A Rodent Model for Forelimb Neuromuscular Stimulation Based Movement Therapy
Prieto, Corina1,1,2; Sanchez, Jenny2; Lambert, Elizabeth2; Borovkov, Alex2; and Sykes, Kathryn2
1Department of Industrial Engineering, Arizona State University; 2Center for Innovations in Medicine, The Biodesign Institute at Arizona State University
Although pro- and eukaryotes use same protein translation code, the frequencies of a particular codon usage can be quite different. As a result, genes carrying high number of such codons are poorly expressed in a heterologous system. To overcome this problem a gene can be recoded by replacing all of its codons with their equivalents commonly used in the system where the gene is going to be expressed. Expression of such synthetic genes is significantly higher then their nature analogues, but they are difficult to assemble. Scientists at the Center for Innovation in Medicine developed a sequence normalization approach, which in addition to the codon optimization, removes repeats, and homopolymeric stretches and normalizes GC content of the gene. Synthetic genes designed by this new algorithm are easier, faster, and cheaper to assemble, but it is not clear yet if they are expressed as efficiently as those recoded by the traditional approach. To evaluate the new algorithm we used a Plasmodium falciparum merozoite surface protein (MSP) encoding gene as a model. In its natural form this gene (MSP-WT) is poorly expressed and weakly immunogenic in mice, but it all drastically change if a traditionally recoded synthetic gene is used instead (MSP-CAI). We constructed three variants of the gene: MSP-WT, MSP-CAI, and MSP-NEW, where the last one was designed by the new algorithm. After sequencing conformation the variants were cloned into a genetic immunization vector and used for ballistic immunization of mice by a gene gun. Sera samples were collected during and after completion of the immunization cycle and used in ELISA to determine titers of the specific antibodies raised against MSP. Results of this study are presented.