Abstract
BackgroundMolecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes. We therefore sought an efficient integrating virus system that could be used to study limb and tail regeneration in salamanders.ResultsWe show that replication-deficient foamy virus (FV) vectors efficiently transduce cells in two different regeneration models in cell culture and in vivo. Injection of EGFP-expressing FV but not lentivirus vector particles into regenerating limbs and tail resulted in widespread expression that persisted throughout regeneration and reamputation pointing to the utility of FV for analyzing adult phenotypes in non-mammalian models. Furthermore, tissue specific transgene expression is achieved using FV vectors during limb regeneration.ConclusionsFV vectors are efficient mean of transferring genes into axolotl limb/tail and infection persists throughout regeneration and reamputation. This is a nontoxic method of delivering genes into axolotls in vivo/ in vitro and can potentially be applied to other salamander species.
Highlights
Molecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes
To test and compare transduction efficiencies of Foamy virus (FV) and Lentivirus (LV) vectors in salamanders, foamy virus (FV) and LV vector particles were generated by transient transfection of 293T cells with transfer vectors (PV, LV) containing an identical human ubiquitin C (UBIC) promoter driven EGFP marker gene cassette including a WPRE posttranscriptional element, and the corresponding packaging expression constructs (PE, PP, PG; LG/P, VG) (Figure 1A, B)
Target cells were exposed to vector particles at a titer of 1.74x106 IU/ml (FV and LV) for 8 hours with an multiplicities of infection (MOI) of 130 and analyzed in weekly intervals by fluorescence-activated cell sorter (FACS) analysis for the percentage of EGFP-positive cells (Figure 1C)
Summary
Molecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes. Salamanders, Newts and Axolotls, are used as model animals in limb regeneration studies. Electroporation of DNA is the fastest and efficient method to introduce exogenous transgenes into salamander limb and spinal cord in vivo but this expression is lost during regeneration as the electroporated DNA is episomal [2]. Infection of cells both in cell culture and in vivo by modified viruses has been a powerful means of expressing exogenous genes in various experimental systems.
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