Injection-mediated transposon transgenesis in Xenopus tropicalis and the identification of integration sites by modified extension primer tag selection (EPTS) linker-mediated PCR.
The generation of transgenic lines is vital to many genetic strategies and provides useful reagents for cell labeling and lineage-tracing experiments. Transposon-based systems offer simple, yet robust, platforms for transgenesis in the frog. Here, we provide a protocol for a microinjection-based transposon transgenesis method using a 'natural breeding' strategy for the collection of Xenopus tropicalis embryos. This method uses co-injection of a plasmid containing a transposon substrate together with synthetic mRNA encoding the transposase to achieve efficient integration of the transgene in the frog genome. We also describe a modified extension primer tag selection linker-mediated PCR technique to identify transposon integration sites within the host genome. This cloning strategy allows rapid identification of genomic sequences flanking the integration sites and multiple independently segregating transposon integration events in a single tadpole can be cloned simultaneously.
Yergeau
Donald A
DA
Department of Pathology, St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, Tennessee 38105, USA.
Kuliyev
Emin
E
Mead
Paul E
PE
D000818:Animals; D030801:Animals, Genetically Modified; D003001:Cloning, Molecular; D017931:DNA Primers; D004251:DNA Transposable Elements; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D008845:Microinjections; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger; D019076:Transgenes; D019895:Transposases / Q000235:genetics; D014981:Xenopus / Q000196:embryology / Q000235:genetics*
D017931:DNA Primers; D004251:DNA Transposable Elements; D012333:RNA, Messenger; D019895:Transposases