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Targeted modification of rat genome

a technology of rat genome and target genome, which is applied in the direction of peptides, viruses/bacteriophages, veterinary instruments, etc., can solve the problems of limited use of rats in human disease modeling, bi-allelic modification of target genomic loci, etc., and achieve the effect of enhancing homologous recombination

Active Publication Date: 2017-07-20
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a method for modifying a genomic locus in a pluripotent cell using a large targeting vector (LTVEC). The LTVEC contains a 5′ homology arm and a 3′ homology arm that can be introduced into the cell. The method allows for the creation of biallelic modifications that can be transmitted through the germline. The pluripotent cell can be a non-human animal or a human induced pluripotent stem (iPS) cell. The method can be used to create new animal models or to develop new treatments for genetic diseases.

Problems solved by technology

While rats have been regarded as an important animal model system that can recapitulate the pathology of various human diseases, including, but not limited to, cardiovascular (e.g., hypertension), metabolic (e.g., obesity, diabetes), neurological (e.g., pain pathologies), and a variety of cancers, the use of rats in modeling human diseases has been limited as compared to mice, due in part to unavailability of germline-transmittable pluripotent rat cells, which can sustain their pluripotency following a series of genetic modifications in vitro, e.g., one or more serial electroporations, and due in part to lack of efficient targeting technologies that allow introduction or deletion of large genomic DNA sequences, or replacement of large endogenous genomic DNA sequences with exogenous nucleic acid sequences in pluripotent rat cells.
Some such methods result in bi-allelic modification of the target genomic locus.

Method used

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  • Targeted modification of rat genome
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  • Targeted modification of rat genome

Examples

Experimental program
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Effect test

example 1

ll Derivation and Characterization

[0603]1.1. Rat ES Cell Characterization

[0604]As shown in FIG. 1, rat ESCs grow as compact spherical colonies, which routinely detach and float in the dish (close-up, FIG. 7). Rat ESCs express pluripotency markers including Oct-4 (FIG. 2A) and Sox2 (FIG. 2B), and express high levels of alkaline phosphatase (FIG. 3, left panel). Karyotype for line DA.2B is 42X,Y (FIG. 3, right panel). Rat ESCs often become tetraploid; thus, lines were pre-screened by counting metaphase chromosome spreads; lines with mostly normal counts were then formally karyotyped.

[0605]ACI blastocysts were collected from super-ovulated females obtained commercially. DA blastocysts were cultured from frozen 8-cell embryos obtained commercially. Zona pellucidae were removed with Acid Tyrodes; and blastocysts were plated onto mitotically inactivated MEFs. Outgrowths were picked and expanded using standard methods. All blastocysts were plated, cultured and expanded using 2i media (Li e...

example 2

ion of Genomic Loci in Rats

[0764]2.1: Inactivation of Endogenous Genomic Loci Using an Endonuclease Agent

[0765]In order to introduce a mutant allele at an endogenous rat genomic locus, the rat ES cells described herein are electroporated with expression vectors (or mRNA) that express ZFNs 1 and 2 (or TALENs 1 and 2). These proteins bind their target sequences on opposite strands, separated by about 6 bp to about 40 bp. A double-stranded break is formed within the target locus, which the cell attempts to repair by Non-Homologous End-Joining (NHEJ). In many cases, NHEJ results in creation of a deletion, which often disrupts the function of the gene (most often by producing a frameshift mutation). In order to identify a positive clone comprising a mutant allele, the electroporated cells are plated at low density, because no drug selection is done. Colonies are picked and assayed at the target site to see if a mutation was produced (e.g., using a modification of allele (MOA) assay descr...

example 3

Modification of Rat Genomic Loci

[0779]3.1: Rat ESC Targeting: The Rat Rosa 26 Locus.

[0780]The rat Rosa26 locus lies between the Setd5 and Thumpd3 genes as in mouse, with the same spacing. The rat Rosa 26 locus (FIG. 12, Panel B) differs from the mouse Rosa 26 locus (FIG. 12, Panel A). The mouse Rosa26 transcripts consist of 2 or 3 exons. The rat locus contains a 2nd exon 1 (Ex1b) in addition to the homologous exon to mouse exon1 (Ex1a). No 3rd exon has been identified in rat. Targeting of a rat Rosa26 allele is depicted in FIG. 12 (bottom), where homology arms of 5 kb each were cloned by PCR using genomic DNA from DA rat ESC. The targeted allele contains a SA (splicing acceptor)-lacZ-hUb-neo cassette replacing a 117 bp deletion in the rat Rosa26 intron.

[0781]Targeting efficiency at the rat Rosa 26 locus was determined (Table 12). Linearized vector was electroporated into DA or ACI rat ESCs, and transfected colonies were cultured in 2i media+G418, using standard techniques. Individua...

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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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of U.S. application Ser. No. 14 / 254,715, filed Apr. 16, 2014, which claims the benefit of U.S. Provisional Application No. 61 / 812,319, filed Apr. 16, 2013, and U.S. Provisional Application No. 61 / 914,768, filed Dec. 11, 2013, all of which are hereby incorporated herein in their entirety by reference.REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB[0002]The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 491907SEQLIST.TXT, created on Jan. 19, 2017, and having a size of 15 kilobytes, and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.FIELD OF INVENTION[0003]Isolated non-human totipotent or pluripotent stem cells, in particular rat embryonic stem ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/85C12N15/90A01K67/027
CPCC12N15/8509A01K67/0278C12N15/907C12N2810/00A01K2227/105A01K2217/07C12N2015/8527A01K67/0276C07K14/7155C07K14/775A01K2267/0362A01K2267/0381C12N2800/30A61D19/04A01K2267/03
Inventor LEE, JEFFREY D.MUJICA, ALEXANDER O.AUERBACH, WOJTEKLAI, KA-MAN VENUSVALENZUELA, DAVID M.YANCOPOULOS, GEORGE D.
Owner REGENERON PHARM INC
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