Mice expressing human voltage-gated sodium channels
Genetically modified non-human animals and methods and compositions for making and using them are provided, wherein the genetic modification comprises a humanization of an extracellular loop of an endogenous Na V channel gene, in particular a humanization of the one or more extracellular pore loops of a Na V 1.7 channel protein. Genetically modified non-human animals are also provided, wherein the genetic modification comprises replacement of an endogenous Na V channel gene, in particular a replacement of the endogenous Na V 1.7 gene with a human Na V 1.7 gene, and wherein the genetically modified non-human animals are capable of generating action potentials and communicating through the excitable cells of the genetically modified non-human animals via the expressed human or humanized Na V 1.7 protein the surface of the excitable cells. Genetically modified mice are described, including mice that express the human or humanized Na V 1.7 gene from the endogenous Na V 1.7 locus, and wherein the mice comprise functional β-subunits.
1. A genetically modified mouse whose genome comprises:
a Na V 1.7 α-subunit gene that is chimeric in that it includes both human and mouse sequences, wherein the human sequence comprises a nucleotide sequence encoding an extracellular pore loop of a domain of a human Na V 1.7 α-subunit selected from a DI/S5-S6 and DIII/S5-S6 loop, and the mouse sequence comprises a nucleotide sequence encoding at least transmembrane segments of a mouse Na V 1.7 α-subunit, wherein the Na V 1.7 α-subunit gene is operably linked to a Na V 1.7 promoter.
2. The genetically modified mouse of claim 1 , wherein the Na V 1.7 promoter is a mouse Na V 1.7 promoter.
3. The genetically modified mouse of claim 1 , wherein the Na V 1.7 promoter is a human Na V 1.7 promoter.
4. The genetically modified mouse of claim 1 , wherein the extracellular pore loop is DI/S5-S6.
5. The genetically modified mouse of claim 1 , wherein the extracellular pore loop is DIII/S5-S6.
6. A genetically modified mouse whose genome comprises:
a Na V 1.7 α-subunit gene that is chimeric in that it includes both human and mouse sequences, wherein the human sequence consists of exons 2-28 of a human Na V 1.7 α-subunit gene encoding a human Na V 1.7 α-subunit, the mouse sequence comprises exon 1 of a mouse Na V 1.7 α-subunit operably linked to a Na V 1.7 promoter, and the mouse sequence comprising exon 1 is upstream and linked contiguously to the human sequence consisting of exons 2-28 of a human Na V 1.7 α-subunit gene encoding a human Na V 1.7 α-subunit.
7. The genetically modified mouse of claim 6 , wherein the Na V 1.7 promoter is a human Na V 1.7 promoter.
8. The genetically modified mouse of claim 6 , wherein the Na V 1.7 promoter is a mouse Na V 1.7 promoter.
9. The genetically modified mouse of claim 6 , wherein the mouse does not detectably express an endogenous Na V 1.7 α-subunit.
10. A cell or tissue derived from the mouse according to any one of claims 4 , 5 and 6 .
11. A genetically modified mouse according to any one of claims 4 , 5 and 6 , wherein the nucleotide sequence encoding a chimeric or full-length human Na V 1.7 α-subunit comprises a substitution associated with a human pain disorder.
12. The genetically modified mouse of claim 11 , wherein the human pain disorder is select from erythromelalgia (IEM), paroxysmal extreme pain disorder (PEPD) and congenital indifference to pain (CIP).
13. A method of making a genetically modified mouse that expresses a Na V 1.7 α-subunit protein from an altered endogenous Na V 1.7 α-subunit locus, the method comprising:
(a) targeting an endogenous Na V 1.7 α-subunit gene in a mouse ES cell with a nucleic acid construct comprising a chimeric Na V 1.7 α-subunit gene that is operably linked to a Na V 1.7 promoter and includes both human and mouse sequences,
wherein the chimeric Na V 1.7 α-subunit gene comprises a human sequence comprising a nucleotide sequence encoding an extracellular pore loop of a domain of a human Na V 1.7 α-subunit selected from a DI/S5-S6 and DIII/S5-S6 loop and a mouse sequence comprising a nucleotide sequence encoding at least transmembrane segments of a mouse Na V 1.7 α-subunit, or
wherein the chimeric Na V 1.7 α-subunit gene comprises a mouse sequence comprising exon 1 of a mouse Na V 1.7 α-subunit upstream and linked contiguously to a human sequence consisting of exons 2-28 of a human Na V 1.7 α-subunit gene;
(b) obtaining a modified mouse ES cell comprising an endogenous Na V 1.7 α-subunit gene that is chimeric and includes the human and mouse sequences of (a); and,
(c) creating a genetically modified mouse using the modified ES cell of (b).
14. The method of claim 13 , wherein the human sequence is exons 2 to 28 of a human Na V 1.7 α-subunit gene.
15. The method of claim 13 , wherein the human sequence is exons 7 to 9 of a human Na V 1.7 α-subunit gene.
16. The method of claim 13 , wherein the human sequence is exons 23 to 25 of a human Na V 1.7 α-subunit gene.
17. A method for generating an immortalized dorsal root ganglion (DRG) neuronal cell line, comprising:
(a) isolating a DRG cell from the mouse according to any one of claims 4 , 5 and 6 ;
(b) introducing into the DRG cell of (a) a vector that encodes an oncogene and a selectable marker;
(c) selecting a cell containing the vector of (b);
(d) maintaining the cell of (c) in culture thereby generating an immortalized DRG neuronal cell line.
18. The method of claim 17 , wherein the vector is a retroviral vector.
19. The method of claim 17 , wherein the oncogene is selected from c-Sis, a receptor tyrosine kinase, a cytoplasmic tyrosine kinase, Raf, a regulatory GTPase, and Myc.
20. The cell or tissue of claim 10 , wherein the cell or tissue detectably expresses a human Na V 1.7 α-subunit.
21. The cell or tissue of claim 10 , wherein the cell or tissue does not detectably express a mouse Na V 1.7 α-subunit.
22. The cell or tissue of claim 21 , wherein the cell is a neuronal cell and the human Na V 1.7 α-subunit is detectably expressed on the cell surface.
23. The cell or tissue of claim 22 , wherein the neuronal cell is a DRG neuron.