Lincrna-deficient non-human animals

Abstract

Genetically modified non-human animals are provided that exhibit a functional lack of one or more IncRNAs. Methods and compositions for disrupting, deleting, and/or replacing IncRNA-encoding sequences are provided. Genetically modified mice that age prematurely are provided. Also provided are cells, tissues and embryos that are genetically modified to comprise a loss-of-function of one or more IncRNAs.

We claim: 
     
         1 . A non-human animal whose genome comprises a knockout of an endogenous long intergenic non coding RNA (lincRNA) locus;
 wherein the knockout comprises a deletion of at least a second exon, a last exon, and all exons between the second and the last exons of the lincRNA locus; and   wherein the non-human animal exhibits a phenotype associated with the knockout.   
     
     
         2 . The non-human animal of  claim 1 , wherein the knockout further comprises
 (a) a replacement of the second exon, last exon, and all exons between the second and the last exons of the lincRNA locus, with an insert nucleic acid, or   (b) a replacement of an entire RNA coding region of the lincRNA locus with an insert nucleic acid.   
     
     
         3 . The non-human animal of  claim 2 , wherein the insert nucleic acid comprises a first nucleotide sequence that encodes a reporter and/or a second nucleotide sequence that encodes a selectable marker. 
     
     
         4 . The non-human animal of  claim 3 , wherein the first nucleotide sequence that encodes the reporter is positioned in the lincRNA locus in operable linkage with an endogenous lincRNA promoter, wherein the endogenous lincRNA promoter drives expression of the first nucleotide sequence, and wherein the expression of the reporter follows an expression pattern of the lincRNA. 
     
     
         5 . The non-human animal of  claim 3 , wherein the first nucleotide sequence comprises a Kozak consensus sequence and/or the second nucleotide sequence is operably linked to a promoter. 
     
     
         6 . The non-human animal of  claim 3 , wherein the insert nucleic acid comprises site-specific recombination sites flanking a segment encoding the reporter and/or site-specific recombination sites flanking a segment encoding the selectable marker. 
     
     
         7 . The non-human animal of  claim 1 , wherein the non-human animal is a mouse. 
     
     
         8 . The non-human animal of  claim 7 , wherein the lincRNA locus is Pantr2, Fendrr, HOTTIP, Haglr, Kantr, Peril or Tug1. 
     
     
         9 . The non-human animal of  claim 8 , wherein the phenotype is
 (a) a premature aging-associated phenotype;   (b) lethality;   (c) a defect in lung development;   (d) a morphological or functional malformation of skeletal and/or muscle tissue;   (e) a loss of muscle mass in one or more tissues;   (f) a defect in brain development; or   (g) a combination thereof of any of (a)-(f).   
     
     
         10 . The non-human animal of  claim 7 , wherein the lincRNA locus and phenotype associated with the knockout respectively comprise:
 (a) Pantr2; and a defect in brain development and/or a premature aging associated phenotype;   (b) Fendrr; and lethality and/or a defect in lung development;   (c) HOTTIP; and at least one morphological or functional defect in skeleton and/or muscle;   (d) Haglr; and lethality;   (e) Kantr; and a defect in brain development and/or a premature aging associated phenotype;   (f) Peril; and a defect in brain development, a premature aging associated phenotype, and/or perinatal lethality;   (g) Tug1; and a defect in brain development and/or a premature aging-associated phenotype;   (h) any combination of (a)-(h).   
     
     
         11 . The non-human animal of  claim 10 , wherein the knockout comprises:
 (a) a deletion of a genomic sequence of a Pantr2 locus between genomic coordinates Chromosome 6: 42707143-42713698;   (b) a deletion of a genomic sequence of a Fendrr locus between genomic coordinates Chromosome 8: 121054882-121074065;   (c) a deletion of a genomic sequence of a HOTTIP locus between genomic coordinates Chromosome 6: 52262834-52267603;   (d) a deletion of a genomic sequence of a HOTTIP locus between genomic coordinates Chromosome 6: 52265374-52267603;   (f) a deletion of a genomic sequence of a Haglr locus between genomic coordinates Chromosome 2: 74750433-74762886;   (g) a deletion of a genomic sequence of a Kantr locus between genomic coordinates Chromosome X: 152298544-152327475;   (h) a deletion of a genomic sequence of a Peril locus between genomic coordinates Chromosome 3: 34767849-34782292;   (i) a deletion of a genomic sequence of a Tug1 locus between genomic coordinates Chromosome 11: 3639794-3648758;   (j) a deletion of a genomic sequence of a Tug1 locus between genomic coordinates Chromosome 11: 3639794-3645518; or   (k) any combination of (a)-(k).   
     
     
         12 . The non-human animal of  claim 10 , wherein the knockout further comprises:
 (a) a replacement of the second exon, last exon, and all exons between the second and the last exons of the lincRNA locus with an insert nucleic acid; or   (b) a replacement of an entire RNA coding region of the IncRNA locus with an insert nucleic acid.   
     
     
         13 . The non-human animal of  claim 10 , wherein the knockout further comprises:
 (a) replacement of the genomic sequence of the Pantr2 locus between genomic coordinates Chromosome 6: 42707143-42713698 with the insert nucleic acid;   (b) replacement of the genomic sequence of the Fendrr locus between genomic coordinates Chromosome 8: 121054882-121074065 with the insert nucleic acid;   (c) replacement of the genomic sequence of the HOTTIP locus between genomic coordinates Chromosome 6: 52262834-52267603 with the insert nucleic acid;   (d) replacement of the genomic sequence of the HOTTIP locus between genomic coordinates Chromosome 6: 52265374-52267603 with the insert nucleic acid;   (f) replacement of the genomic sequence of the Haglr locus between genomic coordinates Chromosome 2: 74750433-74762886 with the insert nucleic acid;   (g) replacement of the genomic sequence of the Kantr locus between genomic coordinates Chromosome X: 152298544-152327475with the insert nucleic acid;   (h) replacement of the genomic sequence of the Peril locus between genomic coordinates Chromosome 3: 34767849-34782292 with the insert nucleic acid;   (i) replacement of the genomic sequence of the Tug1 locus between genomic coordinates Chromosome 11: 3639794-3648758 with the insert nucleic acid;   (j) replacement of the genomic sequence of the Tug1 locus between genomic coordinates Chromosome 11; 3639794-3645518 with the insert nucleic acid; or   (k) any combination of (a)-(j).   
     
     
         14 . The non-human animal of  claim 13 , wherein the insert nucleic acid comprises a first nucleotide sequence that encodes a reporter and/or a second nucleotide sequence that encodes a selectable marker. 
     
     
         15 . The non-human animal of  claim 14 , wherein the first nucleotide sequence comprises a Kozak consensus sequence and/or the second nucleic acid sequence is operably linked to a promoter. 
     
     
         16 . The non-human animal of  claim 14 , wherein the insert nucleic acid comprises site-specific recombination sites flanking a segment encoding the reporter and/or site-specific recombination sites flanking a segment encoding the selectable marker. 
     
     
         17 . A cell, tissue or embryo derived from the non-human animal of  claim 1 . 
     
     
         18 . A method for making the non-human animal of  claim 1 , comprising
 (a) contacting an embryonic stem (ES) cell of the non-human animal with a targeting construct comprising an insert nucleic acid flanked by 5′ and 3′ homology arms; wherein the targeting construct undergoes homologous recombination with the endogenous lincRNA locus in the genome of the non-human animal ES cell to form a modified non-human animal ES cell comprising the endogenous lincRNA locus that comprises a deletion of at least a second exon, a last exon, and all exons between the second and the last exons of the lincRNA locus;   (b) introducing the modified non-human animal ES cell into a host non-human animal embryo; and   (c) gestating the host non-human animal embryo in a surrogate non-human animal mother, wherein the surrogate non-human animal mother produces at least one progeny that comprises the endogenous IncRNA locus that comprises the deletion of at least a second exon, a last exon, and all exons between the second and the last exons of the lincRNA locus and exhibits a phenotype associated with the knockout mutation.   
     
     
         19 . A method for producing a gene expression profile for a long intergenic non coding RNA (lincRNA) locus of a non-human animal that is informative of the structure and/or function of the lincRNA encoded by the lincRNA locus, the method comprising
 obtaining the non-human animal of  claim 1 , wherein the knockout further comprises a replacement of at least the second exon, last exon, and all exons between the second and last exons of the lincRNA locus with a reporter gene, and wherein an endogenous promoter of the lincRNA locus controls expression of the reporter gene;   expressing the reporter gene in the non-human animal; and   producing an expression profile for the lincRNA locus by surveilling expression of the reporter gene, wherein correlation of the spatial and/or temporal expression of the reporter gene with a phenotype that is exhibited by the non-human animal confirms a structure and/or function of the lincRNA locus.   
     
     
         20 . The method of  claim 19 , wherein producing an expression profile for the lincRNA locus comprises specifying at least one cell type expressing the reporter gene. 
     
     
         21 . The method of  claim 19 , wherein producing an expression profile for the lincRNA locus comprises surveilling expression of the reporter gene during embryogenesis of the non-human animal, throughout adulthood of non-human animal, until death of the non-human animal, or a combination thereof.