Foamy virus for efficient gene transfer in regeneration studies
© Khattak et al.; licensee BioMed Central Ltd. 2013
Received: 8 October 2012
Accepted: 26 April 2013
Published: 3 May 2013
Molecular 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.
We 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.
FV 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.
KeywordsFoamy virus Regeneration Salamander in vivo gene transfer
Limb regeneration of salamanders has fascinated scientists for several decades. Salamanders, Newts and Axolotls, are used as model animals in limb regeneration studies. Regeneration of missing structures is achieved by blastema, a pool of restricted progenitors that is formed after amputation .
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 .
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. For example, retroviral infection has been crucial for the molecular analysis of chicken limb development . A corresponding molecular analysis of limb regeneration in salamanders has been limited due to a paucity in gene transduction methods. Vaccinia and adenovirus have been used in limb regeneration studies, but their toxicity and non-integration phenotype limits their effectiveness [4, 5]. Similarly, pseudotyped retroviruses have been used in cultured cells [5–7] and a recent report showing their use in vivo but the issue of viral silencing after second round of regeneration was not investigated . We therefore sought a virus system that efficiently and stably infects salamander cells in vitro and in vivo and does not require pseudotyping and is not prone to silencing during initial and second round of regeneration.
Foamy viruses (FV) are a special type of retroviruses that are endemic to most non-human primates, horses, cattle and cats . They were only lately successfully introduced into the repertoire of vector systems for the correction of inherited diseases, in particular of the hematopoietic lineage, in the mammalian system [10, 11]. However, they have proven to be an efficient, non-toxic and stably integrating gene delivery vector system for mammalian cells. Some of their unique features, including their apparent apathogenicity, infectious particle-associated DNA genome, extremely broad host range as well as their efficient transduction capability for hematopoietic stem cells (HSC), make them one of the most promising tools for various gene therapeutic approaches .
One hallmark of FVs is their extremely broad tropism, including even very distantly related organisms as reptiles or birds [12, 13]. The nature of the broadly expressed, and potentially evolutionary conserved, cellular receptor(s) of FV glycoprotein-mediated attachment and entry remains poorly characterized. Though, several lines of evidence from recent publications suggest that proteoglycans and heparin sulfate function particularly as important attachment factors for FV Env-mediated host cell infection, additional uncharacterized molecules appear to be essential for fusion of viral and cellular lipid membranes during uptake of virions [14, 15]. Based on this feature we tested whether salamander cells would be transduced by FV vectors in vitro and in vivo. We also compared their transduction profile to that of lentiviral (LV) vectors pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-G), which has gained favor in gene delivery methods .
Results and discussion
To determine if viral transduction could be used to achieve cell type-specific expression, we inserted the CarAct:EGFP expression cassette (muscle specific promoter driving EGFP) into a FV vector and produced concentrated vector stocks as described in methods and materials. Subsequently, vector particles were injected in blastemas at day 5 and regeneration was allowed to occur. We observed EGFP expression only in the limb muscle fibers in the regenerated limb and not in any other cell type (Figure 5C-F). Therefore, foamy virus vector-mediated transgene integration results in faithful, cell-type restricted expression.
These results indicate that FV particles can infect blastema cells with stable and persistent expression during regeneration and after amputation. Furthermore, tissue specific expression is achieved when combined with tissue specific promoter. FV vectors have previously been shown to transduce cells from species very distantly related to their natural hosts [12, 13, 15]. Electroporation of plasmid DNA has been successfully established in salamanders but it cannot be used for experiments that require long-term gene expression as electroporated DNA is episomal and dilutes out during cell division. Similarly, establishing germline transgenic axolotls is a tedious process requiring 12-15 months (Khattak et al; Stem Cell Reports-in press). The results of this study show that FV vectors are an effective gene delivery tool for non-mammalian models, such as the salamander, resulting in long-term transgene expression. Interestingly, not only in vitro but also in vivo, their efficiency and the stability of transgene production was superior to the currently most favored retroviral vector system based on lentiviruses pseudotyped by the glycoprotein of the rhabdovirus VSV. Availability of foamy virus gene transfer system together with recently reported psuedotyped retrovirus  for regeneration research, such as the axolotl/salamander system, will greatly expand the ability to test gene function during regeneration.
We here report on the successful application of foamy virus as a vector for transgene integration and sustained expression during regeneration. Upon infection of the blastema, EGFP expression was observed in multiple cell types, and expression lasted throughout regeneration and persisted through a second round of regeneration. Both limb and tail blastema were transduced with foamy virus expressing EGFP/DsRed and resulted in wide range of cells labeled after limb/tail regeneration was complete. We also demonstrated the compatibility of this system with cell-type specific expression by incorporating the Xenopus cardiac actin promoter in the foamy virus vector. Our observation of widespread and stable expression with the foamy virus contrasts with the difficulty to achieve persistent in vivo transgene expression with concentrated lentivirus vectors.
Cells and culture conditions
The human kidney cell line 293T  and the human fibrosarcoma cell line HT1080  were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics. The newt myogenic cell line A1  was cultivated in 70% Eagles minimal essential media supplemented with 10% fetal bovine serum, insulin and penicillin/streptomycin.
For this study a replication-deficient expression-optimized 4-plasmid vector system based on prototype FV (PFV) recently developed by our laboratory was used  (Figure 1A). It comprises three packaging plasmids containing expression-optimized ORFs for the PFV capsid precursor protein Gag (pcoPG4) , the PFV enzymatic precursor protein Pol (pcoPP) as well as the PFV glycoprotein precursor Env (pcoPE)  and the transfer vector puc2MD9 Ubi WPRE . The transfer vector contains the minimal essential cis-acting viral sequences  that are required for vector RNA and enzyme (Pol) encapsidation as well as vDNA synthesis and its integration into the host cell genome mediated by the pol-encoded viral reverse transcriptase and integrase, respectively. With FVs having one of the largest viral genomes (~12 kb) the PFV transfer vector can accommodate up to 9 kb of non-viral genetic material . All packaging plasmids are based on the pczi expression vector, a pcDNA3.1+ variant containing an hCMV intron A sequence resulting in the expression of spliced mRNAs . Expression-optimization and gene synthesis of the PFV ORFs was performed at Geneart and enables production of vector supernatants with 5-10 fold higher titers than achieved with FV vector systems based on authentic viral sequences [22, 24, 25]. The puc2MD9 Ubi WPRE transfer vector used in this study is a variant of puc2MD9 published previously having the original SFFV U3 promoter driving EGFP or DsRed marker gene expression replaced by a human ubiquitin C promoter (hUbiC) . Furthermore the WPRE posttranscriptional active element was inserted downstream of the EGFP gene.
For generation of replication-deficient lentiviral vectors a 3-plasmid system was used (Figure 1B). It consists of two packaging plasmids, pCD/NL-BH , encoding HIV-1 Gag/Pol as well as some accessory proteins; pczVSV-G , encoding the vesicular stomatitis virus glycoprotein (VSV-G); and the HIV-1 transfer vector p6NST90 . p6NST90 is a variant of p6NST50  having the SFFV U3 driven IRES EGFP-Zeo expression cassette replaced by a hUbiC promoter driven EGFP/DsRed cassette. Details on the construction of the vectors are available on request.
Generation of viral supernatants
FV supernatants containing recombinant viral particles were generated essentially as described previously [25, 29]. Briefly, FV supernatants were produced by polyethyleneimine (PEI)-mediated cotransfection of 293T cells with transfer vector (e.g. puc2MD9 Ubi WPRE), Env- (pcoPE), Pol – (pcoPP), and Gag packaging plasmid (pcoPG4) at a ratio of 28:1:2:4 using 16 μg total DNA per 10-cm dish. At 32 h post transfection the medium was replaced, and cell-free viral supernatants were harvested by sterile filtration (0.45 μm pore size) an additional 16 h later. Supernatants were used directly or aliquots snap frozen on dry ice and stored at -80°C until further use. Lentiviral supernatants were generated by cotransfection of transfer vector (p6NST90), Gag/Pol packaging plasmid (pCD/NL-BH), and an Env packaging plasmid (pczVSV-G) at a ratio of 1:1:1 and harvested as described above. For in vivo transduction experiments vector particles were concentrated from cell-free viral supernatant by ultracentrifugation for 1.5 h at 25,500 rpm using a SW28 rotor (35 ml supernatant per bucket). Pelleted vector particles were gently resuspended on ice in PBS with 0.1 % BSA and aliquots were snap frozen and stored in – 80°C until use. Recovery of lentiviral VSV-G pseudotypes was generally above 70% and of PFV vector particles above 50%.
Analysis of in vitro and in vivo transduction efficiency
Infectious titers used for calculation of multiplicities of infection (MOI) were determined on HT1080 cells. In vitro transductions of recombinant EGFP expressing FV or HIV-1 vector particles were performed by infection of 2 × 104 HT1080 cells, plated 24 h in advance in 12-well plates or by infection of 2 × 104 A1 cells, plated 48 h in advance in 6-well plates. For the infection 1 ml (HT1080) or 1.5 ml (A1) of the viral supernatant or dilutions thereof were incubated with the target cells for 8 h. The percentage of EGFP-positive cells was determined by fluorescence-activated cell sorter (FACS) analysis at different time points after infection, as indicated. For double infections, DsRed and EGFP viruses were mixed before putting them on A1 cells as indicated above. All transduction experiments were performed in triplicates and at least three times.
For in vivo infections, 2-3 cm axolotl larvae were used. The animals were bred and raised in our colony and kept in local tap water at 18°C. The larvae were fed with freshly hatched brine shrimps (Artemia) every day. All animal procedures were done according to the animal and biological safety level 2 (S2) license (24D-9168.11-9-2006-1 and 55-8811.72/34-16) issued by “Regierungspräsidium Dresden” and “Sächsisches Staatsministerium für Umwelt und Landwirtschaft (SMUL)” respectively. The ethical approval was obtained from the "Landesdirektion" for animal welfare for the State of Saxony. Before every animal procedure, animals were anesthetized in 0.01% ethyl-p-aminobenzoate (benzocaine; Sigma). For each experiment, lower forelimbs or tails of nine animals were amputated and animals were returned to fresh water in separate boxes for blastema induction. After five days, 5 day blastemas were injected with 0.5 – 2 μl of concentrated foamy and/or lentivirus with help of glass capillaries (Harvard Apparatus) pulled by using a Sutter Flaming Brown P-97 puller to a tip size of approximately 1–2 μm and injected using PV830 Pneumatic PicoPump (WPI). The transduced animals were kept on a wet tissue for 20 min before putting them in fresh water, which was changed every second day. EGFP/DsRed fluorescence was first observed 7-8 days post infection. Amputation of limbs and detection of fluorescence was performed on Olympus SZX16 stereomicroscope. All transduction experiments were repeated three times.
Immunohistochemical analysis of transduced re-regenerated limb tissue
Regenerated limbs were amputated 28 days post infection to harvest the limb tissue. The cut part was fixed in 1% MEMFA for 4 hrs at RT followed by three washes of PBS and then incubated overnight at 4°C in 30% sucrose. The next day, tissue was embedded in Tissue-Tek (OCT compound-Sakura), cryosectioned and slides were allowed to dry at RT for 1-2 hrs. Limb and tail sections were washed 3 times with PBS-Tween (0.3%) and blocked for one hour with 2% BSA, 5% normal serum and 0.3% Triton-X (blocking solution), followed by overnight incubation with the primary antibody diluted in blocking solution. Primary antibodies used were anti-PAX7 mAB (Developmental Studies Hybridoma Bank), anti-MHC (monoclonal antibody 4A1025 kindly provided by S. Hughes), mouse anti-chicken collagen Type II antibody (Millipore) and monoclonal anti-Pan Cytokeratin antibody mixture (Sigma). After several washes with PBS-Tween, sections were then incubated for 1 hour with respective secondary antibodies and Hoechst solution for nuclear staining. Images were taken on Leica confocal microscope and Zeiss Observer.
We are grateful to Stephan Roy for the gift of the axolotl cell line, Heino Andreas for animal care and Maritta Schuez for technical assistance. This work was supported by funding from the Deutsche Forschungsgemeinschaft, TA274/3-1, TA274/4-1 (EMT), LI621/3-3 and LI621/6-1 (DL), the Max Planck Institute of Molecular Cell Biology and Genetics (EMT), and the Center for Regenerative Therapies, Dresden (EMT).
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