Patent applications filed by the National University of Singapore

Technology overview

A research team lead by Dr Zhiyuan Gong at the Department of Biological Sciences, National University of Singapore, identified several homologous gene promoters for zebrafish (Danio rerio), linked them to a modified green fluorescent protein (GFP) gene, and successfully created transgenic zebrafish that express GFP (related publication by Ju et al. (1999).  Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters.  Dev Genet. 25:158-167).  They have filed patent applications concerning this technology, indicating that hormone-inducible or heavy metal-inducible promoter genes can be used to create bioreporter fish that can detect such contaminants in water samples in the patent specification.

Details of patent documents

Patent or Publication no. Title, Independent Claims and Summary Assignee and licensing information
US 2004/143864

  • Earliest priority – 18 Feb 1999
  • Filed – 21 Oct 2003
  • Application pending
  • Expected expiry – not applicable
Title – Chimeric gene constructs for generation of fluorescent transgenic ornamental fish

Claim 1
A method of providing transgenic fish to the ornamental fish market, comprising the steps of:(a) obtaining an ornamental transgenic fish comprising one or more chimeric fluorescence genes positioned under the control of a promoter, wherein

  • the transgenic fish expresses one or more fluorescent proteins encoded by the one or more fluorescence genes at a level sufficient such that said fish fluoresces upon exposure to one or more of a blue light, ultraviolet light or sunlight; and

(b) distributing said fish to the ornamental fish market.

The claims are generally drawn towards:

  • a method of providing transgenic fish to the ornamental fish market (claim 1)

Definitions extracted from the specification are:

  • Ornamental – there is no definition for this term. The Merriam-Webster online dictionary has the following definition: (adj) of, relating to, or serving as ornament; specifically : grown as an ornamental
  • Fish – there is no definition for this term.  Dependent claims 39 and 41 recite the following: zebrafish, medaka, goldfish or carp (claim 39), koi, loach, tilapia, glassfish, catfish, angel fish, discus, eel, tetra, goby, gourami, guppy, Xiphophorus, hatchet fish, Molly fish, or pangasius (claim 41).
  • Chimeric fluorescence gene(s) – … comprising a promoter described herein operatively linked to a heterologous gene.
  • Fluorescence gene(s) – there is no definition for this term.  The Detailed Description provides the following examples: DNA that codes for … fluorescent peptides including GFP, EGFP, BFP, EBFP, YFP, EYFP, CFP, ECFP and enzymes (such as luciferase, β-galactosidase, chloramphenicol acetyltransferase, etc.).
  • Promoter – constitutive (ubiquitous), or that have tissue specificity such as skin specificity or muscle specificity or that are inducible by a chemical substance…

Comments:

Since this is a published application and not a granted patent, currently there are no enforceable rights.

According to USPTO Public PAIR, this application is a continuation of US 09/913898 (filed 3 Oct 2001), which is also still pending.  There is no publication concerning this application.  INPADOC legal status of WO 2000/49150 (see below) indicates that this PCT application entered national phase in the US on the same date of filing of US 09/913898, therefore it is likely that this unpublished US patent application is the original national phase entry of WO 2000/49150 into the US.

Claims in this patent application are not directed towards a product or method concerning a bioreporter system because independent claim 1 states that the use of the transgenic fish is for the ornamental fish market.  However, the PCT application from which this national phase entry arose contains claims reciting use of the transgenic fish for sensing purposes (see below), and therefore was included in the analysis.

National University of Singapore

10 KENT RIDGE CRESCENT
SINGAPORE 0511

Contact on university website:

National University of Singapore

21 Lower Kent Ridge Road, Singapore 119077

Ph +65-6516-6666

WO 2000/49150

  • Earliest priority – 18 Feb 1999
  • Filed – 16 Jul 1999
  • Published – 24 Aug 2000
  • Expected expiry – not applicable
Title – Chimeric gene constructs for generation of fluorescent transgenic ornamental fish

Claim 1

A zebrafish cytokeratin gene promoter which is capable of directing a structural gene to be predominantly expressed in skin epithelia when it is inserted in front of the structural gene and introduced into fish embryos.

Claim 2

A zebrafish muscle creatine kinase gene promoter which is capable of directing a structural gene to be specifically expressed in muscles when it is inserted in front of the structural gene and introduced into fish embryos.

Claim 3

A zebrafish fast skeletal muscle isoform of myosin light chain 2 gene promoter which is capable of directing a structural gene to be predominantly expressed in skeletal muscles when it is inserted in front of the structural gene and introduced into fish embryos.

Claim 4

A zebrafish acidic ribosomal protein PG gene promoter which is capable of directing a structural gene to be expressed ubiquitously in all tissues when it is inserted in front of the structural gene and introduced into fish embryos.

Claim 10

A transgenic fish comprising a DNA that encodes a fluorescent protein under control of a promoter that causes said DNA
(1) to be expressed in predominantly skin epithelia,
(2) to be specifically expressed in muscles,
(3) to be predominantly expressed in skeletal muscles, or
(4) to be expressed ubiquitously in all tissues.

Claim 14

A recombinant DNA vector comprising a promoter DNA that hybridizes under stringent conditions to a polynucleotide of any one of SEQ ID NOS:7, 8, 9, or 22, operatively linked to a structural gene encoding a fluorescent or chemiluminescent protein.

Claim 16

A transgenic fish comprising a chimeric gene in turn comprising a promoter DNA that hybridizes under stringent conditions to a polynucleotide of any one of SEQ ID NOS:7, 8, 9, or 22, operatively linked to a structural gene encoding a fluorescent or a chemiluminescent protein.

Claim 17

A method for sensing a steroid hormone or a steroid hormone derivative in a water sample comprising:
(a) contacting a fish expressing a fluorescent or chemiluminescent protein under control of an estrogen- or other steroid hormone-inducible promoter with a sample of water; and
(b) measuring the amount of fluorescent or chemiluminescent light from said fish.

Claim 18

A method for sensing heavy metals, such as zinc, copper, cadmium, mecury etc., in a water sample comprising:
(a) contacting a fish expressing a fluorescent or chemiluminescent protein under control of a heavy metal-inducible promoter with a sample of water; and
(b) measuring the amount of fluorescent or chemiluminescent light from said fish.

The claims are generally drawn towards:

  • a zebrafish cytokeratin gene promoter (claim 1)
  • a zebrafish muscle creatine kinase gene promoter (claim 2)
  • a zebrafish fast skeletal muscle isoform of myosin light chain 2 gene promoter (claim 3)
  • a zebrafish acidic ribosomal protein PG gene promoter (claim 4)
  • a transgenic fish comprising a DNA that encodes a fluorescent protein under control of a promoter (claim 10)
  • a recombinant DNA vector comprising a promoter DNA operatively linked to a structural gene encoding a fluorescent or chemiluminescent protein (claim 14)
  • a transgenic fish comprising a chimeric gene comprising a promoter DNA operatively linked to a structural gene encoding a fluorescent or chemiluminescent protein (claim 16)
  • a method for sensing a steroid hormone or a steroid hormone derivative in a water sample (claim 17)
  • a method for sensing heavy metals, such as zinc, copper, cadmium, mecury etc., in a water sample (claim 18)

Definitions extracted from the specification are:

  • Ornamental – there is no definition for this term. The Merriam-Webster online dictionary has the following definition: (adj) of, relating to, or serving as ornament; specifically : grown as an ornamental
  • Fish – there is no definition for this term. Dependent claims 39 and 41 recite the following: zebrafish, medaka, goldfish or carp (claim 39), koi, loach, tilapia, glassfish, catfish, angel fish, discus, eel, tetra, goby, gourami, guppy, Xiphophorus, hatchet fish, Molly fish, or pangasius (claim 41).
  • Chimeric fluorescence gene(s) – … comprising a promoter described herein operatively linked to a heterologous gene.
  • Fluorescence gene(s) – there is no definition for this term. The Detailed Description provides the following examples: DNA that codes for … fluorescent peptides including GFP, EGFP, BFP, EBFP, YFP, EYFP, CFP, ECFP and enzymes (such as luciferase, β-galactosidase, chloramphenicol acetyltransferase, etc.).
  • Promoter (unless specified otherwise) – constitutive (ubiquitous), or that have tissue specificity such as skin specificity or muscle specificity or that are inducible by a chemical substance…
  • Structural gene – encode a protein that produces a fluorescent or chemiluminescent light under conditions appropriate to the particular polypeptide in one or more tissues of a fish.
  • SEQ ID NO:7 – complete sequence of the cytokeratin (CK) promoter region
  • SEQ ID NO:8 – complete sequence of the muscle creatine kinase (MCK) promoter region
  • SEQ ID NO:9 – complete sequence of the acidic ribosomal protein P0 (ARP) promoter region including the first intron
  • SEQ ID NO:22 – complete sequence of the fast skeletal muscle isoform of myosin light chain 2 (MLC2f) promoter region
  • Stringent conditions – under salt and temperature conditions providing stringency at least as high as that equivalent to 5×SSC and 42° C.

Comments:

Since this is a published application and not a granted patent, currently there are no enforceable rights.

Unlike the claims recited in the national phase entry of WO 2000/49150 in the US (US 2004/143864, see above), the set of claims recited in this PCT application are not only directed towards ornamental use of these transgenic fish (and concerning promoters and structural genes), but also towards any other method of use, including use in sensing particular substances such as hormones and heavy metals.  The first seven independent claims listed above (claims 1, 2, 3, 4, 10, 14, 16) are not limited to any method of use, therefore the scope of these claims will be broad if they are granted as applied for.

Examples in the specification of WO 2000/49150 provide a chapter on ‘potenatial applications of fluorescent transgenic fish’, which contains a proposal of use of the system to produce transgenic fish that can be used for detection of environemntal pollution, but without actual experimental data to enable the proposal (e.g. promoter/reporter constructs, transgenic fish, response of such transgenic fish to substances).

Remarks Related application filed in Singapore (SG 82624).

Search strategy

Search details
Date of search 02/05/2006
Database searched Patent Lens
Type of search Simple, stemming on
Collections searched AU-B, US-A, US-B, EP-B, WO
Search terms biosensor AND transgenic AND “water quality”
Results 12
Comments Of the 12 results identified using these search terms, 2 results were identified as being of particular interest based on their abstracts and a review of their claims.