Tol2-mediated gene and enhancer trapping in Xenopus tropicalis

自2008年提交修订后的申请和补充数据以来，我们在Tol2项目上取得了重要进展。最重大的进展是展示了可再动员的Tol2转座子的生殖系传递 在受精卵插入事件中注射编码Tol2转座酶的mRNA(图1)。我们之前已经证明了Tol2转座子的体细胞再动员是非常有效的；100%的注射胚胎有新的重新融合事件如下 注射Tol2转座酶基因。在这里，我们证明了再动员事件通过生殖系，并可以产生具有新的GFP表达谱的蝌蚪。图1描述了对单个可再动员的F2动物的分析 (12M2r􀀁5)。在12M2r􀀁5中发现的四个再动员事件中，有三个已整合到感兴趣的基因座(jvh、cen、fo1)。其他F2可重新整合青蛙的异交表明了新的重新整合事件的胚系传递，这一点由 F3代GFP阳性蝌蚪的非孟德尔频率；也就是说，我们观察到与预期的50%GFP阳性后代和Tol2XIG转座子杂合体的可再动员青蛙的异交有显著差异。我们目前正在鉴定来自其他可再动员的F2蛙的后代中的新的整合事件。我们预计很快就会提交一份描述这些数据的手稿。

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.