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FP5

JUMPY Informe resumido

Project ID: QLG2-CT-2000-00821
Financiado con arreglo a: FP5-LIFE QUALITY
País: Germany

Frog Prince transposon system for gene transfer in vertebrates

In an effort to isolate potentially active transposase genes from vertebrate species, transposase coding regions were PCR-amplified from the Rana pipiens (a frog) genome, and cloned into plasmid vectors. Ten transposase genes were aligned to generate a consensus sequence. The individual genes were about 99% identical to the consensus sequence, and one of them differed only in two nucleotides from the consensus, resulting in two amino acid substitutions in its ORF. Site-specific PCR mutagenesis was used to derive the sequence of the consensus transposase gene.
The inverted repeat sequences together with the consensus transposase gene constitute the components of a novel transposable element system that we named Frog Prince (FP).

The initial tests for transpositional activity of the Frog Prince element were done in cultured HeLa cells, using a transposition assay established for Sleeping Beauty. A 17-fold increase in colony number was detected when pFV-FP was cotransfected with its substrate, pFP-neo. This level of activity is similar to that of SB in HeLa cells, and demonstrates that we successfully derived and engineered an active transposon system from the R. pipens genome. Taken together, the data demonstrate that the Frog Prince transposon system can significantly increase the efficiency of transgene integration from plasmid-based vectors to the human genome.

Next we compared the activities of the Sleeping Beauty and Frog Prince systems in cultured cell lines derived from two mammalian, an amphibian and two fish species with the standard transposition assay. FP appeared to be slightly more active than SB in some of the cell lines tested. These data demonstrate that transposition of Frog Prince is not restricted to phylogenetically close taxa, and that it is the most active transposable element in vertebrate species described to date.

High frequency, precise transposition into different genomic loci suggests that genome-wide gene trapping is feasible with FP. For this purpose, an FP-based donor plasmid (pFP/GT-neo) was constructed which contains engrailed-2 intron sequences with the SA, a glycine bridge to allow proper folding of the marker in protein fusions, an ATG-less neo gene, a zeocin resistance gene (zeo) driven by dual eukaryotic/bacterial promoters and a plasmid origin of replication. All chromosomal transposition events give rise to zeocin-resistant cells. A subset of transformant cells will be G418-resistant, if the transposon inserted into an intron of an expressed gene in the proper orientation, and if splicing occurred in-frame with neo. Based on the numbers of zeocin-resistant cell colonies, transposition efficiency of FP/GT-neo was comparable to that of FP-neo. The number of zeocin/G418 double-resistant colonies was about one third of those resistant to zeocin alone, indicating that about 30% of all transposition events occurred in introns of expressed genes and in-frame splicing took place. Five insertion sites of the FP gene trap transposons were identified. All of them mapped to introns of genes in different chromosomes, in the correct orientation. Our results suggest that FP can potentially target a large fraction of genes in the human genome.

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Resultado resumido

Contacto

Zoltan IVICS, (Group leader)
Tel.: +49-309-4062546
Fax: +49-309-4062547
Correo electrónico
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