The ability to use of DNA binding domains (DBDs) able to interact with a specific DNA target in order to develop synthetic proteins capable of modifying gene expression or the functioning of DNA recombination machinery, such as those involving endonucleases, integrases or transposases (Tpase), constitutes a major challenge in the post genomic era. From this perspective, a DBD added to an enzymatic system has to combine several qualities, including i) being bound to a DNA target large enough to ensure specific and high-affinity recognition, ii) having a controllable ability to assemble (or not) as homo-oligomer or with other cellular partners.

The fact that only a few rRNA genes are disrupted accounts for the absence of deleterious effects in genetically-modified cells. Indeed, genetic investigations have demonstrated that the disruption of < 70% of the rRNA genes by integration of DNA segments is not lethal and does not impair the viability of invertebrate genomes. The expression of the transgene also persists for long periods, because integrated DNA fragments are rarely subjected to expression silencing, and their expression can be carried out by the RNA Polymerase I promoter, but also by RNA Polymerase II and III promoters.

Overall, whatever the species and strategy used to integrate a DNA fragment into rRNA genes, it is noticeable that it always requires a protein domain that can target the enzyme and/or the DNA fragment in the proximity of the rRNA genes. To date, we have identified only two kinds of DBD able to bind it as a monomer to a motif present within the conserved 100-bp DNA target, this motif being so large (at least 18-bp) as to be nearly unique in a mammalian genome.

Each of the five fragments encoding a DBD was cloned into the pET14b vector (Novagen, Darmstadt, Germany), in a frame with a Histidine tag (His6 ) at the amino terminal end. The pET14b constructs were transformed in BL21 Escherichia coli bacteria containing a pRARE plasmid (Novagen, Darmstadt, Germany), which encoded rare tRNA E. coli codons. They were also cloned into the pMalc2 system (New England Biolabs France, Every, France), and fused in frame with the Maltose Binding Protein (MBP) at the amino terminal end. The pMalc2 constructs were transformed in JM109 E. coli bacteria using the pRARE plasmid.

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