# US 12,180,510 B2 · Methods and compositions for rapid generation of single and multiplexed reporters in cells

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- Number: US 12,180,510 B2
- Document ID: US12180510B2
- Status: Granted
- Kind: B2
- Application number: 18/080,093
- Filed: 2022-12-13
- Published: 2023-07-06
- Granted: 2024-12-31
- Earliest claimed: 2014-12-15
- Assignees: Rxcell Inc.
- Applicants: Rxcell Inc.
- Inventors: Xianmin Zeng; Mahendra S. Rao
- Web page: https://patentgrep.com/patent/US12180510B2

## CPC

- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 15/907: {in mammalian cells}
- C12N 2510/00: Genetically modified cells
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- C12N 2830/40: being an insulator
- C12N 2840/20: translation of more than one cistron
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods and compositions for rapid development of reporter lines utilizing safe harbor sites in iPSCS, as well as other progenitor cells, pluripotent and multipotent stem cells and differentiated cells, and multiple Lox sites are provided.

## Claims

### Claim 1

A method of generating a new reporter line, said method comprising utilizing a Cre-recombinase induced cassette exchange strategy in a master cell line comprising a first reporter cassette at a safe harbor site on chromosome 13 and a second reporter cassette at a safe harbor site on chromosome 19, wherein said first and second reporter cassettes each comprise a reporter gene driven by a constitutively active promoter and multiple Lox sites to exchange the first or second reporter cassette in the master cell line with a new reporter cassette, thereby generating a new reporter line with the same isogenic background, wherein the master cell line is integrated with the new reporter cassette at a safe harbor site in the cell and wherein the new reporter cassette comprises a reporter gene driven by a constitutively active promoter and multiple Lox sites.

### Claim 2 (depends on 1)

The method of claim 1 wherein the promoter driving the reporter gene is inserted between two Lox sites.

### Claim 3 (depends on 1)

The method of claim 1 wherein the promoter is inserted between lox2272 and lox511.

### Claim 4 (depends on 1)

The method of claim 1 wherein the cell is a progenitor cell.

### Claim 5 (depends on 4)

The method of claim 4 wherein the cell is a pluripotent or multipotent stem cell.

### Claim 6 (depends on 5)

The method of claim 5 wherein the cell is an induced pluripotent stem cell.

### Claim 7 (depends on 4)

The method of claim 4 wherein the cell is a neural stem cell.

### Claim 8 (depends on 1)

The method of claim 1 wherein the cell is a differentiated cell.