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Positive selection based on temperature

The United States of America as represented by the Secretary of Argriculture holds a United States patent on methods for selection of transgenic cells by temperature sensitive marker proteins. The related patent application was also filed in Australia but has lapsed.

Technology overview

Temperature sensitive marker proteins, including heat shock proteins (HSP), heat shock transcription factors (HSTF, also referred to as heat shock factors, HSF), cold regulated proteins (COR, also referred to as cold shock proteins), and cold regulated protein transcription factors, and their corresponding nucleic acid coding sequences have been identified from many different orgnisms in the past decade. This invention uses genes coding for these temperature sensitive proteins as selectable markers for selecting the transformed cells under extreme temperature condition.

Suitable heat shock proteins which may be used as temperature-sensitive markers include those in the families of HSP 100 or HSP110 (those HSPs having a molecular weight range between approximately 100 and 110 kDa), HSP 90 (HSPs ranging in size between approximately 80 to 94 kDa), HSP 70, HSP 60, and low molecular weight (LMW) HSPs (those having a molecular weight between 15 and 30 kDa).

A variety of heat shock transcription factors, cold regulated proteins, or cold regulated protein transcription factors may also be used as potential temperature sensitive markers for selection purpose. These may include heat shock transcription factor or heat shock factor (HSF) proteins derived from humans and Drosophila (see US 5756343), the cold regulation protein COR15 from Arabidopsis (see US 5296462 and US 5356816) and cold regulation protein transcription factor CBF1 from Arabidopsis (see US 5891859 and US 5929305).

By transforming target cells with a gene coding for a temperature sensitive protein as a selectable marker, selection of truly transformed cells can be carried out through the treatment of cells under conditions of extreme temperature. In practice, however, most host cells of interest in fact possess native temperature stress response systems which are functional and could allow even the non-transformed cells to grow at the set extreme temperature and confuse the selection process. To minimize the induction of any of these native proteins that could protect the non-transformed cells and enable their growth at the temperature extremes used, an initial culture on a growth medium under relatively normal conditions, that is, at a temperature suitable to promote the growth of all of the cells but which will not induce the expression of the host cell’s native protective heat shock proteins or cold regulation proteins may be a necessory step prior to the treatment under extreme temperature condition that only the transformed cells are supported.

Specific patent information

Patent/Application Number

Title, Independent Claims and Summary

 Assignee
US 6939676

  • Earliest priority – 30 Dec 2002
  • Filed – 30 Dec 2002
  • Granted – 6 Sep 2005
  • Expected expiry – 30 Dec 2022
 Title – Selection procedure for identifying transgenic cells, embryos, and plants without the use of antibiotics

Claim 1
A method for selecting a cell transformed with one or more nucleic acid coding sequences comprising:(a) transforming a population of cells with a foreign DNA construct comprising at least one first nucleic acid coding sequence and a second nucleic acid coding sequence encoding a temperature sensitive marker protein selected from the group consisting of heat shock proteins, heat shock transcription factors, cold regulated proteins, and cold regulated protein transcription factors, each of said first and second nucleic acid coding sequences being operatively linked to gene expression control sequences, said transforming generating a second population of cells comprising cells which have been successfully transformed with said foreign DNA construct and non-transformed cells,(b) selecting said cells which have been successfully transformed from said non-transformed cells in said second population of cells from said transforming in (a), said selecting consisting essentially of culturing said second population of cells under temperature conditions wherein growth of said non-transformed cells is suppressed or prevented while growth of said cells which have been successfully transformed is supported, and

(c) recovering cells from (b) exhibiting significant growth, wherein the recovered cells exhibiting significant growth are said cells which have been successfully transformed.
Claim 19
A method for selecting a cell transformed with one or more nucleic acid coding sequences comprising:(a) transforming a population of cells with a foreign DNA construct comprising at least one first nucleic acid coding sequence and a second nucleic acid coding sequence encoding a temperature sensitive marker protein selected from the group consisting of heat shock proteins, heat shock transcription factors, cold regulated proteins, and cold regulated protein transcription factors, each of said first and second nucleic acid coding sequences being operatively linked to gene expression control sequences, said transforming generating a second population of cells comprising cells which have been successfully transformed with said foreign DNA construct and non-transformed cells,(b) selecting said cells which have been successfully transformed from said non-transformed cells in said second population from said transforming in (a), wherein said selecting comprises culturing said second population of cells under temperature conditions wherein growth of said non-transformed cells is suppressed or prevented while growth of said cells which have been successfully transformed is supported, and said selecting does not comprise any of: culturing said second population of cells transformed in (a) on culture media comprising an antibiotic or pesticide, or detection of toxins, enzyme products, or pigments produced by said cells, and

(c) recovering cells from (b) exhibiting significant growth, wherein the recovered cells exhibiting significant growth are said cells which have been successfully transformed.
Claim 20
A method for selecting a cell transformed with one or more nucleic acid coding sequences comprising:(a) transforming a population of cells with a foreign DNA construct comprising at least one first nucleic acid coding sequence and a second nucleic acid coding sequence encoding a temperature sensitive marker protein selected from the group consisting of heat shock proteins, heat shock transcription factors, cold regulated proteins, and cold regulated protein transcription factors, each of said first and second nucleic acid coding sequences being operatively linked to gene expression contrcl sequences,(b) culturing said populatior. of cells transformed in (a) under temperature conditions wherein growth of non-transformed cells is suppressed or prevented while growth of cells transformed with said DNA construct is supported, and

(c) recovering cells from (b) exhibiting significant growth as presumptive positive transformants,

wherein said DNA construct does not comprise an antibiotic resistance marker or a pesticide resistance marker.

The United States of America as represented by the Secretary of Agriculture

Remarks

 The related patent application in Australia (AU 2003300183) lapsed. A PCT application (WO 2004/061128 A1) was also filed.

Search strategy

Search details
Date of search 31/05/2006
Database searched Patent Lens
Type of search Simple, stemming on
Collections searched AU-B, US-A, US-B, EP-B, WO
Search terms “Positive selection transformation cell” in abstract
Results 117 hits
Comments The “The United States of America as represented by the Secretary of Argriculture” family represented by  WO 2004/61128 titled ” Selection procedure for identifying transgenic cells, embryos, and plants without the use of antibiotics”. This patent family claims methods for selection of transgenc cells using temperature sensitive marker proteins.