Alternative methods to antibiotic resistance marker genes

Researchers have devised selection methods that avoid the use of antibiotic or herbicide resistance genes or eliminate them in the final transgenic product. The development of these methods are in part in response to the concerns about horizontal transfer of antibiotic resistance genes, the public perception of risk and the consumer acceptance of the marketed products. Another motivation has been the need for multiple selectable marker genes because the use of a selectable marker gene in a particular line precludes further use of the same selectable gene in subsequent transformations of the same line. The generation of a cultivar with several distinct desirable traits may require repeated transformations events, which would require the use of a different selectable marker for each transformation event. The number of suitable, multiple selectable markers available is limited at present. In addition, the presence of multiple homologous sequences in the same genome may cause instability of the transgenes.

Two general strategies have been pursued to avoid the use of antibiotic resistance genes:

  1. elimination of the selectable marker gene in the resultant transgenic organism and
  2. use of a non−toxic compound that favors or promotes the regeneration and growth of transformed cells expressing a transgene product that acts on the compound (positive selection).

In the first strategy, the methods currently employed are:

  • Co−transformation. Two separate DNA constructs, one containing a gene of interest and the other having a selectable marker gene are co−transformed into the target cell. As the transformed genes are physically separated, the selectable marker can be eliminated by a variety of mechanisms after assessing the integration and expression of the gene of interest. The system depends on a high efficiency of co−transformation and on the integration of the co−transformed DNAs in distant loci. Co−transformation can be achieved by using either two separate plasmid molecules with the different DNAs in the same transformation agent, e.g. one Agrobacterium strain, or by using two different bacterial strains, each containing one of the two constructs.
  • Site−specific recombination systems. These systems require an enzyme that acts in trans (it does not need to be operatively linked to the molecule upon which it acts) to catalyze recombination between two short, cognate DNA sequences. The marker gene, which is cloned between the short cognate sequences, is eliminated when the enzyme, a recombinase, acts upon the sequences. Some of the site−specific recombination systems used are FRT(asymmetric inverted repeat sequences)/FLP (specific recombinase) from Saccharomyces cerevisiae and loxP/Cre (causing recombination) from bacteriophage P1.
  • Intra−genomic relocation of transgenes via transposable elements. Transposition of the maize element families Ac/Ds and the Spm/dSpm is a cut and paste mechanism that results in the excision of the elements of one locus prior to the reinsertion into a second locus. In this way, the selectable marker is removed via transposition of the transposable element that is flanking the marker gene. Failure of the transposable element to reintegrate will cause the selectable marker to be lost.

The second strategy, known as positive selection;

uses selective, non−toxic compounds to exploit the auxotrophies of the transformed material, i.e., the material is unable to regenerate and grow in the absence of an external supply of specific compounds. These methods of positive selection are based on complementing the transformed cells with a gene(s) that enables them:

  • to produce the essential substance themselves, or
  • to be able to utilize a substrate, which confers a metabolic advantage over the non−transformed cells.

Examples of positive selection systems are a:

  • transgenic glucuronidase in combination with the use of cytokinin glucuronides or other phytohormone glucuronides, and
  • transgenes such as phosphomannose isomerase and xylose isomerase that confer the ability to metabolize alternative carbohydrate sources such as mannose and xylose.