This disclosure provides methods and landing pad constructs for generation of parental cell lines suitable for targeted integration. A method is provided by the parental cell line development; this is, the introduction of binding sites of BPV1 E2 protein to landing pad vectors so that expressed BPV1 E2 protein could locate the vector to transcriptionally active region in the genome. Cells with high expression level of reporter genes are selected for the next stage and will be used in the development of cell lines expressing another recombinant protein by recombination mediated cassette exchange (RMCE). Landing pad constructs include recombination target sites for site-specific recombinases, and therefore, it could be replaced with gene-of-interest expression construct containing the same set of recombination target sites. This yields the generation of producer cell lines with less effort compared to traditional cell line development by random integration.

The invention relates to transgenic plants comprising an inverted-repeat construct which triggers post-transcriptional gene silencing of an endogenous visual reporter gene driven by a tissue-specific promoter wherein said tissue is relevant for pathogen entry, propagation or replication and their uses for screening natural or synthetic molecules, microorganisms or extracts from micro- or macro-organisms for their potential ability to inhibit pathogen entry, propagation or replication in plants by enhancing PTGS or for characterizing the mode of action of natural or synthetic molecules that are known to enhance plant disease resistance through an ill-defined mode of action.

The invention relates to transgenic plants comprising an inverted-repeat construct which triggers post-transcriptional gene silencing of an endogenous visual reporter gene driven by a tissue-specific promoter wherein said tissue is relevant for pathogen entry, propagation or replication and their uses for screening natural or synthetic molecules, microorganisms or extracts from micro- or macro-organisms for their potential ability to inhibit pathogen entry, propagation or replication in plants by enhancing PTGS or for characterizing the mode of action of natural or synthetic molecules that are known to enhance plant disease resistance through an ill-defined mode of action.

The present invention relates to a shuttle plasmid replicable in Clostridium and E. coli, the shuttle plasmid comprising: a nucleotide sequence of the first replication origin allowing replication in E. coli; a nucleotide sequence coding for a replication protein region derived from pUB110 plasmid; and an expression terminator sequence of a gene.

The present invention relates to a shuttle plasmid replicable in Clostridium and E. coli, the shuttle plasmid comprising: a nucleotide sequence of the first replication origin allowing replication in E. coli; a nucleotide sequence coding for a replication protein region derived from pUB110 plasmid; and an expression terminator sequence of a gene.

The technology relates to a method for producing a transformed and fully-segregated cyanobacteria, the method comprising incubating the cyanobacteria and a nucleic acid comprising a selectable marker under conditions suitable for transformation of the cyanobacteria with the nucleic acid; further incubating the cyanobacteria in growth media under conditions suitable for recovery of the cyanobacteria; and selecting the transformed and fully-segregated cyanobacteria using a selection agent.

The technology relates to a method for producing a transformed and fully-segregated cyanobacteria, the method comprising incubating the cyanobacteria and a nucleic acid comprising a selectable marker under conditions suitable for transformation of the cyanobacteria with the nucleic acid; further incubating the cyanobacteria in growth media under conditions suitable for recovery of the cyanobacteria; and selecting the transformed and fully-segregated cyanobacteria using a selection agent.

Recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

Recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

The present invention is within the field of industrial protein production. The invention provides a novel expression system using dihydroorotate dehydrogenase (DHODH) as a selection marker in combination with leflunomide or a metabolite thereof, notably for use in mammalian cell lines. Expression vectors encoding DHODH, cell lines comprising said vectors and methods of producing recombinant proteins are also provided.