The use of transgenics in the laboratory axolotl

Abstract

The ability to generate transgenic animals sparked a wave of research committed to implementing such technology in a wide variety of model organisms. Building a solid base of ubiquitous and tissue-specific reporter lines has set the stage for later interrogations of individual cells or genetic elements. Compared to other widely used model organisms such as mice, zebrafish and fruit flies, there are only a few transgenic lines available in the laboratory axolotl (Ambystoma mexicanum), although their number is steadily expanding. In this review, we discuss a brief history of the transgenic methodologies in axolotl and their advantages and disadvantages. Next, we discuss available transgenic lines and insights we have been able to glean from them. Finally, we list challenges when developing transgenic axolotl, and where further work is needed in order to improve their standing as both a developmental and regenerative model.

Keywords: Ambystoma mexicanum; genome editing; laboratory axolotl; transgenics.

FIGURE 1

A typical example of a cassette for random transgenesis. The gene of interest (green) is driven by a gene regulatory element (promoter/enhancer) that allows either a ubiquitous or tissue-specific expression depending on the regulatory element (blue). The flanking I-Sce-I site or transposon element (eg, Tol2) facilitates integration of the cassette into the genome when co-injected with the I-Sce-I meganuclease or transposase (eg, Tol2 transposase), respectively

FIGURE 2

Methods for generating a transgenic animal using CRISPR/Cas9. A, Strategy to generate c-NHEJ dependent knock-out animal. Once a target genomic sequence (cyan) has been selected, guide RNA (gRNA) can be synthesized and co-injected with the CAS9 enzyme to generate a transgenic knock-out animal. B, Strategy to generate c-NHEJ-dependent knock-in animal. Injecting a plasmid that contains the same target genomic sequence followed by the gene of interest (green) alongside the gRNA and Cas9 enzyme will generate a transgenic knock-in animal. PAM (protospacer adjacent motif) (orange) sequences are required for a Cas9 nuclease to create double strand break. Indels (purple)

FIGURE 3

Effect of double ERT2 and single ERT2 sequence, and promoter activity on conversion efficiency in axolotl. Stereoscopic DIC images overlaid with fluorescence of, A-C, Prrx1:TFPnls-T2a-ERT2-Cre-ERT2; CAGGs:loxP-eGFP-loxP-Cherry, (double ERT2), D-F, Prrx1:TFPnls-T2A-ERT2-Cre; CAGGs:loxP-eGFP-loxPCherry (single ERT2) transgenic axolotl. A,B,D,E, Unamputated limb, A,D, before 4-OH-tamoxifen (4-OHT) treatment, B,E, 30 days after first 4-OHT treatment. C,F, 30 days post amputation (Amp.) regenerated limb. 4-OHT treatment was given during early stages (first treatment at 3 days post amputation) of blastema formation. 2 μM 4-OHT treatment was given three times by bathing on every alternate day. Converted cells (cherry), Scale bar: 500 μm

FIGURE 4

Schematic model of the IPTG/LacI system. A, Lac repressor binds to the lac operator and blocks movement of RNA polymerase II. B, In the presence of IPTG, the lac repressor fails to bind to the lac operator and allows RNA polymerase II mediated transcription of the gene of interest

FIGURE 5

A possible strategy to generate conditional point mutation knock-in using the Flex system. Using the CRISPR/Cas9 system the wild type allele is replaced by a targeting vector containing wild type exon2, point mutated exon2 and two sets of different lox sites (eg, loxP (blue) and lox2272 (grey)). Such a line could be combined with tissue-specific inducible Cre line to achieve spatiotemporal control of the mutant genotype. Upon 4-hydroxy tamoxifen treatment Cre activity causes two recombination events, leading to replacement of wild type exon2 by the point mutated exon2′. In step 1, the blue set of lox sites facing each other causes flipping of the flanked sequence. This leaves the grey lox sites with the same orientation causing the excision of the intervening sequence, containing the wt exon2

FIGURE 6

Possible strategies to perform cell ablation in a targeted cell type. A, A tissue specific promoter drives expression of a protein (eg, Caspase3) or a toxin (diphtheria toxin) that is responsible for cell death. In such strategy cell ablation occurs as soon as certain cell types are specified and expresses toxin. As an alternative strategy, B, receptor (diphtheria toxin receptor) that binds to toxin (diphtheria) or, C, enzyme (nitroreductase) that produces toxin upon drug (metronidazole) administration is expressed under tissue specific promoter. In such strategies, administration of a toxin or a drug is done at a timepoint when cell ablation is needed, thus providing temporal control over cell ablation. Tissue-specific promoter provides spatial control over cell ablation in all three strategies