Non-human animal exhibiting diminished upper and lower motor neuron function and sensory perception

Abstract

An animal model for motor neuron dysfunction in disease, e.g., amyotrophic lateral sclerosis (ALS), comprising a genetically modified non-human animal that comprises a genetically modified DR6 allele and exhibits normal phenotypes at birth and for a few weeks or months after birth. However, as the non-human animal ages, it develops motor neuron dysfunction that presents as one or more ALS-like symptoms, which may progress rapidly after onset. Methods of identifying candidate agents that may be used to prevent, delay or treat ALS are also provided.

1 .- 21 . (canceled) 
     
     
         22 . A method of making a population of motor neurons that exhibits increased oxidative stress compared to wildtype motor neuron cells, the method comprising
 establishing from embryoid bodies derived from a genetically engineered rodent embryonic stem (ES) cell comprising a modified endogenous DR6 locus lacking a nucleotide sequence that encodes the cytoplasmic domain of a DR6 protein in its entirety, and   differentiating the embryoid bodies into the population of motor neurons,   wherein the population of motor neurons releases an increased amount of reactive oxygen species compared to wildtype motor neuron cells comprising a wildtype endogenous DR6 locus.   
     
     
         23 .- 37 . (canceled) 
     
     
         38 . A method for screening a candidate agent for reducing oxidative stress in a motor neuron comprising
 (a) culturing the population of motor neurons made according to the method of  claim 22  in the presence or absence of an agent;   (b) determining whether the agent prevents, inhibits, and/or reduces oxidative stress in the population of motor neurons as compared to a control population of motor neurons cultured in the absence of the agent; wherein the prevention, inhibition, and/or reduction of oxidative stress in the motor neurons is indicative of a candidate agent for reducing oxidative stress in a motor neuron.   
     
     
         39 . The method of  claim 22 , wherein the modified endogenous DR6 locus comprises a replacement of the endogenous genomic sequence comprising part of exon 2 to the entirety of exon 6 with a heterologous polynucleotide comprising an ROR1 transmembrane domain encoding sequence operably linked to a β-galactosidase gene such that the locus (i) lacks a nucleotide sequence encoding amino acids 1-614 of SEQ ID NO:15 and (ii) comprises a nucleic acid sequence set forth as SEQ ID NO:17, or a degenerate variant thereof, operably linked to endogenous DR6 transcriptional regulatory sequences. 
     
     
         40 . The method of  claim 22 , wherein the ES cells are heterozygous for the modified endogenous DR6 locus. 
     
     
         41 . The method of  claim 22 , wherein the ES cells are rat ES cells. 
     
     
         42 . The method of  claim 22 , wherein the ES cells are mouse ES cells. 
     
     
         43 . The method of  claim 42 , wherein the mouse ES cells are from a strain selected from the group consisting of a 129 strain, a C57BL/6 strain, and a mixed C57BL/6×129 strain. 
     
     
         44 . The method of  claim 38 , wherein the population of motor neurons are made from genetically engineered mouse embryonic stem (ES) cell comprising a modified endogenous DR6 locus comprising a replacement of the endogenous genomic sequence comprising part of exon 2 to the entirety of exon 6 with a heterologous polynucleotide comprising an ROR1 transmembrane domain encoding sequence operably linked to a β-galactosidase gene such that the locus (i) lacks a nucleotide sequence encoding amino acids 1-614 of SEQ ID NO:15 and (ii) comprises a nucleic acid sequence set forth as SEQ ID NO:17, or a degenerate variant thereof, operably linked to endogenous DR6 transcriptional regulatory sequences. 
     
     
         45 . The method of  claim 38 , wherein the ES cells are heterozygous for the modified endogenous DR6 locus. 
     
     
         46 . The method of  claim 38 , wherein the ES cells are rat ES cells. 
     
     
         47 . The method of  claim 38 , wherein the ES cells are mouse ES cells. 
     
     
         48 . The method of  claim 42 , wherein the mouse ES cells are from a strain selected from the group consisting of a 129 strain, a C57BL/6 strain, and a mixed C57BL/6×129 strain. 
     
     
         49 . A motor neuron cell made according to the method of  claim 22 . 
     
     
         50 . The motor neuron cell of  claim 49 , wherein motor neuron is made from a genetically engineered mouse embryonic stem (ES) cell comprising a modified endogenous DR6 locus comprising a replacement of the endogenous genomic sequence comprising part of exon 2 to the entirety of exon 6 with a heterologous polynucleotide comprising an ROR1 transmembrane domain encoding sequence operably linked to a β-galactosidase gene such that the locus (i) lacks a nucleotide sequence encoding amino acids 1-614 of SEQ ID NO:15 and (ii) comprises a nucleic acid sequence set forth as SEQ ID NO:17, or a degenerate variant thereof, operably linked to endogenous DR6 transcriptional regulatory sequences. 
     
     
         51 . The method of  claim 50 , wherein the ES cell is heterozygous for the modified endogenous DR6 locus. 
     
     
         52 . The method of  claim 50 , wherein the ES cells are rat ES cells. 
     
     
         53 . The method of  claim 50 , wherein the ES cells are mouse ES cells. 
     
     
         54 . The method of  claim 53 , wherein the mouse ES cells are from a strain selected from the group consisting of a 129 strain, a C57BL/6 strain, and a mixed C57BL/6×129 strain.