Positive selection based on trehalose

Expressive Rsearch B.V. obtained an European patent claiming a method for the selection of transformed plant cells using trehalose. Related patent applications were also filed in the United States, Canada and some other jurisdictions such as Japan and China.

Technology overview

Trehalose is a nonreducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi, and invertebrates. As a fact, trehalose does not accumulate to detectable levels in most plants. Cells, in particular plant cells, can not normally develop in a medium in which an increased concentration of trehalose is present without another metabolizable carbon source being present.

Positive selection methods were developed by transforming target plant cells with genes coding for trehalase, which is able to hydrolyze intracellular trehalose and/or its derivative to glucose for plant use, and selecting transformed cells on a medium containing trehalose as the sole carbon source. The genes coding for intracellular trehalases can be of bacterial origin (e.g. the TreF gene from E. coli) or of plant origin (e.g. the AtTRE1 gene from Arabidopsis).

The gene coding for an endogenous trehalase exists in the genomes of higher plants such as soybeen and Arabidopsis thaliana. However, these endogenous trehalase genes generally code for an extracellular trehalase, which is not active in the cell. Such endogenous genes can be modified by a number of ways such as deactivating the protein-secretion signal via deletion or mutagenesis, changing the protein targeting sequences, or the pH sensitivity of the enzymatically active site to make the modified trehalases intracellularly active.

Detailed patent information

Patent/Application Number Title, Independent Claims and Summary Assignee
EP 1442126 B1

  • Earliest priority – 6 Nov 2001 (NL)
  • Filed – 06 Nov 2002
  • Granted – 08 Feb 2006
  • Expected expiry – 06 Nov 2022
Title – Method for selecting genetically transformed cells

Claim 1
Method for selecting genetically transformed cells from a population of cells, comprising ofa) introducing into a cell at least one desired nucleotide sequence and at least one selection-nucleotide sequence to obtain a genetically transformed cell, wherein the selection-nucleotide sequence comprises a region which codes for a protein involved in the metabolizing of trehalose;
b) placing a population with transformed and non-transformed cells into contact with trehalose and/or derivative thereof; and
c) selecting the transformed cells from the population on the basis of the capacity of the transformed cells to metabolize the trehalose and/or derivative.
Claim 19
Use of trehalose and/or derivative thereof for the selection of transformed cells from a population of transformed and non-transformed cells, wherein the genome of the transformed cells comprises at least one selection-nucleotide sequence comprising a region which codes for a protein involved in the metabolizing of trehalose.

 Original applicant: Stichting Voor De Technische Wetenschappen

Reassigned to: Expressive Research B.V. (NL)

US 2005/084971 A1

  • Earliest priority – 6 Nov 2001 (NL)
  • Filed – 06 Nov 2002
  • Granted – Pending
  • Expected expiry – N/A
Title – Method for selecting genetically transformed cells

Claim 1
The same as  Claim 1 in EP 1442126 B1.
Claim 17
Method for using trehalose and/or a derivative thereof for the selection of transformed cells from a population of transformed and non-transformed cells, wherein the genome of the transformed cells comprises at least one selection-nucleotide sequence comprising a region which codes for a protein involved in the metabolizing of trehalose.

Remarks

Related patent application were also filed in Canada (CA 2466088 AA), China (CN 1622999 A), Japan (JP 2005508190 T2), Isreal (IL 161773 A0) and South Africa (ZA 200403307 A). The PCT application is WO 2003/040377 A1.

Search strategy

Search details
Date of search 7/06/2006
Database searched Patent Lens
Type of search Simple, stemming on
Collections searched AU-B, US-A, US-B, EP-B, WO
Search terms “selecting transformed cells” in title
Results 30 hits
Comments Relevant patents/applications:

EP 1442126 B1

US 2005/84971