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Fluorescent protein activation-based assay for transposon excision

Our aim was to use the transposon Sleeping Beauty with transitory expression of the transposase to insert the transgene into Xenopus genome on a somatic basis. To follow the transposition event, we constructed the 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 then giving a red fluorescence. Thus, emission of red fluorescence allows one to identify cells in which excision (and possibly integration) has occurred. Two other molecules that function on the same model as the pRed-pTGFP molecule, were designed as pRed-pTAmpiORI, used to identify stable insertion of a transposon in the genome sequence of X. tropicalis, and pLUC-pTGFP, used to quantify the efficiency of excision events in different conditions.

Figure (email the contact person for schema) : Schematic representation of constructs carrying the transposon Sleeping Beauty.

- To follow excision/transposition, we constructed the pRed-pTGFP plasmid 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) sequence. Following transfection in the absence of any transposase activity, transcription of the unmodified plasmid produces green fluorescence and no red signal (GFP+RFP-). However, after transfection in presence of transposase activity, if there is an excision event, the transposon is removed placing CMV in front of the RFP gene (new plasmid) resulting in transcription of both green fluorescence (GFP+) and red fluorescence (RFP+). Thus, emission of red fluorescence allows one to identify cells in which excision (and possibly integration) has occurred.

- Two others constructs were designed to identify transposition events, the pRed-pTAmpiORI molecule (top) and the pLUC -pTGFP molecule (bottom). Both function on the same model as the the pRed-pTGFP molecule. The pRed-pTAmpiORI construct was used to demonstrate that stable insertion of a transposon can occur in the genome sequence of X. tropicalis, from RFP positive cells that are obtained after an excision event. Presence of ampicilin selection (Ampi) and an origin of replication (ORI) allow the use of the plasmid rescue method to clone insertional events. The pLUC -pTGFP construct was used to quantify the efficiency of excision events following transfection performed in different conditions. Removal of the transposon after an excision event places the CMV promoter in front of the LUC gene, luciferase activity thus providing an indicator of the excision/transposition efficiency.

We used a combination of the transposon/transposase system using these constructs and the Somatic Gene Transfer technique to show that transposase-dependent insertion events can be detected in vivo after brain or muscle transfections.
A publication on this subject (Zincelle et al) has been submitted.

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