Selection procedure for identifying transgenic cells, embryos, and plants without the use of antibiotics

Abstract

Transgenic cells may be selected using temperature sensitive marker proteins. In this method, a population of host cells are transformed with a foreign DNA construct which includes at least one first nucleic acid coding sequence and a second nucleic acid sequence encoding a temperature sensitive marker protein, wherein each of the first and second nucleic acid coding sequences are operatively linked to gene expression control sequences. Suitable temperature sensitive marker proteins which may be used herein include heat shock proteins, heat shock transcription factors, cold regulated proteins (COR), or cold regulated protein transcription factors. Following transformation, the population of cells are cultured under temperature conditions wherein growth of non-transformed cells is suppressed or prevented while growth of cells transformed with the DNA construct is supported or promoted. Thus, survival and/or significant growth of a cell is an indication that the cell has been successfully transformed with the DNA construct. Those cells are considered presumptively positive transformants, and may be recovered.