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Tol2-mediated gene and enhancer trapping in Xenopus tropicalis

热带爪蟾中 Tol2 介导的基因和增强子捕获

基本信息

批准号：
7826691
负责人：
PAUL E MEAD
金额：
$ 43.71万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-05 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7826691
关键词：
African Amphibia Animals Biological Sciences Breeding Cells DNA Data Development Developmental Biology Diploidy Embryo Enhancers Enzymes Event Explosion Expressed Sequence Tags Figs - dietary Frequencies Gene Expression Generations Genes Genetic Genome Germ Cells Germ Lines Goals Harvest Humulus Injection of therapeutic agent Insertional Mutagenesis Investigation Laboratories Lesion Libraries Limited Stage Longevity Manuscripts Mediating Medicine Messenger RNA Methods Modeling Modification Molecular Molecular Genetics Molecular Profiling Mutagenesis Mutagenicity Tests Organogenesis Pathway interactions Pattern Plasmids Polyadenylation Production RNA Splicing Rana Reagent Recovery Reporter Reporter Genes Resources Signal Transduction System Tadpoles Testing Time Tissues Toxic effect Transgenic Organisms Transposase Xenopus Xenopus laevis cell growth genetic manipulation genome sequencing improved insight interest male mutant novel plasmid DNA research study transmission process vector zygote

项目摘要

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.

自2008年提交修订申请和补充数据以来，我们在Tol 2项目上取得了重要进展。最重要的进展是证明了在重组后重新活化的Tol 2转座子的生殖系传播。在受精卵插入事件中注射编码Tol 2转座酶的mRNA（图1）。我们之前已经证明Tol 2转座子的体细胞再动员是非常有效的; 100%的注射胚胎在以下情况下具有新的重新整合事件：注射Tol 2转座酶mRNA。在这里，我们证明了再动员事件是通过生殖系，可以产生蝌蚪与新的GFP表达谱。图1描述了单个再动员的F2动物的分析（12M2r5）.在12 M2 r15中发现的四个再动员事件中，有三个整合到感兴趣的基因位点（jvh，cen，fol）。􀀁其他F2再动员青蛙的异交表明了新的再整合事件的种系传播， F3代中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阳性后代的显著偏差，所述预期的50%GFP阳性后代来自Tol 2XIG转座子杂合的再动员青蛙的异交。目前，我们正在表征的新的整合事件的后代从其他再动员的F2青蛙。我们预计很快就会提交一份描述这些数据的手稿。