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.
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
Title, Independent Claims and Summary
||Title – Selection procedure for identifying transgenic cells, embryos, and plants without the use of antibiotics
The United States of America as represented by the Secretary of Agriculture
|The related patent application in Australia (AU 2003300183) lapsed. A PCT application (WO 2004/061128 A1) was also filed.|
|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|
|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.|