Pseudotyped retroviruses for infecting axolotl in vivo and in vitro

Abstract

SUMMARYAxolotls are poised to become the premiere model system for studying vertebrate appendage regeneration. However, very few molecular tools exist for studying crucial cell lineage relationships over regeneration or for robust and sustained misexpression of genetic elements to test their function. Furthermore, targeting specific cell types will be necessary to understand how regeneration of the diverse tissues within the limb is accomplished. We report that pseudotyped, replication-incompetent retroviruses can be used in axolotls to permanently express markers or genetic elements for functional study. These viruses, when modified by changing their coat protein, can infect axolotl cells only when they have been experimentally manipulated to express the receptor for that coat protein, thus allowing for the possibility of targeting specific cell types. Using viral vectors, we have found that progenitor populations for many different cell types within the blastema are present at all stages of limb regeneration, although their relative proportions change with time.

A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors

“…To test this, we used a replication-incompetent retrovirus (Whited et al, 2013) to constitutively overexpress kazald1 in regenerating limbs (Figure S6A). A majority (15/22) of kazald1 -infected limbs regenerated abnormally compared to control EGFP infections (0/18) (Figure S6B).…”

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). …”

“…Recently, several transgenic axolotl lines have been created to track cell fates and visualize the activation of signaling pathways during regeneration (Khattak et al, 2013a;Sobkow et al, 2006). The study of gene function in regeneration now benefits from several improvements in the delivery of exogenous DNA constructs into adult axolotl tissues (Khattak et al, 2013b;Whited et al, 2013); however, to date, no studies describe the targeted induction of mutations to perturb the function of endogenous genes. Such targeted loss-of-function mutations might enable the identification of genes that are essential for regeneration from the expanding lists of those specifically upregulated in regenerating tissues.…”

Advances in Decoding Axolotl Limb Regeneration

“…In the past eleven years, both transgenesis [4] and genome editing [5–8] have been successfully performed in axolotls in several laboratories. Retroviral infection, which results in genomic integration, has also been demonstrated in embryos and in larval and adult limbs [9, 10]. Localized genome editing has now been developed, allowing for study of mutant cells in vivo without necessarily waiting for homozygous loss-of-function mutants [10].…”