Genetic modification of rats

Patent metadata

Field	Value
Publication number	US-10894965-B2
Application number	US-201916401539-A
Country	US
Kind code	B2
Filing date	May 2, 2019
Priority date	Feb 20, 2013
Publication date	Jan 19, 2021
Grant date	Jan 19, 2021

Abstract

Compositions and methods are provided for making rat pluripotent and totipotent cells, including rat embryonic stem (ES) cells. Compositions and methods for improving efficiency or frequency of germline transmission of genetic modifications in rats are provided. Such methods and compositions comprise an in vitro culture comprising a feeder cell layer and a population of rat ES cells or a rat ES cell line, wherein the in vitro culture conditions maintain pluripotency of the ES cell and comprises a media having mouse leukemia inhibitory factor (LIF) or an active variant or fragment thereof. Various methods of establishing such rat ES cell lines are further provided. Methods of selecting genetically modified rat ES cells are also provided, along with various methods to generate a transgenic rat from the genetically modified rat ES cells provided herein. Various kits and articles of manufacture are further provided.

First claim

That which is claimed:

1. A method of making a genetically modified rat embryonic stem (ES) cell clone comprising a targeted genetic modification and capable of generating a genetically modified rat comprising the targeted genetic modification and transmitting the targeted genetic modification through the germline, comprising:
(a) culturing a population of rat ES cells under conditions comprising a layer of feeder cells that are not modified to express leukemia inhibitory factor (LIF) and a medium comprising about 50 U/mL to about 150 U/mL LIF, N2 supplement, B27 supplement, and a combination of inhibitors consisting of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021;
(b) introducing into the rat ES cells a polynucleotide or polypeptide encoding a nuclease agent that generates a single or double strand break at a targeted genomic locus and/or a targeting vector comprising upstream and downstream homology arms flanking an insert polynucleotide, wherein the homology arms correspond to genomic regions within the targeted genomic locus; and
(c) obtaining the genetically modified rat ES cell clone comprising the targeted genetic modification, wherein the obtaining consists of identifying in a single cloning step the genetically modified rat ES cell clone comprising the targeted genetic modification and capable of generating the genetically modified rat comprising the targeted genetic modification and transmitting the targeted genetic modification through the germline.

2. The method of claim 1 , wherein the LIF is a mouse LIF or has at least 91% sequence identity to SEQ ID NO: 1.

3. The method of claim 1 , wherein the concentration of LIF in the medium is between about 75 U/mL to about 125 U/mL.

4. The method of claim 3 , wherein the concentration of LIF in the medium is between about 90 U/mL to about 110 U/mL.

5. The method of claim 4 , wherein the concentration of LIF in the medium is about 100 U/mL.

6. The method of claim 5 , wherein the concentration of the MEK inhibitor PD0325901 is about 1 μM, and the concentration of the GSK3 inhibitor CHIR99021 is about 3 μM.

7. The method of claim 1 , wherein the concentration of the MEK inhibitor PD0325901 is 0.8 μM to about 1.2 μM, and the concentration of the GSK3 inhibitor CHIR99021 is about 2.5 μM to about 3 μM or 3 μM to about 3.5 μM.

8. The method of claim 7 , wherein the concentration of the MEK inhibitor PD0325901 is about 1 μM, and the concentration of the GSK3 inhibitor CHIR99021 is about 3 μM.

9. The method of claim 1 , wherein the layer of feeder cells comprises a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs).

10. The method of claim 1 , wherein the medium further comprises DMEM/F12 basal medium and Neurobasal medium.

11. The method of claim 10 , wherein the layer of feeder cells comprises mitotically inactivated mouse embryonic fibroblasts, and the medium is a 2i medium comprising DMEM/F12 basal medium, Neurobasal medium, N2 supplement, B27 supplement, LIF, and a combination of inhibitors consisting of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021.

12. The method of claim 11 , wherein the concentration of the LIF is 100 U/mL, the concentration of the MEK inhibitor PD0325901 is about 1 μM, and the concentration of the GSK3 inhibitor CHIR99021 is about 3 μM.

13. The method of claim 1 , wherein:
(I) the rat ES cells have a normal karyotype; and/or
(II) the rat ES cells form spherical, free-floating colonies in culture.

14. The method of claim 1 , wherein the rat ES cells are derived from an ACI rat or a Dark Agouti (DA) rat.

15. The method of claim 1 , wherein the rat ES cells are male (XY) rat ES cells.

16. The method of claim 1 , wherein the rat ES cells are female (XX) rat ES cells.

17. The method of claim 1 , wherein the rat ES cells have one or more of the following characteristics:
(a) the rat ES cells express at least one pluripotency marker selected from Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, and Utf1;
(b) the rat ES cells do not express one or more pluripotency markers selected from c-Myc, Ecat1, and Rexo1;
(c) the rat ES cells do not express one or more mesodermal markers selected from Brachyury and Bmpr2;
(d) the rat ES cells do not express one or more endodermal markers selected from Gata6, Sox17, and Sox7;
(e) the rat ES cells do not express one or more neural markers selected from Nestin and Pax6;
(f) the rat ES cells express one or more pluripotency markers selected from Oct-4, Sox2, and alkaline phosphatase; and
(g) the rat ES cells express one or more rat ES-cell-specific genes selected from Adherens Junctions Associated Protein 1 (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1 (Igfbpl1), Interleukin 36 beta Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1 (Lefty1), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid transcription factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1), and miR-632.

18. The method of claim 1 , wherein the step (b) comprises one or more rounds of electroporation.

19. The method of claim 1 , wherein the targeted genetic modification comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof.

20. The method of claim 19 , wherein the targeted genetic modification comprises an insertion of a heterologous polynucleotide into the genome of the rat ES cells.

21. The method of claim 1 , wherein the method comprises modifying the population of rat ES cells to comprise two or more targeted modifications, wherein the modified rat ES cells can transmit the two or more targeted genetic modifications through the germline.

22. The method of claim 1 , wherein the targeted genetic modification is generated via a homologous recombination event.

23. The method of claim 22 , wherein the targeted genetic modification is generated by employing the targeting vector.

24. The method of claim 1 , wherein the targeted genetic modification is generated using the nuclease agent.

25. The method of claim 24 , wherein the nuclease agent is a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, or a CRISPR/Cas system.

26. The method of claim 1 , wherein:
(i) step (b) comprises introducing into the population of rat ES cells a heterologous polynucleotide comprising a selection marker, wherein the selection marker is operably linked to a promoter active in the rat ES cells, and wherein the selection marker is present in the targeting vector; and
(ii) step (c) comprises culturing in vitro the rat ES cells produced by step (b) in alternating first and second culture media, wherein the first culture medium comprises an eff

Assignees

• Regeneron Pharma

Inventors

• Lee Jeffrey D
• Auerbach Wojtek
• Heslin David
• Frendewey David
• Lai Ka-Man Venus
• Valenzuela David M

Classifications

• C12N5/0606 Primary

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title

• C12N15/8775

  Murine embryos · CPC title

• C12N15/8509 Primary

  for producing genetically modified animals, e.g. transgenic · CPC title

• A01K67/0271

  Chimeric vertebrates, e.g. comprising exogenous cells · CPC title

• A01K67/02 Primary

  Breeding vertebrates · CPC title