Targeted modification of rat genome

Abstract

Compositions and methods are provided for modifying a rat genomic locus of interest using a large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences as described herein. Compositions and methods for generating a genetically modified rat comprising one or more targeted genetic modifications in their germline are also provided. Compositions and methods are provided which comprise a genetically modified rat or rat cell comprising a targeted genetic modification in the rat interleukin-2 receptor gamma locus, the rat ApoE locus, the rat Rag2 locus, the rat Rag1 locus and/or the rat Rag2/Rag1 locus. The various methods and compositions provided herein allows for these modified loci to be transmitted through the germline.

We claim: 
     
       1. A method for targeted modification of a genomic locus of interest in a population of pluripotent rat cells to produce a genetically modified rat whose genome comprises a targeted genetic modification that is transmitted through the germline, comprising:
 (a) providing a population of pluripotent rat cells obtained by culturing isolated rat embryonic stem cells on a feeder cell layer that is not modified to express leukemia inhibitory factor (LIF) with a medium comprising N2 supplement, B27 supplement, about 50 U/mL to about 150 U/mL LIF, and a combination of inhibitors consisting of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021, wherein the pluripotent rat cells:
 lack expression of c-Myc; 
 form spherical, free-floating colonies in culture; 
 are diploid; and 
 are germline competent; 
 
 (b) obtaining a pluripotent rat cell clone comprising the targeted genetic modification at the genomic locus of interest, wherein the obtaining consists of:
 (i) introducing into the pluripotent rat cells a large targeting vector (LTVEC) comprising an insert nucleic acid flanked by a 5′ homology arm homologous to a first nucleic acid sequence at the genomic locus of interest and a 3′ homology arm homologous to a second nucleic acid sequence at the genomic locus of interest to produce the targeted genetic modification via homologous recombination, wherein the sum total of the 5′ and the 3′ homology arms is at least 10 kb; and 
 (ii) performing a loss of allele (LOA) assay or a modification of allele (MOA) assay to identify in a single cloning step a pluripotent rat cell clone comprising the targeted genetic modification at the genomic locus of interest and capable of transmitting the targeted genetic modification through the germline; 
 
 (c) introducing the pluripotent rat cell clone into a rat host embryo; 
 (d) gestating the rat host embryo comprising the pluripotent rat cell clone in a surrogate mother, wherein the surrogate mother produces an F0 progeny genetically modified rat comprising the targeted genetic modification; and 
 (e) breeding the F0 progeny genetically modified rat with another rat to produce an F1 progeny genetically modified rat comprising the targeted genetic modification, wherein the targeted genetic modification is transmitted through the germline. 
 
     
     
       2. The method of  claim 1 , wherein the pluripotent rat cells are rat embryonic stem (ES) cells. 
     
     
       3. The method of  claim 1 , wherein the pluripotent rat cells are derived from a DA strain or an ACI strain. 
     
     
       4. The method of  claim 1 , wherein the pluripotent rat cells are characterized by expression of at least one pluripotency marker selected from the group consisting of Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, and Utf1. 
     
     
       5. The method of  claim 1 , wherein the pluripotent rat cells are characterized by one or more of the following characteristics:
 (I) lack of expression of one or more of the pluripotency markers Ecat 1 and Rexo1; 
 (II) lack of expression of one or more of the mesodermal markers Brachyury and Bmpr2; 
 (III) lack of expression of one or more of the endodermal markers Gata6, Sox17, and Sox7; and 
 (IV) lack of expression of one or more of the neural markers Nestin and Pax6. 
 
     
     
       6. The method of  claim 5 , wherein the pluripotent rat cells lack expression of Ecat1. 
     
     
       7. The method of  claim 1 , wherein the sum total of the 5′ and the 3′ homology arms of the LTVEC is from 10 kb to about 150 kb, or
 wherein the 5′ homology arm ranges from about 5 kb to about 100 kb or the 3′ homology arm ranges from about 5 kb to about 100 kb. 
 
     
     
       8. The method of  claim 1 , wherein the LTVEC is from about 20 kb to about 400 kb. 
     
     
       9. The method of  claim 1 , wherein the insert nucleic acid is from about 5 kb to about 400 kb. 
     
     
       10. The method of  claim 1 , wherein the targeted genetic modification is biallelic. 
     
     
       11. The method of  claim 1 , wherein the targeted genetic modification comprises deletion of an endogenous rat nucleic acid sequence, wherein the deletion ranges from about 5 kb to about 3 Mb. 
     
     
       12. The method of  claim 1 , wherein the targeted genetic modification comprises insertion of an exogenous nucleic acid sequence ranging from about 5 kb to about 400 kb. 
     
     
       13. The method of  claim 1 , wherein the targeted genetic modification comprises:
 (I) replacement of an endogenous rat nucleic acid sequence with a homologous or an orthologous nucleic acid sequence; 
 (II) deletion of an endogenous rat nucleic acid sequence; 
 (III) insertion of an exogenous nucleic acid sequence comprising a nucleic acid sequence that is homologous or orthologous to an endogenous rat nucleic acid sequence; 
 (IV) insertion of a chimeric nucleic acid sequence comprising a human nucleic acid sequence and a rat nucleic acid sequence; 
 (V) insertion of a conditional allele flanked by site-specific recombinase target sequences; or 
 (VI) insertion of a reporter gene operably linked to a promoter active in a rat cell. 
 
     
     
       14. The method of  claim 1 , wherein the targeted genetic modification comprises:
 (I) an insertion of the insert nucleic acid, wherein the insert nucleic acid comprises a human nucleic acid sequence; 
 (II) a replacement of a rat nucleic acid sequence at the genomic locus of interest with a human nucleic acid sequence, wherein the replaced rat nucleic acid sequence is homologous or orthologous to the human nucleic acid sequence; 
 (III) a chimeric nucleic acid sequence comprising a human nucleic acid sequence and a rat nucleic acid sequence; or 
 (IV) a combination thereof. 
 
     
     
       15. The method of  claim 1 , wherein introducing step (b)(i) further comprises introducing a nucleic acid encoding a nuclease agent that promotes homologous recombination between the LTVEC and the genomic locus of interest in the pluripotent rat cells. 
     
     
       16. The method of  claim 15 , wherein the nuclease agent comprises:
 (I) a chimeric protein comprising a zinc finger-based DNA binding domain fused to a FokI endonuclease; or 
 (II) a chimeric protein comprising a Transcription Activator-Like Effector Nuclease (TALEN). 
 
     
     
       17. The method of  claim 15 , wherein the nuclease agent comprises a CRISPR/Cas system. 
     
     
       18. The method of  claim 17 , wherein the CRISPR/Cas system comprises a Cas9 nuclease and a guide RNA comprising a fused crRNA-tracrRNA. 
     
     
       19. The method of  claim 18 , wherein the guide RNA comprises SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8. 
     
     
       20. The method of  claim 1 , wherein the genomic locus of interest comprises:
 (i) an Interleukin-2 receptor gamma locus, an ApoE locus, a Rag1 locus, a Rag2 locus, or a Rag2/Rag1 locus; 
 (ii) an immunoglobulin locus; or 
 (iii) a T cell receptor locus. 
 
     
     
       21. The method of  claim 1 , wherein the concentration of LIF in the medium is between about 75 U/mL to about 125 U/mL. 
     
     
       22. The method of  claim 21 , wherein the concentration of LIF in the medium is between about 90 U/mL to about 110 U/mL. 
     
     
       23. The method of  claim 22 , wherein the concentration of LIF in the medium is about 100 U/mL. 
     
     
       24. The method of  claim 1 , wherein the concentration of the MEK inhibitor is 0.8 μM to about 1.2 μM, and the concentration of the GSK3 inhibitor is about 2.5 μM to about 3.5 μM. 
     
     
       25. The method of  claim 24 , wherein the concentration of the MEK inhibitor is about 1 μM, and the concentration of the GSK3 inhibitor is about 3 μM. 
     
     
       26. The method of  claim 23 , wherein the concentration of the MEK inhibitor is about 1 μM and the concentration of the GSK3 inhibitor is 3 μM. 
     
     
       27. The method of  claim 1 , wherein the feeder cell layer comprises mitotically inactivated mouse embryonic fibroblasts, and the medium is a 2i medium comprising DMEM/F12 basal medium at a concentration of 1x; neurobasal medium at a concentration of 1x; penicillin/streptomycin at a concentration of 1%; L-Glutamine at a concentration of 4 mM; 2-mercaptoethanol at a concentration of 0.1 mM; N2 supplement at a concentration of 1x; B27 supplement at a concentration 1x; LIF at a concentration of 100U/mL; PD0325901 at a concentration of 1 μM, and CHIR99021 at a concentration of 3 μM. 
     
     
       28. The method of  claim 1 , wherein step (b)(i) produces a population of modified pluripotent rat cells, wherein the modified pluripotent rat cells retain the ability to form sphere-like colonies when cultured on the feeder cell layer in vitro such that at least 50% of the colonies that form when the modified pluripotent rat cells are plated on the feeder cell layer in vitro have a sphere-like morphology following modification of the genomic locus of interest. 
     
     
       29. The method of  claim 1 , wherein step (b)(i) produces a population of modified pluripotent rat cells, wherein at least 50% of the modified pluripotent rat cells remain diploid following modification of the genomic locus of interest. 
     
     
       30. The method of  claim 20 , wherein the immunoglobulin locus is an immunoglobulin heavy chain locus or an immunoglobulin light chain locus. 
     
     
       31. The method of  claim 30 , wherein the immunoglobulin light chain locus is a λ or κ immunoglobulin light chain locus. 
     
     
       32. The method of  claim 20 , wherein the genomic locus of interest comprises a T cell receptor alpha locus. 
     
     
       33. The method of  claim 1 , wherein the insert nucleic acid:
 (i) comprises a polynucleotide of interest comprising a genomic nucleic acid sequence that encodes a human immunoglobulin heavy chain variable region amino acid sequence; 
 (ii) comprises a polynucleotide of interest comprising a genomic nucleic acid sequence that encodes a human immunoglobulin light chain variable region amino acid sequence; 
 (iii) comprises a polynucleotide of interest comprising a polynucleotide encoding at least a region of a T cell receptor; or 
 (iv) comprises a polynucleotide of interest comprising at least one disease allele. 
 
     
     
       34. The method of  claim 33 , wherein the insert nucleic acid comprises a polynucleotide of interest comprising a polynucleotide encoding at least a region of a T cell receptor alpha. 
     
     
       35. The method of  claim 1 , wherein the insert nucleic acid:
 (i) is from a human; 
 (ii) comprises a knock-in allele of at least one exon of an endogenous gene; 
 (iii) comprises a regulatory element; 
 (iv) comprises a conditional allele; 
 (v) comprises a nucleic acid flanked by site-specific recombination target sequences; 
 (vi) comprises a polynucleotide encoding a selection marker; or 
 (vii) comprises a reporter gene operably linked to a promoter. 
 
     
     
       36. The method of  claim 35 , wherein the insert nucleic acid comprises a polynucleotide encoding a selection marker and/or a reporter gene flanked by site-specific recombination target sequences or comprises a self-deleting selection cassette. 
     
     
       37. The method of  claim 1 , wherein the introducing step is mediated by electroporation. 
     
     
       38. The method of  claim 1 , wherein steps (a) and (b) are sequentially repeated to allow for the targeted integration of at least two insert nucleic acids into the genomic locus of interest. 
     
     
       39. The method of  claim 14 , wherein the size of the insertion is from about 5 kb to about 500 kb, or wherein the size of the replacement is from about 5 kb to about 500 kb.