• Apoliprotein A-I
• Apolipoprotein A-I Milano
• multicistronic vectors
• 2A peptides

Apolipoprotein A-I (ApoA-I), encoded by the APOA1 gene, is the main structural and functional constituent of High Density Lipoproteins (HDL). Its principal role is in reverse cholesterol transport from peripheral tissues to liver for catabolism. Epidemiological evidence has correlated high levels of HDL in plasma and general positive outcomes not only in cardiovascular disease, field where traditionally the protein role has been studied, but also in other pathological conditions belonging to metabolic and inflammatory disorders. Many in vitro and in vivo studies have then pointed out the anti-atherosclerotic, anti-inflammatory and antioxidant properties of ApoA-I, making it an attractive therapeutic molecule. In this context, there is a strong interest in a natural variant of the gene encoding for a protein known as ApoA-I Milano (AIM) which shows increased beneficial effects as compared to the wild type protein. Despite the knowledge about some pathways and enzymes involved in the processes, many details about molecular mechanisms that lead to the ApoA-I protective effect are still unclear, especially those underlying the differences in variants effectiveness. The aim of this thesis is to design and construct a tool to study those in vitro molecular mechanisms underlying the protective effect. In detail, the tool that has been designed and developed consists of a set of multicistronic vectors and appropriate control plasmids in order to allow overexpression of ApoA-I or AIM in eukaryotic cells. Multicistronic vectors allow the expression of two or more proteins from a single transcript, which is a desirable situation in applications that require simultaneous expression of multiple proteins. The system here used exploits the 2A peptides, self-cleaving oligopeptides of about 20 amino acids of viral origin, whose coding sequence is inserted between coding sequences of proteins to be expressed (without stop codon), obtaining a single transcript. During the translation, a cleavage occurs between two amino acids in 2A peptides resulting in the release from the ribosome of the protein product, which maintains the N-terminus region of 2A. The C-terminus region remains at the start of the downstream protein.
Multiple proteins in equimolar quantities are obtained. A common application is the expression of the protein of interest along with a reporter gene to quickly and easily detect it, avoiding fusion proteins that could affect folding and functionality of the studied protein. The series of plasmid we produced presents EGFP as reporter gene, in bicistronic and tricistronic vectors containing ApoA-I or AIM coding sequence, under the transcriptional control of a strong promoter. As first step, site directed mutagenesis was performed to introduce the Milano mutation in the ApoA-I cDNA sequence, through PCR with a primer carrying the mutation. Bicistronic plasmids containing ApoA-I or AIM upstream and EGFP downstream were produced using Gibson Assembly, a cloning method which exploits overlapping ends of adjacent fragments added by PCR. With the same cloning system we produced plasmids carrying only AIM, ApoA-I, F2A or EGFP, as control plasmids. We also designed “inverted” plasmids, bicistronic plasmids containing EGFP upstream of 2A sequence, and AIM or ApoA-I downstream, in order to be able to assess the possible interference of 2A residues at N- or C-terminus of the protein, and as intermediate plasmids for the construction of other plasmids. Again, these were produced by Gibson Assembly. Tricistronic vectors were finally assembled, containing two copies of ApoA-I or AIM downstream of EGFP. As the repetitions of the sequence make impossible to use Gibson Assembly, these plasmids were obtained by digestion with restriction enzyme of inverted bicistronic plasmids and ligation of appropriate insert. With the same strategy we added a third AIM copy, successfully obtaining a tetracistronic vector. Validations by means of restriction analysis, PCR and sequencing confirmed the right insertion and sequence in each plasmid. Use of this series of vectors will allow to perform different kind of studies that could provide new insight about ApoA-I and AIM protective effects.