# US 2010/0169986 A1 · Conditional Mst Overexpressing Construct and Conditional Myostatin Overexpressing Transgenic Mouse

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- Number: US 2010/0169986 A1
- Document ID: US20100169986A1
- Status: Published application
- Kind: A1
- Application number: 12/398,994
- Filed: 2009-03-05
- Published: 2010-07-01
- Earliest claimed: 2008-03-05
- Assignees: Charles Drew University of Medicine and Science
- Inventors: Suzanne Porszasz-Reisz
- Web page: https://patentgrep.com/patent/US20100169986A1

## CPC

- A61K 49/008: {lipoprotein vesicle, e.g. HDL or LDL proteins}
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- A61K 49/0008: {Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure}
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- G01N 33/5023: {on expression patterns}
- G01N 33/5088: {of vertebrates}
- G01N 33/6887: {from muscle, cartilage or connective tissue}
- A01K 2217/15: Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
- A01K 2217/203: Animal model comprising inducible/conditional expression system, e.g. hormones, tet
- A01K 2267/03: Animal model, e.g. for test or diseases
- G01N 2800/10: Musculoskeletal or connective tissue disorders
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Provided herein are novel nucleic acid sequences, vectors comprising such nucleic acid sequences, host cells comprising such vectors, and transgenic animals comprising such nucleic acid sequences, and related molecules and methods relating thereto. Such novel nucleic acid sequences, vectors comprising such nucleic acid sequences, host cells comprising such vectors, and transgenic animals comprising such nucleic acid sequences, and related molecules and methods provide conditional overexpression of genes, such as myostatin, and transgenic animals conditionally overexpression genes, such as myostatin.

## Claims

### Claim 1

A conditional bicistronic myostatin expression construct comprising a construct selected from the group of constructs consisting of polyA-EGFP/IRES-Mst/HA-TRE//MCK-rtTA/BFP-polyA, polyA-BFP/rtTA-MCK//TRE-HA/Mst-IRES/EGFP-polyA, TRE-HA/Mst-IRES/EGFP-polyA//MCK-rtTA/BFP-polyA, MCK-rtTA/BFP-polyA//TRE-HA/Mst-IRES/EGFP-polyA, and polyA-EGFP/IRES-Mst/HA-TRE//MCK-rtTA/BFP-polyA, where MCK is a promoter selected from MCK and MCK-3E.

### Claim 2 (depends on 1)

A conditional monocistronic construct according to claim 1 comprising two sequences: a: the regulatory construct MCK-rtTA/BFP-polyA, and b: the response construct TRE-HA/Mst-IRES/EGFP-polyA.

### Claim 3 (depends on 1)

A conditional bicistronic myostatin expression construct according to claim 1, comprising a construct of claims 1 lacking a HA tag at the 5′ end of Mst sequence.

### Claim 4 (depends on 1)

A conditional monocistronic construct according to claim 1 where the response construct lacks a HA tag on Mst sequence, lacks an IRES/EGFP, or lacks BFP fusion to the rtTA sequence.

### Claim 5 (depends on 1)

A construct of claim 1 wherein said construct comprises a plasmid.

### Claim 6 (depends on 1)

A method of producing a transgenic non-human animal comprising introducing the construct of claim 1 into a non-human animal.

### Claim 7

A transgenic non-human animal produced by: (a) crossing a first transgenic non-human animal comprising a first (regulatory) construct that expresses rtTA operably linked to promoter MCK or MCK-3E with a second non-human animal comprising a second (response) construct comprising Mst or Mst cDNA operably linked to a promoter TRE and (b) selecting from the first generation offspring a transgenic non-human animal having a genome comprising both the first regulatory construct and the second response construct.

### Claim 8 (depends on 1, 7)

A transgenic non-human animal for conditionally overexpressing Mst of claim 7, comprising cells comprising a construct of claim 1.

### Claim 9 (depends on 7)

A method for screening for therapeutic agents that inhibit Mst activity comprising (a) administering test agents to the transgenic animal of claim 7, (b) evaluating the effects of the test agent on the Mst associated phenotype of the animal.

### Claim 10 (depends on 1)

A conditional bicistronic myostatin expression construct of claim 1 comprising a regulatory sequence and a myostatin (Mst) response sequence, wherein said bicistronic myostatin expression construct comprises a DNA transgene, said DNA transgene comprising SEQ ID NO. 13, or variants thereof with greater than 80%, 90%, 95%, 99% sequence identity operably linked to the TRE promoter, as a response sequence, and a regulatory sequence comprising a tissue specific promoter.

### Claim 11 (depends on 1)

The conditional bicistronic myostatin expression construct of claim 1 wherein the regulatory sequence comprises reverse tetracycline transcription activator (rtTA).

### Claim 12 (depends on 1)

The conditional bicistronic myostatin expression construct of claim 1 wherein the response sequence comprises TRE promoter.

### Claim 13 (depends on 1)

The conditional bicistronic myostatin expression construct of claim 1 wherein the tissue specific promoter is selected from MCK, MCK-3E and Troponin I.

### Claim 14 (depends on 7)

A transgenic non-human animal for conditionally overexpressing Mst of claim 7 comprising cells comprising a bicistronic myostatin expression construct comprising a regulatory sequence and a myostatin response sequence, wherein said bicistronic myostatin expression construct comprises a DNA transgene, said DNA transgene comprising SEQ ID NO. 13, or variants thereof with greater than 80%, 90%, 95%, 99% sequence identity operably linked to the TRE promoter, as a response sequence, and a regulatory sequence comprising a tissue specific promoter.

### Claim 15 (depends on 14)

The transgenic non-human animal of claim 14 wherein the regulatory sequence comprises reverse tetracycline transcription activator (rtTA).

### Claim 16 (depends on 14)

The transgenic non-human animal of claim 14 wherein the response sequence comprises TRE.

### Claim 17 (depends on 14)

The transgenic non-human animal of claim 14 wherein the tissue specific promoter is MCK, MCK-3E or Troponin I.

### Claim 17 (depends on 1, 7)

A method of producing a transgenic non-human animal of claim 7 comprising introducing a conditional bicistronic myostatin expression vector into a non-human animal, wherein said conditional bicistronic myostatin expression vector comprises a myostatin expression cloned into the construct of claim 1 into a vector.

### Claim 18 (depends on 1, 2, 3, 4, 6, 8, 10)

A method of producing a conditional bicistronic myostatin expression vector comprising cloning the myostatin expression construct of any of claims 1-4, 6, 8 and 10 into a vector.

### Claim 18 (depends on 1)

A construct of claim 1, further comprising a fluorescent marker coding sequence selected from the group consisting of GFP, EGFP, dsRed, dsRed2, CFP, ECFP, YFP, EYFP, BFP, dsRed1, dsRed2, DsRed-Express, AsRed2, HcRed1, AmCyan, ZsYellow, ZsGreen, AcGFP-1, luciferase, and aequorin.

### Claim 19 (depends on 18)

The construct of claim 18, comprising EGFP.

### Claim 20 (depends on 1)

The conditional bicistronic myostatin expression response construct of claim 1 wherein the transgenic nucleotide sequence comprises SEQ ID NO. 5 or Mst cDNA operably linked to a TRE2 promoter in cells engineered to express rtTA protein in the presence of tetracycline or doxycycline.