Generation of pre-implantation pig SCNT embryos harboring the amyotrophic lateral sclerosis related hSOD1G93A gene

Introduction. ALS is a fatal neurodegenerative disease that occurs in two forms: sporadic and familial, the latter linked to mutations in the SOD1 gene. Pathogenic hypotheses mainly rely on transgenic rodent models however doubts have been raised about their suitability to faithfully reproduce the human disease. Therefore, a more suitable model is strongly demanded to provide a better tool to study the disease and to facilitate the preclinical findings extrapolation. Swine model plays an emerging role in biomedical research as its anatomical, physiological and biochemical features are more closely related to human species. On this basis, our group produced, by Genetic Engineering and SCNT, transgenic swine blastocysts carrying the hSOD1G93A mutation, which is the most frequently studied in rodents, since it reproduces the ALS patients phenotype progression. Material and Methods. Using the Multisite Gateway System (Invitrogen) we obtained the “EntryClone” pENTRL1L2- hSODG93A, containing the cDNA coding the hSOD1G93A gene whose open reading frame was confirmed by sequencing, and the “DestinationVector” pMGOrfA5¢3’MARpuro5171. The exchange reaction between the “DestinationVector” and the “EntryClone” was mediated by LR Clonase-Invitrogen, and was used to transform chemically competent E.coli cells (OneShotMatch1-Invitrogen). The resulting “ExpressionVector” pMG5¢3’MARPuro-hSODG93A was purified and analyzed. The same construct was successfully used to develop an ubiquitous EGFP expression vector that maintains high expression level through the next generation of pigs. This vector carries the pCAGGS hybrid promoter (CMV-IE enhancer + chicken β-actin promoter) inserted between two insulators (5′MAR of chicken lysozyme gene) to prevent positional or copy number silencing effects. 5 µg of linearized vector were used to transfect 1 × 106 Pig Adult Fibroblast (PAF) cultured in DMEM/ M199[1:1] + 10%FCS+1% β-FGF by Nucleofector (Amaxa Biosystem). Transgene expression was detected by ICC using an anti-hSOD1 antibody ([1:200]07–403 Millipore) to select PAF colonies to be employed as donor cells. The endogenous expression level was evaluated in Huvec cells. SCNT-embryos were reconstructed following a zona-free method, as described previously. Transgenic hSOD1G93A-embryos were finally grown in vitro to assess morphology, development and survival. Results. PAFs showed a transgene expression level ranging from lower (about 0.5 times) to higher (about 3–4 times) than the endogenous one. Thirteen hSOD1G93A-PAF colonies carrying the complete range of expression levels were employed in eight SCNT experiments. A total of 1,630 oocytes were enucleated and 1430 SCNT-embryos were obtained. The average viable embryos percentage was 39.16% of the total manipulated oocytes. These results, consistent with those obtained from similar experiments conducted with other transgenes, have ruled out the hSOD1G93A lethality in the pre-implantation embryonic phases, regardless of its expression level. Conclusion. On the basis of these results the hSOD1G93Ablastocysts can be generated by SCNT. The next step will be to implant them in recipient sows to generate offspring. Taking into account the similarity in size and physiology of neuromuscolar system, a swine ALS model may represent a more suitable model in reproducing the human disease than current transgenic rodents.