Method for stable inversion of dna sequence by site-specific recombination and dna vectors and transgenic cells thereof

Abstract

The invention relates to a method for the stable inversion of a DNA fragment upon recombinase-mediated rearrangements using two sets of two incompatible site-specific recombinase targeting sites (SSRTS) in the same order but in reverse orientation flanking said DNA fragment to be inverted. The invention also relates to a method for the stable inversion of said DNA fragment upon rearrangement mediated by a recombinase such as Cre recombinase. The invention also relates to a method for obtaining a transgenic cell of which at least one allele of a DNA sequence of interest is invalidated by a process of conditional deletion and the genome of which comprises a reporter gene inserted at the place of the DNA fragment deleted by said processor conditional deletion. The invention also concerns a method to generate targeting sites to perform site-specific recombination mediated cassette exchange. The corresponding vectors, host cells, and transgenic animals are claimed.

1 . An isolated DNA molecule comprising at least a sequence A flanked by at least site specific recombinase targeting sequences (SSRTS) L1, and at least a sequence B flanked by at least site specific recombinase targeting sequences (SSRTS) L2, said SSRTS L1 and SSRTS L2 being unable to recombine with one another, wherein: 
 (i) sequences L1 are in an orientation opposite to one another,    (ii) sequences L2 are in an orientation opposite to one another, and    (iii) the order of SSRTS sequences in said DNA molecule is 5′-L1-L2-L1-L2-3′.    
     
     
         2 . The DNA molecule according to  claim 1 , wherein the order of sequences in said DNA molecule is: 5′-L1-sequence A-L2-sequence B-L1-L2-3′.  
     
     
         3 . The DNA molecule according to  claim 1 , wherein the order of sequences in said DNA molecule is: 5′-L1-L2-sequence A-sequence B-L1-L2-3′.  
     
     
         4 . The DNA molecule according to  claim 1 , wherein the order of sequences in said DNA molecule is: 5′-L1-L2-sequence A-L1-sequence B-L2-3′.  
     
     
         5 . The DNA molecule according to  claim 1 , wherein sequences A and B are in an orientation opposite to each other.  
     
     
         6 . The DNA molecule according to  claim 1 , wherein the recombinase specific of said SSRTS L1 and the recombinase specific of said SSRTS L2 are the same.  
     
     
         7 . (Canceled)  
     
     
         8 . The DNA molecule according to  claim 6 , wherein said recombinase specific of said SSRTS is selected from the group consisting of Cre recombinase of bacteriophage P1, the FLP recombinase of  Saccharomyces cerevisiae , the R recombinase of  Zygosaccharomyces rouxii  pSR1, the A recombinase of  Kluyveromyces drosophilarium  pKD1, the A recombinase of  Kluyveromyces waltii  pKW1, the integrase λ Int, the recombinase of the GIN recombination system of the Mu phage, and bacterial β recombinase.  
     
     
         9 . The DNA molecule according to  claim 8 , wherein said recombinase is said Cre recombinase of bacteriophage P1.  
     
     
         10 . The DNA molecule according to  claim 9 , wherein said SSRTS L1 and/or L2 specific for said Cre recombinase are selected from the group consisting of Lox P1, Lox 66, Lox 71, Lox 511, Lox 512, Lox 514 and mutated of Lox P1 sequences, wherein said mutated Lox P1 sequences comprise at least one point mutation in the spacer sequence.  
     
     
         11 . The DNA molecule according to  claim 10 , wherein either SSRTS L1 comprises the Lox P1 nucleotide sequence (SEQ ID NO. 52) and SSRTS L2 comprises the Lox 511 nucleotide sequence (SEQ ID NO. 53) or SSRTS L1 comprises the Lox 511 sequence and SSRTS L2 comprises Lox P1 sequence.  
     
     
         12 . (Canceled)  
     
     
         13 . The DNA molecule according to  claim 12 , characterized in that said SSRTS L1 and/or L2 specific for said FLP recombinase are chosen from the group composed of the sequences FRT-S and FRT-F3 0.88 .  
     
     
         14 . (Canceled)  
     
     
         15 . The DNA molecule according to  claim 1 , wherein said sequences A and B are selected in the group consisting of non transcribed sequence, transcribed but not translated sequence, transcribed and translated sequence.  
     
     
         16 . The DNA molecule according to  claim 15 , wherein at least the sequences A and/or B are transcribed and translated, wherein said translated sequences code for at least one protein.  
     
     
         17 . The DNA molecule according to  claim 16 , wherein said protein is selected in the group consisting of reporter protein, selection marker and protein of interest.  
     
     
         18 . The DNA molecule according to  claim 16 , wherein sequences A and/or B are coding for at least one exon, or a fragment thereof.  
     
     
         19 . The DNA molecule according to  claim 18 , wherein said exon differs from the wild type exon of a protein of interest by one or more point mutations.  
     
     
         20 . The DNA molecule according to  claim 16 , wherein said protein is encoded by a cDNA sequence, and wherein an IRES sequence is inserted 5′, or 3′, or 5′ and 3′ to said cDNA sequence.  
     
     
         21 . The DNA molecule according to  claim 17 , wherein said reporter protein is selected in the group consisting of autofluorescent proteins and enzymes detectable by a histochemical process.  
     
     
         22 . The DNA molecule according to  claim 21 , wherein said autofluorescent protein is selected from the group consisting of the green fluorescent protein (GFP), the enhanced green fluorescent protein (EGFP), the red fluorescent protein (RFP), the blue fluorescent protein (BFP), and the yellow fluorescent protein (YFP).  
     
     
         23 . The DNA molecule according to  claim 21 , wherein said enzyme, detectable by a histochemical process, is selected in the group consisting of β-galactosidase, β-glucoronidase, alcaline phosphatase, luciferase, alcohol deshyd rogenase, chloramphenicol-acetyl transferase.  
     
     
         24 . A vector comprising the isolated DNA molecule of  claim 1 .  
     
     
         25 . (Canceled)  
     
     
         26 . An isolated transgenic host cell transformed by an isolated DNA molecule according to  claim 1 .  
     
     
         27 . (Canceled)  
     
     
         28 . The isolated transgenic host cell according to  claim 26  wherein said isolated DNA molecule or said vector is integrated by homologous recombination in at least one targeted locus of the genome of said cell.  
     
     
         29 . The isolated transgenic host cell according to  claim 26  wherein said isolated DNA molecule or said vector is integrated in sites of the genome chosen among polyA sites and gene promoters.  
     
     
         30 . The isolated transgenic host cell according to  claim 26  wherein said isolated DNA molecule or said vector is randomly integrated in at least one locus of the genome of said cell.  
     
     
         31 . The isolated transgenic host cell according to  claim 26  wherein said isolated DNA molecule or said vector is maintained in said cell in an episomal form.  
     
     
         32 . A transgenic non-human organism comprising at least one cell according to  claim 26 .  
     
     
         33 . A method for the stable inversion of a DNA sequence comprising the steps of: 
 (i) contacting a DNA molecule according to  claim 1  with at least one site specific recombinase targeting sequence SSRTS L1 and SSRTS L2;    (ii) inversion of said sequences A and B or sequence A or sequence B by recombination catalyzed by said recombinase at either SSRTS L1 or L2 sequences; and    (iii) excision by recombination catalyzed by said recombinase of a DNA fragment comprised in between the SSRTS L1 or L2 sequences that are now present in direct orientation following the inversion of step (ii), and that are able to recombine with one another.    
     
     
         34 . The method according to  claim 33  wherein said DNA fragment excised in step (iii) comprises the sequence A.  
     
     
         35 . A method for obtaining a transgenic cell of which at least one allele of a DNA sequence of interest is invalidated by a process of conditional deletion and the genome of which comprises a gene selected among reporter gene, marker gene and gene encoding a protein of interest, inserted at the place of the DNA fragment deleted by said process of conditional deletion, said method comprises the steps of: 
 (i) preparation of a DNA molecule according to  claim 1  wherein sequence A or sequence B is coding at least for part of the DNA fragment of interest to be invalidated and sequence B or sequence A is coding at least for a reporter gene;    (ii) obtention of a transgenic cell genetically modified by the targeted insertion by homologous recombination at the place of said DNA sequence of interest, of a DNA molecule prepared at step (i);    (iii) contacting said DNA molecule with at least one site specific recombinase targeting sequence SSRTS L1 and one recombinase specific of SSRTS L2;    (iv) inversion of sequences A and B or sequence A or sequence B by recombination catalyzed by said recombinase at either SSRTS L1 or SSRTS L2 sequences; and    (v) excision of a DNA sequence by recombination catalyzed by said recombinase at SSRTS L2 or SSRTS L1 respectively, these SSRTS L2 or SSRTS L1 sequences being now present in direct orientation following to the inversion of step (iii), and being to recombine with one another.    
     
     
         36 . The method of  claim 35 , wherein the order of sequences in said DNA molecule is 5′-L1-sequence A-L2-sequence B-L1-L2-3′ and wherein a sequence of homology with the DNA sequence of interest are present at both extremities of said DNA molecule and wherein, the DNA fragment excised in step (v) comprises sequence A.  
     
     
         37 . The method of  claim 35 , wherein the order of sequences in said DNA molecule is 5′-L1-L2-sequence A-sequence B-L1-L2-3′ and wherein a sequence of homology with the DNA sequence of interest are present at both extremities of said DNA molecule.  
     
     
         38 . The method of  claim 35  wherein the order of sequences in said DNA molecule is 5′-L1-L2-sequence A-L1-sequence B-L2-3′ and wherein a sequence of homology with the DNA sequence of interest are present at both extremities of said DNA molecule.  
     
     
         39 . A method to perform site-specific recombination mediated cassette exchange (RMCE), said method comprising the steps of: 
 (i) preparation of a first DNA molecule comprising a first DNA sequence of interest flanked by incompatible site specific recombinase targeting sequences SSRTS L1 and L2 in an opposite direction, obtainable by the method of  claim 33;     (ii) preparation of a second DNA molecule comprising a second DNA sequence of interest flanked by the same incompatible SSRTS L1 and L2 as in step (i) in an opposite direction, by in vitro DNA cloning;    (iii) contacting said first and said second DNA molecule with at least one site specific recombinase targeting sequence SSRTS L1 and L2;    (iv) exchange by recombination catalysed by said recombinase of said first and said second DNA sequence of interest comprised in between the SSRTS L1 and L2.    
     
     
         40 . The method according to  claim 39  wherein said second DNA molecule of step (ii) is obtainable by the method of  claim 33 .  
     
     
         41 . The method according to  claim 33  wherein the steps are made in a cell free system.  
     
     
         42 . The method according to  claim 39  wherein the steps are made in an isolated transgenic host cell transformed by an isolated DNA molecule comprising at least a sequence A flanked by at least site specific recombinase targeting sequences (SSRTS) L1, and at least a sequence B flanked by at least site specific recombinase targeting sequences (SSRTS) L2, said SSRTS L1 and SSRTS L2 being unable to recombine with one another, and wherein: 
 (i) sequences L1 are in an orientation opposite to one another,  
 (ii) sequences L2 are in an orientation opposite to one another, and  
 (iii) the order of SSRTS sequences in said DNA molecule is 5′-L1-L2-L1-L2-3′.  
 
     
     
         43 . The method according to  claim 42 , further comprising the step of introducing into the cell a gene encoding the corresponding site-specific recombinase.  
     
     
         44 . The method according to  claim 43 , wherein the gene encoding said site-specific recombinase is contained in an expression vector.  
     
     
         45 . The method according to  claim 43 , wherein the gene encoding said site-specific recombinase is stably inserted into the genome of said cell.  
     
     
         46 . The method according to  claim 39 , wherein either SSRTS L1 comprises the Lox P1 sequence and SSRTS L2 comprises the Lox 511 sequence, or SSRTS L1 comprises the Lox 511 sequence and SSRTS L2 comprises Lox P1 sequence, and wherein the corresponding site-specific recombinase is Cre.  
     
     
         47 . (Canceled)  
     
     
         48 . (Canceled)  
     
     
         49 . A non-human living organism, except humans, that comprises at least one transgenic cell obtainable by the method of  claim 39 .  
     
     
         50 . The non-human living organism of  claim 49 , wherein said organism is selected from the group consisting of bacteria, yeast,  Caenorhabditis elegans, Drosophila melanogaster , zebrafish, mice, rat, rabbit, hamster, Guinea pig, cow, pig, goat, sheep, horse, and primate.  
     
     
         51 . The non-human living organism of  claim 50 , wherein said organism is a mouse.  
     
     
         52 . The non-human living organism of  claim 50 , wherein said organism is a yeast.  
     
     
         53 . The DNA molecule according to  claim 1 , wherein sequences A and B are in the same orientation.  
     
     
         54 . The method according to  claim 33 , wherein said DNA fragment excised in step (iii) comprises the sequence B.