Genetic modification of rats

Patent metadata

Field	Value
Publication number	US-10329582-B2
Application number	US-201615242025-A
Country	US
Kind code	B2
Filing date	Aug 19, 2016
Priority date	Feb 20, 2013
Publication date	Jun 25, 2019
Grant date	Jun 25, 2019

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 whose genome comprises a targeted genetic modification that is transmitted through the germline, comprising:
(a) providing a rat ES cell line comprising a population of rat ES cells obtained by culturing isolated rat ES 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 population of rat ES cells:
lacks expression of c-Myc;
forms spherical, free-floating colonies in culture;
is diploid; and
is germline competent;
(b) obtaining a rat ES cell clone comprising the targeted genetic modification, wherein the obtaining consists of:
(i) modifying the population of rat ES cells to comprise the targeted genetic modification; and
(ii) culturing the rat ES cells from step (b)(i) under the conditions set forth in step (a) and identifying in a single cloning step a rat ES cell clone comprising the targeted genetic modification and capable of transmitting the targeted genetic modification through the germline;

(c) introducing the rat ES cell clone into a rat host embryo, wherein the rat ES cell clone is from a different rat strain than the rat host embryo;
(d) gestating the rat host embryo comprising the rat ES 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 providing step (a) comprises:
(i) culturing in vitro a first feeder cell layer that is not modified to express leukemia inhibitory factor (LIF) and a morula or a blastocyst stage rat embryo,
wherein the zona pellucida of the morula or blastocyst-stage rat embryo has been removed, and
wherein the culture conditions maintain pluripotency of a rat ES cell and comprise 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 said culturing produces an outgrowth of undifferentiated rat ES cells; and
(ii) transferring the outgrowth from (i) to an in vitro culture well comprising a second feeder cell layer that is not modified to express LIF; and
(iii) culturing the outgrowth from (ii) under conditions comprising the 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, thereby establishing a pluripotent rat ES cell line.

3. The method of claim 2 , wherein the morula or blastocyst stage rat embryo is from a superovulated rat.

4. The method of claim 1 , wherein the rat ES cell line is derived from an ACI rat or a Dark Agouti (DA) rat.

5. The method of claim 1 , wherein the rat ES cell line is a male (XY) rat ES cell line.

6. The method of claim 1 , wherein the rat ES cell line is a female (XX) rat ES cell line.

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

8. 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.

9. The method of claim 8 , wherein the targeted genetic modification comprises an insertion of a heterologous polynucleotide into the genome of the rat ES cells.

10. The method of claim 1 , wherein the modifying step (b)(i) 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.

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

12. The method of claim 11 , wherein the targeted genetic modification is generated by employing a targeting vector comprising upstream and downstream homology arms flanking an insert polynucleotide, wherein the homology arms correspond to genomic regions within a targeted genomic locus.

13. The method of claim 12 , wherein the modifying step (b)(i) comprises:
(i) 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) culturing in vitro the rat ES cells produced by step (a) in alternating first and second culture media, wherein the first culture medium comprises an effective amount of a selection agent for a first time period and the second culture medium does not comprise the selection agent, wherein the in vitro culture conditions are sufficient to maintain pluripotency, thereby selecting modified rat ES cells having the heterologous polynucleotide stably integrated into the genome.

14. The method of claim 13 , wherein the first and second culture media are alternated every 24 hours.

15. The method of claim 13 , wherein the selection marker imparts resistance to an antibiotic.

16. The method of claim 13 , wherein the selection marker comprises one or more of neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r ), puromycin-N-acetyltransferase (puro r ), blasticidin S deaminase (bsr r ), xanthine/guanine phosphoribosyl transferase (gpt), and herpes simplex virus thymidine kinase (HSV-tk).

17. The method of claim 13 , wherein the selection marker has one or more of the following characteristics:
(a) the selection marker comprises a non-attenuated selection marker gene; and
(b) the selection marker has an increased activity compared to a wild type selection marker.

18. The method of claim 13 , wherein the modified rat ES cells comprise multiple copies of the selection marker stably incorporated into the genome.

19. The method of claim 1 , wherein the targeted genetic modification is generated using a nuclease agent that generates a single or double strand break at a targeted genomic locus.

20. The method of claim 19 , wherein the nuclease agent is a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, or a CRISPR/Cas system.

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

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

23. The method of claim 22 , wherein the concentration of LIF in the medium is between about 90 U/mL to about 110 U/mL.

24. The method of claim 23 , wherein the concentration of LIF in the medium is about 100 U/mL.

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 1 , wherein the concentration of the MEK inhibitor is 0.8 μM to about 1.2 μM, and the concentration of the GSK3 inhibitor

Assignees

• Regeneron Pharma

Inventors

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

Classifications

• A01K67/0275

  Genetically modified vertebrates, e.g. transgenic · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title

• A01K67/0271

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

• A01K2217/15

  Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding · CPC title

• C12N15/8509 Primary

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