Abstract: The present invention relates, in general, to a method for introducing a heterologous replacement gene sequence into a host embryonic stem cell to replace an endogenous host gene target sequence. In particular, the invention relates to a method for inserting large pieces of DNA into embryonic stem cells with improved efficiency, by first deleting the endogenous host gene target sequence, and subsequently utilising two proximally positioned site-specific recombinase target (RT) sites to insert a heterologous replacement gene sequence into the host chromosome.

Abstract: The present invention relates to a biological entity carrying a regulator construct comprising a specific repressor gene and a responder construct comprising at least one segment corresponding to a short hairpin RNA (shRNA) or corresponding to complementary short interfering RNA (siRNA) strands, said at least one segment being under control of a promoter which contains an operator sequence corresponding to the repressor. The invention further relates to a method for preparing said biological entity and its use.

Abstract: The present invention relates to a biological entity, notably a rat, carrying a regulator construct comprising a specific repressor gene and a responder construct comprising at least one segment corresponding to a short hairpin RNA (shRNA) or corresponding to complementary short interfering RNA (siRNA) strands or corresponding to miRNA, said at least one segment being under control of a promoter which contains an operator sequence corresponding to the repressor. The invention further relates to a method for preparing said biological entity and its use.

Abstract: The invention provides a method for targeted transgenesis of short hairpin RNA expression cassettes using recombinase mediated cassette exchange. Suitable nucleotide acid sequences and vectors for the targeted transgenesis and recombinase mediated transgenesis are provided.

Abstract: The present invention relates to a process that enables constitutive and inducible gene knock down in living organisms using a short hairpin RNA expression vector integrated into the genome of the organism.

Abstract: The invention provides a method for targeted transgenesis using the Rosa26 locus. Suitable nucleotide acid sequences and vectors for the targeted transgenesis and recombinase mediated transgenesis are provided. The Rosa26 locus proved to be a suitable integration site allowing strong and predictable expression of inserted transgenes carrying exogenous promoters.

Abstract: The invention relates to a method that enables automated identification and isolation of cells harbouring a predetermined genetic modification (homologous recombination) using detectable/sortable markers, e.g. fluorescence markers, to identify homologous DNA modifications. Suitable vectors are also provided.

Abstract: The invention relates to a method for marker-free DNA expression cassette exchange in the genome of cells or parts of cells by using the FLP recombinase mediated cassette exchange. A first DNA expression cassette carrying a positive-negative selection marker flanked by two FLP recombinase recognition target (FRT) sites is integrated into a chromosomal locus of the genome. Following selection of cell clones surviving the conditions for positive selection, the first DNA cassette is exchanged by an incoming second DNA expression cassette located on a circular vector and carrying a transgene flanked by the same FRT sites as the first DNA cassette by using FLP-recombinase. The cell clones surviving the conditions for negative selection contain specifically inserted the gene of the incoming DNA cassette without inserted unwanted vector sequences or positive selectable markers.

Abstract: The present invention relates to a process that enables constitutive and inducible gene knock down in living organisms using a short hairpin RNA expression vector integrated into the genome of the organism.

Abstract: The invention relates to a method for marker-free repetitive DNA expression cassette exchange in the genome of cells or parts of cells by using the FLP recombinase mediated cassette exchange. In a first step a first DNA expression cassette carrying a positive-negative selection marker flanked by a wild type FLP recombinase recognition target (FRT) site on one end and a modified heterospecific FRT on the other end is integrated into a chromosomal locus of the genome for tagging. Following selection of cell clones surviving the conditions for positive selection said first DNA cassette as a second step is exchanged by an incoming second DNA expression cassette located on a circular vector and carrying a homologous or heterologous gene (transgene) of any coding sequence flanked by the same FRT sites as the first DNA cassette by using FLP-recombinase.