Method for genetically modifying mouse embryonic stem cell by homologous recombination

• US 8,759,105 B2
• Filed: 06/01/2007
• Issued: 06/24/2014
• Est. Priority Date: 10/31/2000
• Status: Expired due to Fees

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Economides, Aris N., Murphy, Andrew J., Valenzuela, David M., Frendewey, David, Yancopoulos, George D.
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): Moloye, Titilayo
• Application Number:
  US11/809,473
• Publication Number:
  US 20090055943A1
• Time in Patent Office: 2,580 Days
• Field of Search: None
• US Class Current: 435/463
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2810/10 Vectors comprising a non-peptidic targeting moiety
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

Abstract

A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.

Claims

1. A method for genetically modifying a mouse embryonic stem (ES) cell, comprising:

(a) targeting a mouse ES cell by homologous recombination with a targeting vector that comprises a human cloned genomic fragment larger than 20 kb containing a DNA sequence of interest that is orthologous to an endogenous mouse allele;

(b) using a quantitative assay that detects modification of allele (MOA) in the mouse ES cell of (a) to identify a mouse ES cell in which the endogenous mouse allele has been genetically modified, the assay comprising;

(i) exposing the DNA of the mouse ES cell of (a) with a first probe and a second probe, wherein the first probe binds within the endogenous mouse allele but not within the DNA sequences of interest, and the second probe binds within a reference gene but not within the endogenous mouse allele and not within the DNA sequence of interest, and wherein both probes generate a detectable signal upon binding, and wherein the reference gene is of known copy number;

(ii) detecting the signal from the binding of the first probe and the signal from the binding of the second probe; and

,

(iii) comparing the signal from the binding of the second probe to the signal from the binding of the first probe, and determining from the comparison a copy number of the endogenous mouse allele;

wherein the modification comprises replacing the endogenous mouse allele with the orthologous DNA sequence of interest.

2. The method of claim 1, wherein the targeting vector is capable of accommodating DNA fragments greater than 100 kb.

3. The method of claim 1, wherein the signal from the binding of the first probe is used to determine a first threshold cycle (Ct) value for the endogenous mouse allele and the signal from the binding of the second probe is used to determine a second value for the reference gene, and wherein copy number of the endogenous mouse allele is determined by comparing the first Ct value and the second Ct value.

4. The method of claim 1, wherein the copy number of the endogenous mouse allele is one.

5. The method of claim 1, wherein the copy number of the endogenous mouse allele is zero.

6. The method of claim 1, wherein the Ct value of the second probe is the same in targeted ES cells as compared to the Ct value in non-targeted cells.

7. The method of claim 1, wherein the DNA sequence of interest comprises regulatory elements.

8. The method of claim 1, wherein the DNA sequence of interest comprises protein-coding sequences.

9. The method of claim 8, wherein the protein-coding sequence contains an alteration as compared to a parental protein-coding sequence.

10. The method of claim 9, wherein the alteration is selected from the group consisting of an addition, a substitution, and a fusion.