Inducible genetic system for the axolotl

Abstract

Transgenesis promises a powerful means for assessing gene function during amphibian limb regeneration. This approach is complicated, however, by the need for embryonic appendage development to proceed unimpeded despite the genetic alterations one wishes to test later in the context of regeneration. Achieving conditional gene regulation in this amphibian has not proved to be as straightforward as in many other systems. In this report we describe a unique method for obtaining temporal control over exogenous gene expression in the axolotl. Based on technology derived from the Escherichia coli Lac operon, uninduced transgenes are kept in a repressed state by the binding of constitutively expressed Lac repressor protein (LacI) to operator sequences within the expression construct. Addition of a lactose analog, IPTG, to the swimming water of the axolotl is sufficient for the sugar to be taken up by cells, where it binds the LacI protein, thereby inducing expression of the repressed gene. We use this system to demonstrate an in vivo role for thrombospondin-4 in limb regeneration. This inducible system will allow for systematic analysis of phenotypes at defined developmental or regenerative time points. The tight regulation and robustness of gene induction combined with the simplicity of this strategy will prove invaluable for studying many aspects of axolotl biology.

Pseudotyped retroviruses for infecting axolotl in vivo and in vitro

“…Hence, we injected single-cell axolotl embryos with this plasmid to generate transgenic F 0 animals. Though embryos were injected when they were single cells, transgenics show mosaic expression of transgenes (Monaghan and Maden, 2012;Sobkow et al, 2006;Whited et al, 2012) that is likely to be due to integration of the transgenic element into the genome after cell division has commenced; we observed a similar phenomenon. EGFP fluorescence was evident in embryos within 5-6 days post-injection.…”

“…In the absence of Cre, cells with the reporter only express EGFP. When co-transfected with a plasmid encoding a constitutively active Cre, AL1 cells and limb blastemas express tdTomato because the floxed EGFP-stop cassette has been eliminated through recombination at the LoxP sites (Whited et al, 2012). Al1 cells transfected with the Cre reporter plasmid were infected with a VSV-pseudotyped QCAG-Cre virus (titer 10 8 ) 3 days later.…”

“…Although the biological processes underlying this remarkable regenerative ability have been investigated for decades, a mechanistic understanding at a genetic or molecular level has remained elusive, owing in large part to a lack of modern tools that can be applied to salamanders (reviewed by Whited and Tabin, 2009). Transgenesis is possible (Sobkow et al, 2006) but is relatively inefficient, suffers from the long generation time of the axolotl, and has only recently been coupled with any induction system (Whited et al, 2012) or tissue-specific promoters (Monaghan and Maden, 2012). Electroporation is currently the predominant means with which to mis-express genetic elements in salamanders (Berg et al, 2010;Echeverri and Tanaka, 2002;Echeverri and Tanaka, 2003;Echeverri and Tanaka, 2005;Kumar et al, 2007;Mercader et al, 2005), yet this method has drawbacks, such as dilution of plasmid with cell division, and the procedure itself has been shown to cause some degree of cellular dedifferentiation in newts (Atkinson et al, 2006) akin to the dedifferentiation that is a hallmark of limb regeneration.…”

Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

“…Although off-target mutations can be selected against by outcrossing mutant lines for several generations, the time frame required by this approach is not practical for experiments with an organism with a long generation time. Thus, phenotypes should be confirmed in mutants created with sgRNAs directed against different targets within the same gene, and, when possible, rescue experiments should be conducted using recent innovations in exogenous gene delivery and inducible gene expression in the axolotl (Khattak et al, 2013b;Whited et al, 2012Whited et al, , 2013). …”

Mapping hematopoiesis in a fully regenerative vertebrate: the axolotl

“…This suggests that adult regeneration may be an acquired trait in axolotls that might be inducible in mammals if the required genetic pathways are mapped. Recent advances in production of transgenic axolotls, [6][7][8][9][10] complete mapping of the axolotl transcriptome, 11 and production of gene expression arrays [12][13][14][15][16] finally allow molecular mapping of regeneration pathways. Additionally, given the extensive conservation of synteny between axolotls and humans, this amphibian may be a powerful genetic model to study hematopoietic cell function in scar-free wound healing and tissue regeneration.…”