# US 11,078,494 B2 · Vector for targeting the human Rosa26 gene

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- Number: US 11,078,494 B2
- Document ID: US11078494B2
- Status: Granted
- Kind: B2
- Application number: 16/664,291
- Filed: 2019-10-25
- Published: 2020-03-05
- Granted: 2021-08-03
- Earliest claimed: 2007-01-19
- Assignees: Icahn School of Medicine at Mount Sinai
- Applicants: Icahn School of Medicine at Mount Sinai
- Inventors: Gordon Keller; Stefan Irion; Herve Luche; Paul Gadue; Hans J. Fehling
- Web page: https://patentgrep.com/patent/US11078494B2

## CPC

- C12N 15/85: for animal cells
- C07K 14/4702: {Regulators; Modulating activity}
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 15/907: {in mammalian cells}
- A01K 2207/15: Humanized animals
- A01K 2217/00: Genetically modified animals
- C12N 2510/00: Genetically modified cells
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- C12N 2830/85: mammalian
- C12N 2840/44: being a specific part of the splice mechanism, e.g. donor, acceptor
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## 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.

## Claims

### 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

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.

### Claim 4 (depends on 2)

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

### Claim 5 (depends on 2)

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

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 1)

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.

### Claim 11 (depends on 1)

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.