Rodents With Conditional ACVR1 Mutant Alleles

Abstract

A genetically modified mouse is provided that comprises a conditional Acvr1 allele that comprises a mutated exon that, upon induction, converts to a mutant exon phenotype, wherein the mutant exon phenotype includes ectopic bone formation. Mice comprising a mutant Acvr1 exon 5 in antisense orientation, flanked by site-specific recombinase recognition sites, are provided, wherein the mice further comprise a site-specific recombinase that recognizes the site-specific recombinase recognitions sites, wherein the recombinase is induced upon exposure of the mouse to tamoxifen. Upon exposure to tamoxifen, the recombinase is expressed and acts on the RRS-flanked mutant exon 5 and places the mutant exon 5 in sense orientation and deletes the wild-type exon.

1 - 7 . (canceled) 
     
     
         8 . A set of oligonucleotides for detecting a loss of a ACVR1 allele in a genetically modified mouse comprising:
 a forward primer oligonucleotide,   a reverse primer oligonucleotide, and   a detection oligonucleotide, wherein the detection oligonucleotide is labelled,   wherein the forward primer oligonucleotide and the reverse primer oligonucleotide are configured to amplify in an amplification reaction a mouse native ACVR1 nucleic acid sequence to generate an amplification product detectable by the detection oligonucleotide.   
     
     
         9 . The set of oligonucleotides of  claim 8 , wherein the forward primer oligonucleotide is SEQ ID NO: 1. 
     
     
         10 . The set of oligonucleotides of  claim 8 , wherein the forward primer oligonucleotide is SEQ ID NO: 2. 
     
     
         11 . The set of oligonucleotides of  claim 8 , wherein the reverse primer oligonucleotide is SEQ ID NO: 3. 
     
     
         12 . The set of oligonucleotides of  claim 8 , wherein the reverse primer oligonucleotide is SEQ ID NO: 4. 
     
     
         13 . The set of oligonucleotides of  claim 8 , wherein the probe oligonucleotide is SEQ ID NO: 5. 
     
     
         14 . The set of oligonucleotides of  claim 8 , wherein the probe oligonucleotide is SEQ ID NO: 6. 
     
     
         15 . The set of oligonucleotides of  claim 8 , wherein the forward primer oligonucleotide is SEQ ID NO: 1, the reverse primer oligonucleotide is SEQ ID NO: 3, and the detection oligonucleotide is SEQ ID NO 5. 
     
     
         16 . The set of oligonucleotides of  claim 8 , wherein the forward primer oligonucleotide is SEQ ID NO: 2, the reverse primer oligonucleotide is SEQ ID NO: 4, and the detection oligonucleotide is SEQ ID NO 6. 
     
     
         17 . A method for detecting a loss of an ACVR1 allele in a genetically modified mouse, the method comprising:
 performing a nucleic acid amplification assay on a sample from the genetically modified mouse using a set of oligonucleotides comprising a forward primer oligonucleotide, a reverse primer oligonucleotide, and a detection oligonucleotide, wherein the forward primer oligonucleotide and the reverse primer oligonucleotide are configured to amplify in an amplification reaction a mouse native ACVR1 nucleic acid sequence, and   detecting a level of a amplification product with the detection oligonucleotide,   wherein the presence of the amplification product at a level that corresponds to one gene copy number indicates that the mouse carries a targeted genetic modification.   
     
     
         18 . The method of  claim 17 , wherein the forward primer oligonucleotide is SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         19 . The method of  claim 17 , wherein the reverse primer oligonucleotide is SEQ ID NO: 3 or SEQ ID NO: 4. 
     
     
         20 . The method of  claim 17 , wherein the detection oligonucleotide is SEQ ID NO: 5 or SEQ ID NO: 6. 
     
     
         21 . The method of  claim 17 , wherein the forward primer oligonucleotide is SEQ ID NO: 1, the reverse primer oligonucleotide is SEQ ID NO: 3, and the detection oligonucleotide is SEQ ID NO 5. 
     
     
         22 . The method of  claim 17 , wherein the forward primer oligonucleotide is SEQ ID NO: 2, the reverse primer oligonucleotide is SEQ ID NO: 4, and the detection oligonucleotide is SEQ ID NO 6. 
     
     
         23 . The method of  claim 17 , wherein the presence of the amplification product at a level that corresponds to two gene copies number indicates that the mouse carries a targeted genetic modification. 
     
     
         24 . The method of  claim 17 , wherein the targeted genetic modification is a conditional mutation in the Activin A receptor type 1 (Acvr1) gene. 
     
     
         25 . The method of  claim 24 , wherein the conditional mutation is a modified mouse Activin A receptor type 1 (Acvr1) gene comprising:
 i) a human exon 5 of the Acvr1 gene encoding glutamic acid at the first codon instead of aspartic acid in sense orientation flanked upstream and downstream by first pair of recombination recognition sites; and   ii) a mutant exon 5 of a mouse Acvr1 gene comprising an R206H mutation in antisense orientation flanked upstream and downstream by second pair of recombination recognition sites that are different than the first pair of recombination recognition sites;   wherein the first and second recombination recognition sites are oriented so that a recombinase can invert the mutant exon 5 into sense orientation, delete the human exon 5, and allow a mutant Acvr1 comprising the mutant exon 5 to be expressed resulting in ectopic bone formation.   
     
     
         26 . A genetically modified mouse whose genome comprises a modified mouse Activin A receptor type 1 (Acvr1) gene comprising:
 i) a human exon 5 of the Acvr1 gene encoding glutamic acid at the first codon instead of aspartic acid in sense orientation flanked upstream and downstream by first pair of recombination recognition sites; and   ii) a mutant exon 5 of a mouse Acvr1 gene in antisense orientation flanked upstream and downstream by second pair of recombination recognition sites that are different than the first pair of recombination recognition sites;   wherein the first and second recombination recognition sites are oriented so that a recombinase can invert the mutant exon 5 into sense orientation, delete the human exon 5, and allow a mutant Acvr1 comprising the mutant exon 5 to be expressed resulting in ectopic bone formation.   
     
     
         27 . The genetically modified mouse of  claim 26 , wherein the mutant exon 5 comprises an R206H mutation.