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FP5

JUMPY Résumé de rapport

Project ID: QLG2-CT-2000-00821
Financé au titre de: FP5-LIFE QUALITY
Pays: France

Method of somatic gene transfer into the muscle of frogs

We developed somatic gene transfer (SGT) techniques applied to brain and muscle of Xenopus tropicalis tadpoles (Rowe et al. 2002. Dev. Dyn. 224, pp 381-390). so as to follow transcriptional regulations, protein function and cell fate within the constraints of an integrated in vivo system. Stable integration should extend gene expression and allow lineage and fate studies during and after metamorphosis. To this end, we used the transposon Sleeping Beauty with transitory expression of the transposase to insert the transgene in Xenopus genomic sequence.

- Visualisation of transposon/transposase events with fluorescent proteins. To follow the transposition event, we constructed a pRed-pTGFP molecule where a transposon containing the transgene CMV-GFP expressing the Green Fluorescent Protein (GFP) is inserted between another CMV promoter and the Red Fluorescent Protein (RFP).

Thus, after following transfection in presence of transposase activity, we observe a green fluorescence, and if there is an excision event the transposon is removed placing CMV in front of the RFP gene. Emission of red fluorescence allows one to identify cells in which excision (and possibly integration) has occurred. Red fluorescence was never seen in absence of transposase activity.

We used a combination of the transposon/transposase system using the pRed-pTGFP construct and the SGT technique to show that transposase-dependent insertion events can be detected in vivo after transfections. The following results were obtained:
-- Comparison of SGT in X. laevis and X. tropicalis, shows that the efficiency of excision event in tail muscle (increase in red fluorescence) is greater for X. laevis and furthermore is more rapid being detectable after 3 days whereas 15 days were required for X. tropicalis.

-- Transposase activity was introduced into the target tissue using RNA-SB (RNA transposase synthesized in vitro) or plasmid DNA (with the construct CMV-SB where the transposase is under the control of CMV promoter). In muscle, we show that excision is very efficient using RNA-SB and obtained 1 day after injection.

- Molecular characterisation of excision/insertion events. In order to characterise excision and integration events during transposition, another molecule was prepared. In this construct, the pRed-pTAmpiORI molecule, the transposon unit contains the Ampicillin resistance gene (Amp) and an origin of replication (ORI). This construct can be used to clone putative insertion in the genomic sequence after transposition, using a plasmid rescue method.

Using the two constructs, pRed-pTGFP and pRed-pTAmpiORI, the following results have been obtained:
-- To analyse genomic insertion events at the molecular level, we first tried a plasmid rescue method using the pRed-pTAmpiORI molecule. The construct was injected in presence of two kinds of transposase (RNA-SB or expression of RNA from plamsid CMV-SB), and genomic DNAs from different samples were analysed at 15 days post-injection. DNAs were EcoRI-digested, religated and transformed in bacteria, and 36 ampicillin resistant clones were sequenced (8 clones from pRed-pTAmpiORI + CMV-SB samples; 28 clones from pRed-pTAmpiORI + RNA-SB samples). Result shows that no positive clone could be found, i.e. no transposon/transposase dependent insertion event was found, only 2 random insertions in genomic sequence have been characterised with this method.

-- Then, the same DNA samples were tested with a more sensitive technique, the 5'-RACE PCR method. Genomic DNAs were EcoRI-digested, and a compatible adaptator was ligated at the free extremities. Thus, PCR and nested PCR using two couples of primer were performed to amplify specifically genomic sequence where the transposon is integrated in the Xenopus genome. However, for each sample tested no discrete band could be isolated and analysed.

-- Finally, we used LAM-PCR, (Schmidt et al 2001 Hum Gene Ther 12, ), a novel direct genomic sequencing technique for identification of vector insertion sites. We applied the LAM PCR on our different samples, either the pRed-pTAmpiORI or the pRed-pTGFP constructs in presence of RNA- or CMV-transposase). However all PCR bands sequenced were negative, representing only single primer products.

-- We also analysed genomic excision events at the molecular level, using PCR and nested PCR with specific primers taken outside of the transposon unit. We were unable to find the expected sequence signature of excision transposase-dependent event in our conditions.

It has not been possible to demonstrate that insertion of a transposon has occurred in the genome sequence of X. tropicalis or X. laevis species using the SGT technique to express transposon/transposase Sleeping Beauty system. These results have been submitted to Genesis for publication.

Contact

Barbara DEMENEIX, (Project Coordinator)
Tél.: +33-140-793607
Fax: +33-140-793618
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