We have made important progress on the Tol2 project since the revised application and the supplemental data were submitted in 2008. The most significant advance is the demonstration of germ line transmission of remobilized Tol2 transposons following the injection of mRNA encoding Tol2 transposase in fertilized eggs insertion event (Fig. 1). We had previously shown that somatic remobilization of Tol2 transposons is very efficient;100% of injected embryos have novel reintegration events following injection of Tol2 transposase mRNA. Here, we demonstrate that the remobilization events are passed through the germ line and can produce tadpoles with novel GFP expression profiles. Figure 1 depicts the analysis of a single remobilized F2 animal (12M2r??5). Three of the four remobilization events found in 12M2r??5 have integrated into interesting gene loci (jvh, cen, fol). Outcross of other F2 remobilized frogs has indicated germ line transmission of novel re-integration events highlighted by the non-Mendelian frequencies of GFP-positive tadpoles in the F3 generation;that is, we observed a significant deviation from the expected 50% GFP-positive progeny from the outcross of remobilized frogs heterozygous for the Tol2XIG transposon. We are currently characterizing the novel integration events in the progeny from the other remobilized F2 frogs. We anticipate submitting a manuscript describing this data soon.
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