Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Targeted modification of rat genome

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

Inactive Publication Date: 2014-10-16
REGENERON PHARM INC
View PDF6 Cites 319 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a method for modifying a genomic locus in a pluripotent cell using a targeted genetic modification approach. The method involves introducing a large targeting vector (LTVEC) into the cell, which contains an insert nucleic acid flanked with homology arms. The LTVEC can be introduced into the cell using a pluripotent cell derived from a non-human animal, such as a rodent or human. The method can be used to create biallelic modifications or to transmit the genetic modification through the germline. The patent also provides information on the size of the LTVEC and the expression of pluripotency markers in different cell types. The technical effect of this method is the ability to modify the genome of a pluripotent cell with precision, which can be useful in research and development of new therapies.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Targeted modification of rat genome
  • Targeted modification of rat genome
  • Targeted modification of rat genome

Examples

Experimental program
Comparison scheme
Effect test

example 1

Rat ES Cell Derivation and Characterization

1.1. Rat ES Cell Characterization

[0602]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.

[0603]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 (L...

example 2

Inactivation of Genomic Loci in Rats

2.1: Inactivation of Endogenous Genomic Loci Using an Endonuclease Agent

[0756]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 de...

example 3

Targeted Modification of Rat Genomic Loci

3.1: Rat ESC Targeting: the Rat Rosa 26 Locus

[0767]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.

[0768]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. Individ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 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, both 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 444253SEQLIST.TXT, created on Apr. 16, 2014, 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 cells, that are capable of sustaining pluripotency following one or more serial genetic modific...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C12N15/85
CPCC12N15/8509A01K67/0278A61D19/04A01K67/0276C07K14/7155C07K14/775A01K2227/105A01K2267/0362A01K2267/0381C12N2800/30A01K2217/07C12N15/907C12N2015/8527C12N2810/00A01K2267/03
Inventor LEE, JEFFREY D.MUJICA, ALEXANDER O.AUERBACH, WOJTEKLAI, KA-MAN VENUSVALENZUELA, DAVID M.YANCOPOULOS, GEORGE D.
Owner REGENERON PHARM INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com