The frog Xenopus
has been a valuable model for the elucidation of gene function during early vertebrate development due to the combined use of the cell lineage fate map, cell microinjection, and embryo manipulation (Warkman and Krieg, 2007
). Study of later embryonic stages is important for understanding organ development and metamorphic tissue remodeling (corresponding to events that occur during the fetal period in mammals), which may then help elucidate the developmental orgins of adult diseases (Blitz et al, 2006
). However, inducible methods for altering gene expression in later development are limited in frogs compared to other model organisms. Thus, the utility of Xenopus
as a developmental model would be greatly increased by further development of transgenic tools to manipulate post-embryonic gene expression.
Many post-embryological studies require simultaneous and precise temporal and spatial control of transgene expression. Binary transgene expression systems are capable of such controlled expression and are comprised of 1) a ligand-activated transgenic transcription factor that acts on 2) a cognate transgenic promoter controlling a gene of interest. These two components precisely regulate transgene expression by virtue of tissue-specific expression of the transgenic transcription factor activated by exogenous addition of the activating ligand. Two such binary methods have successfully expressed transgenes in a tissue-specific and inducible manner in frogs, the GAL4/UAS and Tet-On systems (Hartley et al, 2002
; Das and Brown, 2004
). The GAL4 transcripiton factor from yeast can bind and activate the transgenic UAS promoter. GAL4 can be controlled from ubiquitous (Hartley et al, 2002
; Das and Brown, 2004
) or tissue-specific promoters (Chae et al, 2002
). Inducibility has been achieved by fusing Gal4 to the progesterone (Chae et al, 2002
) or estrogen ((Das and Brown, 2004
) receptor ligand binding domain. Commonly used in flies, the tissue-specific inducible versions of the Gal4/UAS system in frogs have not gone beyond proof of principle. A third binary strategy using Cre/lox has been developed, where Cre is inducibly expressed with the ubiquitous heat shock inducible promoter (Roose et al, 2009
) or constitutively expressed from a muscle-specific promoter (Waldner et al, 2006
) and acts on a separate transgene with appropriately placed lox sites to activate that transgene.
The Tet-On system, used extensively in mice, has been used to delimit the timing of thyroid hormone influence in hind limb innervation (Das and Brown, 2004
), examine genes important for limb muscle development (Cai et al, 2007
), and detect gene switching during liver metamorphosis (Mukhi et al, 2010
). In the Tet-On system, the transgenic transcription factor rtTA (a re-engineered version of the bacterial Tet repressor fused to three copies of the minimal viral transactivation domain of VP16 to make a ligand-inducible transcriptional activator) regulates expression via a tetracycline-inducible promoter (seven copies of the bacterial tet operator upstream of a minimal CMV promoter) upon addition of the tetracycline mimic doxycycline (Dox).
Transgenic lines available from previous studies using the Tet-On system (Das and Brown, 2004
; Cai et al, 2007
) are of limited future use because the rtTA and TRE components are not separable, i.e., these components co-integrated into the same chromosomal position (the tet-inducible L-FABP:TRDN (Mukhi et al, 2010
) were not reared to adulthood). Thus, no Tet-On lines are available that can be flexibly applied to a wide variety of research questions, highlighting the need to characterize new Dox-inducible transgenic Xenopus
lines. Here, we characterize two transgenic Xenopus
lines incorporating the Tet-On system, one inducer line with ubiquitous expression of rtTA and one responder line for Dox-dependent expression of a dominant positive thyroid hormone receptor (Buchholz et al, 2004
). We examined expression induction characteristics to determine parameters important for overexpression studies. Development of these and future transgenic frog lines and depostion into the Xenopus
stock center will significantly augment the utility of Xenopus
as a model system by incorporating transgenic strategies into the major advantages of the Xenopus
model system for studies of development and regeneration.