Site-directed integration of transgenes: transposons revisited using DNA-binding-domain technologies.

In the last 20 years, tools derived from DNA transposons have made major contributions to genetic studies from gene delivery to gene discovery. Various complementary and fairly ubiquitous DNA vehicles have been developed. Although many transposons are efficient DNA vehicles, they appear to have limited ability to target specific sequences, since all that is required at the integration locus is the presence of a short 2- to 4-bp sequence. Consequently, insertions mediated by transposon-based vectors occur somewhat randomly. In the past 5 years, strategies have emerged to enhance the site-specificity of transposon-based vectors, and to avoid random integrations. The first proposes that new target site specificity could be grafted onto a transposase by adding a new DNA-binding domain. Alternative strategies consist of indirectly targeting either the transposase or the transposon to a chosen genomic locus. The most important information available about each strategy are presented, and limitations and future prospects are discussed.

The mariner Mos1 transposase produced in tobacco is active in vitro.

The mariner-like transposon Mos1 is used for insertional mutagenesis and transgenesis in different animals (insects, nematodes), but has never been used in plants. In this paper, the transposition activity of Mos1 was tested in Nicotiana tabacum, but no transposition event was detected. In an attempt to understand the absence of in planta transposition, Mos1 transposase (MOS1) was produced and purified from transgenic tobacco (HMNtMOS1). HMNtMOS1 was able to perform all transposition reaction steps in vitro: binding to ITR, excision and integration of the same pseudo-transposon used in in planta transposition assays. The in vitro transposition reaction was not inhibited by tobacco nuclear proteins, and did not depend on the temperature used for plant growth. Several hypotheses are proposed that could explain the inhibition of HMNtMOS1 activity in planta.

Sustained transgene expression using non-viral enzymatic systems for stable chromosomal integration.

Gene delivery technologies have been developed for various biotechnology applications. In gene therapy, they are promising for the treatment of several inherited and acquired human diseases. When therapies require the transfection of a transgene, the vector integration is one of the solutions that is used for maintaining and sustaining expression. On the basis of their origin, vectorisation technologies are currently divided in two fields, gathering on one hand viral vectors and, on the other hand, non-viral approaches. In the case of the non-viral therapies, three main sub-fields are in progress to integrate transgenes. The first uses oligonucleotides to stimulate targeted gene repair by homologous recombination. The second is based on site-specific endonucleases for which the cleavage activity is used to stimulate the host recombination mechanisms in the presence of a DNA vector. The third one is developed from phage and transposon enzymatic systems. The two lasts sub-fields use non-viral enzymes and are the scope of this review. Here, our objective was to overview the main non-viral enzymatic systems able to integrate DNA cassettes. Their molecular and functional characteristics are summarized, and their properties and limits in the current state of the art highlighted. An overview of the safety and quality issues is also presented and discussed, taking into account the solutions that might circumvent problems, intellectual property and economic status for each system. As a conclusion, we propose projections of the future technological developments in the context of the different interests for public and private bodies.

TCP transcription factors predate the emergence of land plants.

TCP proteins are plant-specific transcription factors identified so far only in angiosperms and shown to be involved in specifying plant morphologies. However, the functions of these proteins remain largely unknown. Our study is the first phylogenetic analysis comparing the TCP genes from higher and lower plants, and it dates the emergence of the TCP family to before the split of the Zygnemophyta. EST database analysis and CODEHOP PCR amplification revealed TCP genes in basal land plant genomes and also in their close freshwater algal relatives. Based on an extensive survey of TCP genes, families of TCP proteins were characterized in the Arabidopsis thaliana, poplar, rice, club-moss, and moss genomes. The phylogenetic trees indicate a continuous expansion of the TCP family during the diversification of the Phragmoplastophyta and a similar degree of expansion in several angiosperm lineages. TCP paralogues were identified in all genomes studied, and Ks values indicate that TCP genes expanded during genome duplication events. MEME and SIMPLE analyses detected conserved motifs and low-complexity regions, respectively, outside of the TCP domain, which reinforced the previous description of a "mosaic" structure of TCP proteins.