Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease that may occur in two forms: sporadic and familial, the latter linked to a mutation in the Cu/Zn superoxide dismutase (SOD1) gene. Rodents expressing the human mutated form of SOD1 (hSOD1G93A) fail to faithfully reproduce the biology of the disease. Moreover, results of drug tests in mice have never been translated into humans. Aim of this work is to create a novel model for ALS, closer related to man, by overexpressing the hSOD1G93A cDNA in pig. We have previously developed an ubiquitous EGFP expression vector, driven by the pCAGGS hybrid promoter (CMV-IE enhancer ? chicken beta actin promoter) that maintains high level of expression through the next generation of pigs (Brunetti et al. 2008). We created a Destination Vector (pMGMARneoP-OrfA) inserting the Conversion cassette (OrfA) of Multisite Gateway system (Invitrogen) into the ubiquitous expression vector. This vector had the pCAGGS promoter inserted between two insulators (MAR of chicken lysozyme gene) to prevent different silencing effects (positional or copy number effects). On the same structure there is a ﬂoxed PGK-neo cassette to select the transfected clones but keeping the possibility to remove the selection cassette using the Cre recombinase. We initially inserted the hSOD1G93A cDNA into the Entry clone of Gateway system obtaining the pENTRL1L2-hSODG93A. The SalI-BamHI fragment of this construct was removed and the resulting pENTRL1L2-hSODG93AdelSB was veriﬁed by sequencing before using it in a LR exchange reaction with the Destination Vector pMG MARneoP-OrfA mediated by the LR Clonase. The obtained exchange reaction was used to transform chemically competent E.coli cells (One Shot Mach1-Invitrogen) and the resulting pMGMARneoP-CXhSODG93A vector was puriﬁed and analyzed by different restriction enzymes and conﬁrmed by sequencing. Speciﬁc PCR reaction and DIGlabelled probe have been developed and validated. The linearized expression vector, with the veriﬁed correct sequence is currently being transfected to immortalized PK15 pig cells. We have also screened by ICC an antibody (human anti-SOD1, Millipore) and found that it does not cross react with wild type pig cells and therefore it will be used to detect and quantify the hSOD1G93A expression in combination with RT-PCR, Northern Blot, Southern Blot, Western Blot. Data will be presented on the expression levels of hSOD1G93A in PK15 cells and in ﬁbroblasts that will be subsequently destined for SCNT to create the swine model.
- Transgenic pig