Elimination of endogenous porcine retrovirus

Abstract

Porcine nucleic acid sequences flanking potentially infectious porcine endogenous retroviral (PERV) insertion sites have been identified and isolated. The unique flanking sequences include porcine nucleic acid sequences that flank the 3′ end and porcine nucleic acid sequences that flank the 5′ end of PERV insertion sites. The present invention provides compositions and methods for detecting presence of PERV in a sample, particularly those with infectious potential. In addition, the invention relates to breeding of pigs or selection of porcine tissue that is free of infectious PERV for use as a xenotransplant tissue.

1 . An isolated nucleic acid sequence comprising a member selected from the group consisting of (i) a porcine nucleic acid sequence comprising a 3′ end flanking sequence of an infectious porcine endogenous retrovirus (PERV) insertion site, (ii) a porcine nucleic acid sequence comprising a 5′ end flanking sequence of an infectious porcine endogenous retrovirus (PERV) insertion site, (iii) a nucleic acid sequence capable of hybridizing under moderately stringent conditions to a porcine 3′ end flanking sequence of an infectious PERV insertion site, (iv) a nucleic acid sequence capable of hybridizing under moderately stringent conditions to a porcine 5′ end flanking sequence of an infectious PERV insertion site, (v) a nucleic acid sequence having at least 80% sequence identity to a porcine 3′ end flanking sequence of an infectious PERV insertion site, (vi) a nucleic acid sequence having at least 80% sequence identity to a porcine 5′ end flanking sequence of an infectious PERV insertion site, and (vii) a complement of any of (i)-(vi).  
     
     
         2 . The nucleic acid sequence of  claim 1  wherein the nucleic acid sequence is a porcine nucleic acid sequence comprising a 3′ end flanking sequence of an infectious porcine endogenous retrovirus (PERV) insertion site selected from the group consisting of SEQ ID NO: 6 to 35.  
     
     
         3 . The nucleic acid sequence of  claim 1  wherein the nucleic acid sequence is a porcine nucleic acid sequence comprising a 5′ end flanking sequence of an infectious porcine endogenous retrovirus (PERV) insertion site selected from the group consisting of SEQ ID NO: 101 to 118.  
     
     
         4 . The nucleic acid sequence of  claim 1  wherein the nucleic acid sequence is capable of hybridizing under moderately stringent conditions to a porcine 3′ end flanking sequence of an infectious PERV insertion site selected from the group consisting of SEQ ID NO:8, SEQ ID NO: 24, and SEQ ID NO: 33.  
     
     
         5 . An isolated 3′ end flanking sequence of infectious PERV insertion sites comprising a member selected from the group consisting of (i) SEQ ID NO: 8 or as identified by ATCC No. ______, (ii) SEQ ID NO: 24 or as identified by ATCC No. ______, (iii) SEQ ID NO: 33 or as identified by ATCC No. ______, and (iv) a complement of any of (i)-(iii).  
     
     
         6 . A vector comprising a nucleic acid sequence of  claim 1 .  
     
     
         7 . The vector of  claim 6 , wherein the vector comprises a porcine 3′ or 5′ end flanking sequence of an infectious PERV insertion site.  
     
     
         8 . The vector of  claim 7  wherein the porcine 3′ end flanking sequence is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 24, and SEQ ID NO: 33.  
     
     
         9 . A host cell comprising the vector of  claim 7 .  
     
     
         10 . The nucleic acid sequence of  claim 1 , wherein the nucleic acid sequence comprises a porcine 3′ or 5′ end flanking sequence of a infectious PERV insertion site.  
     
     
         11 . The nucleic acid sequence of  claim 10  further comprising porcine genomic DNA or cDNA.  
     
     
         12 . A method for detecting porcine endogenous retroviruses (PERV) comprising: 
 providing a nucleic acid probe specific for a PERV genomic 3′ or 5′ target region;    forming a target region: nucleic acid probe duplex; and    detecting said target region: nucleic acid probe duplex.    
     
     
         13 . The method of  claim 12  wherein said target region further comprises a porcine 3′ or 5′ end flanking sequence of a infectious PERV insertion site.  
     
     
         14 . A method for making a probe suitable for detecting presence of potentially infectious PERV comprising: 
 isolating of a nucleic acid sequence flanking a PERV integration site; and    identifying of the nucleic acid sequence as a unique flanking sequence.    
     
     
         15 . The method of  claim 14  wherein the unique flanking sequence is a unique 3′ or 5′ flanking sequence.  
     
     
         16 . The method of  claim 14  wherein the unique flanking sequence is identified using DNA sequence analysis.  
     
     
         17 . The method of  claim 14  wherein the nucleic acid sequence flanking a PERV integration site is isolated from a lambda library.  
     
     
         18 . The method of  claim 14  wherein isolation of the nucleic acid sequence flanking a PERV integration site includes using a conserved PERV sequence.  
     
     
         19 . The method of  claim 18  wherein the conserved PERV sequence is derived from a envelope region of a PERV sequence.  
     
     
         20 . An isolated porcine nucleic acid sequence or its complement wherein the nucleic acid sequence hybridizes under stringent conditions to a nucleic acid sequence of SEQ ID NO: 137, 138, or 139, or a subsequence thereof.  
     
     
         21 . The porcine nucleic acid sequence of  claim 20  comprising a nucleic acid sequence of SEQ ID NO: 137, 138, or 139, or a subsequence thereof.  
     
     
         22 . A vector comprising the nucleic acid of  claim 20 .  
     
     
         23 . A host cell comprising the vector of  claim 22.