GENETIC CONSTRUCT FOR SELECTION OF HOMOLOGOUS RECOMBINANTS ON A SINGLE SELECTIVE MEDIUM

摘要

Novel genetic constructs and methods for their use in transforming target cells are disclosed. The genetic constructs, which are particularly adapted for homologous recombination with target-cell genomic DNAs, comprise a positively selectable genetic marker and a negative selection system 'antagonistic' to the expression of the positively selectable marker. The positively selectable marker is situated in a region of the construct between a first and a second flanking sequence homologous to sequences flanking a desired integration site in the target-cell genome. The negative selection system is situated outside the region. The negative selection system preferably comprises an antisense gene that prevents expression of the positively selectable marker. The positively selectable marker preferably encodes an antibiotic resistance factor. The construct can also include a 'gene of interest', also situated between the homologous flanking sequences adjacent the positively selectable marker, for introduction via homologous recombination into the target genome at the desired integration site. Transformed target cells whose genomes homologously recombine with the genetic construct acquire the positively selectable marker and (if present) the gene of interest. Non-homologous recombinants acquire both the positively selectable marker and the negative selection system, thereby losing the ability to survive exposure to the corresponding positive selection agent. Non-recombinants acquire no part of the construct. Thus, homologous recombinants have the exclusive ability to survive exposure to the positive selection agent and can be easily isolated from all other target cells.