Drosophila recombination-associated protein and methods of use

Abstract

The invention encompasses Drosophila Recombination Associated Protein (DRAP) isolated from D. melanogaster and a nucleic acid sequence encoding DRAP. The Drosophila Recombination Associated Protein, its homologues from other organisms or active peptides derived therefrom, as well as DNA encoding such protein are useful for homology-dependent pairing of three DNA strands. The combination of strand-transfer and topoisomerase activities associated with DRAP permits directed pairing and cleavage at defined site(s) within DNA. This in turn makes possible the isolation and/or removal of a defined segment of DNA. DRAP is also useful in cloning, genomic cloning and gene mapping, in promoting gene disruptions or “knockout” mutations, in carrying out targeted mutagenesis of specific genes and in generating transgenic animals. The invention further encompasses a method for experimental and therapeutic application of DRAP driven knockouts or other modifications of genes responsible for genetic diseases as well as the use of DRAP driven genetic manipulation of genes in gene therapy.

1 - 17 . (canceled)  
     
     
         18 . A method of making a transgenic non-human animal, comprising: 
 introducing a protein and a first DNA segment into a cell, said cell being capable of developing into said transgenic non-human animal, and said first DNA segment being homologous to target DNA in said cell, wherein said protein exhibits recombinase and topoisomerase activities and is encoded by a nucleic acid that hybridizes under high stringency hybridization conditions to an isolated nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3;    implanting said cell or progeny thereof into a non-human animal capable of bearing the cell or progeny thereof to term; and    allowing the implanted cell or cells to develop to term,    thereby obtaining said transgenic non-human animal.    
     
     
         19 . The method of  claim 18  wherein said protein comprises the amino acid sequence of SEQ ID NO: 4.  
     
     
         20 . The method of  claim 18  wherein the first DNA segment is an oligonucleotide.  
     
     
         21 . The method of  claim 18  wherein said protein is introduced into said cell by electroporation, injection, or a ballistic method.  
     
     
         22 . The method of  claim 21  wherein said protein is introduced into said cell by injection.  
     
     
         23 . The method of  claim 21  wherein said protein and said first DNA segment are co-introduced into said cell.  
     
     
         24 . The method of  claim 22  wherein said protein and said first DNA segment are co-injected into said cell.  
     
     
         25 . The method of  claim 18  wherein said protein is introduced into said cell by expression of a gene present in said cell.  
     
     
         26 . The method of  claim 25  wherein said gene is under control of an inducible promoter.  
     
     
         27 . The method of  claim 18  wherein said first DNA segment is in an intron.  
     
     
         28 . The method of  claim 18  wherein said first DNA segment is in an exon.  
     
     
         29 . The method of  claim 18  wherein said non-human animal is a mammal.  
     
     
         30 . The method of  claim 29  wherein said mammal is a member selected from the group consisting of mouse, cat, dog, cow, sheep, and goat.  
     
     
         31 . The method of  claim 18  wherein said introduction of said first DNA segment and said protein yields a heterozygous mutation.  
     
     
         32 . The method of  claim 18  wherein said introduction of said first DNA segment and said protein yields a homozygous mutation.  
     
     
         33 . The method of  claim 18  wherein said first DNA segment comprises a sequence from a disease-related gene.  
     
     
         34 . The method of  claim 18  wherein said first DNA segment comprises a marker sequence.  
     
     
         35 . A method of generating a transgenic tissue or organ, comprising: 
 introducing a protein and a first DNA segment into a cell, said cell being capable of developing into said transgenic tissue or organ, and said first DNA segment being homologous to target DNA in said cell, wherein said protein exhibits recombinase and topoisomerase activities and is encoded by a nucleic acid that hybridizes under high stringency hybridization conditions to an isolated nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3,    implanting said cell or progeny thereof into a recipient tissue or organ;    thereby obtaining said transgenic tissue or organ.    
     
     
         36 . The method of  claim 35  wherein said protein comprises the amino acid sequence of SEQ ID NO: 4.  
     
     
         37 . The method of  claim 35  wherein the first DNA segment is an oligonucleotide.  
     
     
         38 . The method of  claim 35  wherein said protein is introduced into said cell by electroporation, injection, or a ballistic method.  
     
     
         39 . The method of  claim 38  wherein said protein is introduced into said cell by injection.  
     
     
         40 . The method of  claim 38  wherein said protein and said first DNA segment are co-introduced into said cell.  
     
     
         41 . The method of  claim 39  wherein said protein and said first DNA segment are co-injected into said cell.  
     
     
         42 . The method of  claim 35  wherein said protein is introduced into said cell by expression of a gene present in said cell.  
     
     
         43 . The method of  claim 42  wherein said gene is under control of an inducible promoter.  
     
     
         44 . The method of  claim 35  wherein said first DNA segment is in an intron.  
     
     
         45 . The method of  claim 35  wherein said first DNA segment is in an exon.  
     
     
         46 . The method of  claim 35  wherein said tissue or organ is from a mammal.  
     
     
         47 . The method of  claim 46  wherein said mammal is a member selected from the group consisting of mouse, cat, dog, cow, sheep, and goat.  
     
     
         48 . The method of  claim 35  wherein said introduction of said first DNA segment and said protein yields a heterozygous mutation.  
     
     
         49 . The method of  claim 35  wherein said introduction of said first DNA segment and said protein yields a homozygous mutation.  
     
     
         50 . The method of  claim 35  wherein said first DNA segment comprises a sequence from a disease-related gene.  
     
     
         51 . The method of  claim 35  wherein said first DNA segment comprises a marker sequence.  
     
     
         52 . A method of making a transgenic non-human animal, comprising: 
 introducing a protein and a first DNA segment into an embryonic stem (ES) cell, said first DNA segment being homologous to target DNA in said cell, wherein said protein exhibits recombinase and topoisomerase activities and is encoded by a nucleic acid that hybridizes under high stringency hybridization conditions to an isolated nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3;    implanting said ES cell into an embryo;    implanting said embryo into a non-human animal capable of bearing the embryo to term; and    allowing the embryo to develop to term,    thereby obtaining said transgenic non-human animal.