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Transgenesis in Xenopus and AAV-ITRs 16714Transient Transgenesisin Xenopus laevis Facilitated by AAV-ITRsYuchang Fu, Donghui Kan, and Sylvia Evans1. IntroductionA common approach to the study of gene function in Xenopus embryos is toectopically express the gene of interest, or a mutant of that gene, by injectingeither RNA or DNA, encoding the gene into early-stage embryos. Both of theseapproaches have disadvantages (1). RNA injection results in little temporal orspatial control, as the RNA begins to be translated immediately followinginjection. In addition, RNA is unstable; therefore, transgene expression is relativelytransient. In principle, DNA injections could ameliorate these problems.DNA does not begin to be translated until embryonic stage 8, at the mid-blastulatransition, when zygotic genes begin to be expressed. Promoters can be utilized,which should enable more spatial and temporal control of gene expression,and DNA is more stable than RNA, prolonging the time frame in whichtransgenes can be expressed. However, for reasons that are not clear, expressionof DNA plasmids following direct injection into embryos is extremelymosaic, and tissue specificity of promoters is often lost.Recently, a method for the generation of stably transgenic frogs has beendeveloped (2), referred to as restriction enzyme-mediated integration (REMI).This method overcomes the shortcomings of transient transgene expression,enabling efficient and tissue-specific expression of DNAs following their integrationinto sperm DNA, and the subsequent fertilization of eggs by thetransgenic sperm nuclei. This method, however, is technically very demanding.We have recently developed an alternative, technically simpler, method forthe expression of DNA transgenes in Xenopus embryos, which also results inan increased efficiency of transgene expression and, at least in the case of onepromoter, tissue-specific transgene expression (3). This method involves theFrom: Methods in Molecular Biology, Vol. 127: Molecular Methods in Developmental Biology:Xenopus and Zebrafish Edited by: M. Guille © Humana Press Inc., Totowa, NJ167
168 Fu, Kan, and Evansconstruction of a vector DNA containing inverted terminal repeat sequences(ITRs) from an adeno-associated virus (AAV) (4) bracketing an expressioncassette. The resulting constructs are directly injected into Xenopusembryos utilizing standard techniques (see refs. 1 and 5; Chapters 10 and 12of this volume).We initially considered investigating the ability of AAV-ITRs to improvetransgene expression in Xenopus following reports that plasmid DNAs containingthese sequences (rAAV) exhibited enhanced and prolonged transgeneexpression in mammalian cells (6). Adeno-associated virus is a nonautonomousparvovirus requiring coinfection with another virus for productive infection ofcells and is a completely nonpathogenic integrating virus (7). AAV vectorshave been developed in which most of the viral genome is replaced by atransgenic expression cassette, retaining only the viral terminal repeatsequences of 145 nucleotides. The terminal 125 nucleotides of each ITR formpalindromic hairpin structures that are required for DNA replication, integration,and excision of proviral sequences following integration. Although rAAVplasmids are capable of integration, enhanced transgene expression does notappear to require integration (8).Our results in Xenopus embryos have demonstrated that inclusion of AAV-ITRs on DNA plasmids can significantly increase transgene expression from aubiquitous viral enhancer (see Fig. 1). With ITR constructs, up to 65% oftransgenic embryos expressed the reporter transgene in more than 50% ofdescendents of injected cells, whereas only 16% of embryos showed comparabletransgene expression when injected with non-ITR-containing plasmids.Up to 20% of ITR-plasmid-injected embryos expressed the transgenein 90–100% of the progeny of the injected cell, versus 3% with non-ITRcontrol plasmids. These efficiencies of transgene expression are comparableto those obtained with REMI (2).Increased transgene expression does not appear to be a reflection of differencesin DNA replication or stability, but rather appears to reflect, in part,improved segregation of plasmid DNA. Southern blot analysis provided littleevidence that significant integration of transgene DNA was occurring (3).In addition to enhancing expression of a transgene driven by a ubiquitouslyexpressed enhancer, the inclusion of ITRs enabled efficient and tissue-specificexpression of a reporter transgene driven by a striated muscle specific promoter, thatof the alpha cardiac actin gene (9). Ectopic expression of the transgene outside of thedomain of the endogenous gene was greatly reduced by the presence of the ITRs.The ability of the ITRs to enhance both ubiquitous and tissue-specifictransgene expression and to restrict non-tissue specific expression of the tissue-specificpromoter is reminiscent of the ability of matrix attachment sites toovercome position effects (10). In this regard, it is of interest to note that plas-
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