Vector for targeting the human Rosa26 gene

Patent metadata

Field	Value
Publication number	US-11078494-B2
Application number	US-201916664291-A
Country	US
Kind code	B2
Filing date	Oct 25, 2019
Priority date	Jan 19, 2007
Publication date	Aug 3, 2021
Grant date	Aug 3, 2021

Abstract

The invention provides a method for generating a transgenic eukaryotic cell population having a modified human Rosa26 locus, which method includes introducing a functional DNA sequence into the human Rosa26 locus of starting eukaryotic cells. Also provided are targeting vectors useful in the method, as well as a cell population and a transgenic non-human animal comprising a modified human Rosa26 locus. Finally, the invention provides an isolated DNA sequence corresponding to the human Rosa26 locus.

First claim

We claim:

1. A targeting vector comprising an expression cassette comprising a nucleic acid encoding a protein, wherein said nucleic acid is heterologous to a human Rosa 26 gene, said expression cassette flanked by DNA sequences homologous to the human Rosa26 gene.

2. The targeting vector of claim 1 wherein the expression cassette further comprises a promoter operably linked to the nucleic acid encoding the protein.

3. The targeting vector of claim 2 wherein the promoter is selected from the group consisting of a constitutive ubiquitous promoter, a constitutive tissue specific promoter, an inducible ubiquitous promoter and an inducible tissue specific promoter.

4. The targeting vector of claim 2 wherein the promoter is heterologous to the human Rosa26 gene.

5. The targeting vector of claim 2 wherein the promoter is the endogenous human Rosa26 promoter.

6. The targeting vector of claim 1 wherein the sequences homologous to the human Rosa26 gene are derived from the 5′ and 3′ flanking arms of the human Rosa26 gene.

7. The targeting vector of claim 1 further comprising tags for protein detection, enhancers, selection markers, and combinations thereof.

8. The targeting vector of claim 1 wherein the nucleic acid encodes a recombinase or a reporter.

9. The targeting vector of claim 1 wherein the expression cassette further comprises a marker gene, one or more recombinase recognition sites, a poly A signal, an intron, or combinations thereof.

10. The targeting vector of claim 1 wherein the expression cassette further comprises a viral splice acceptor, a loxP-flanked promoterless neomycin resistance gene, an inverted RFP variant, loxP2272 sites, or combinations thereof.

11. The targeting vector of claim 1 wherein the expression cassette comprises the following elements in sequential order: (a) a viral splice acceptor, (b) a loxP site, (c) a promoterless neomycin resistance gene, (d) a loxP2272 site, (e) an inverted nucleic acid sequence encoding the protein, (f) a loxP site, and (g) a loxP2272 site.

Assignees

• Icahn School Med Mount Sinai

Inventors

• Keller Gordon
• Irion Stefan
• Luche Herve
• Gadue Paul
• Fehling Hans J

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

• C12N15/907

  in mammalian cells · CPC title

• C12N2830/85

  mammalian · CPC title

• C12N15/85 Primary

  for animal cells · CPC title

• C07K14/4702

  Regulators; Modulating activity · CPC title