
     
     
         76 . The targeting vector of  claim 68  wherein the first or second nucleotide sequence comprises at least 150 contiguous nucleotides of the porcine genomic CMP-Neu5Ac hydroxylase sequence. 
     
     
         77 . A cell transfected with the targeting vector of  claim 67 . 
     
     
         78 . The cell of  claim 77  wherein at least one allele of a porcine CMP-Neu5Ac gene has been rendered inactive via homologous recombination. 
     
     
         79 . A porcine animal comprising the cell of  claim 78 . 
     
     
         80 . The animal of  claim 79  wherein at least one allele of a porcine CMP-Neu5Ac hydroxylase gene has been rendered inactive via homologous recombination. 
     
     
         81 . An organ obtained from the animal of  claim 80 . 
     
     
         82 . A tissue obtained from the animal of  claim 80 . 
     
     
         83 . The organ of  claim 81  wherein the organ is selected from the group consisting of heart, lung, kidney and liver. 
     
     
         84 . A method to produce genetically modified cells comprising: (a) transfecting a porcine cell with the targeting vector of  claim 67 ; and (b) selecting a transfected cell in which at least one allele of a porcine CMP-N-acetylneuraminic-acid (CMP-Neu5Ac) hydroxylase gene has been rendered inactive. 
     
     
         85 . The method of  claim 84  further comprising: (c) transferring the nucleus of the selected transfected cell into an enucleated oocyte to produce an embryo; and (d) allowing the embryo to develop into a non-human animal wherein at least one allele of a porcine CMP-Neu5Ac hydroxylase gene has been rendered inactive.