br Problem setting PS M V

Problem setting PS1-M146V, a presenilin model implying the presence of a mutation in the gene encoding the presenilin protein [7]; APPKI, an amyloid model implying the presence of a mutation in the gene encoding APP [9]. Recently, our laboratory has developed one more model which, in ampa with the previous ones, is cellular, named the low amyloid toxicity model [8].
Test subjects and experimental methods Mice. The 5×FAD (5 Familial Alzheimer\'s Disease, MMRC, # 34840) model is a line of transgenic mice with five mutations: three in the genes of the human APP protein (Swedish (K670N/M671L), Florida (I716V), London (V717I)), and two in the presenilin 1 genes (M146L, L286V). The mice were kept in the vivarium of the Molecular Neurodegeneration Laboratory (Peter the Great St. Petersburg Polytechnic University) with a 12-hour light cycle. Viruses. We used type 2 adeno-associated viruses (AAV2 /1) in the study. The first virus, AAV2/1CMVSTIM2WThrGFP, is a type 2 adeno-associated virus (AAV2 stands for adeno-associated dependoparvovirus 2) carrying the mouse STIM2 gene conjugated to the green fluorescent protein (GFP), with a titer of 1013 viral particles per ml. The adeno-associated virus cannot replicate independently but can integrate its genome into the host genome. AAV2 is strictly neuron-specific. This virus was obtained in the Gene Transfer Vector Core facility of the Iowa State University (Ames, USA) in a baculovirus expression system. When injected, it causes STIM2 overexpression. The second virus, AAV2/1CMVNLS-GFP, is a type 2 adeno-associated virus carrying the GFP gene with a titer of 1013 viral particles per ml. NLS (Nuclear Localization Signal) indicates that the GFP protein is expressed only in the neuronal nucleus. This virus was designed by the same facility as the first one, and was used as control in this study. Mice genotyping. The presence of the transgene in 5×FAD mice was checked using a genotyping technique. The necessary DNA material was obtained from the tip of the animal\'s tail. The tip was then placed in 125 µl of a SNET buffer (20 mM Tris–HCl, 5 mM EDTA, 400 mM NaCl, 1% SDS (pH 8)) with an addition of 10 µl of K proteinase dissolved in water at a concentration of 10 mg/ml and incubated at a temperature of 55 °C for 2 to 16 h. Next 160 µl of phenol/chloroform/isoamyl alcohol was added to the reaction mixture in a ratio of 25:24:1. The content of the test tube was mixed thoroughly and centrifuged at 14,000 rpm for 10 min at room temperature. The upper fraction containing the DNA was transferred to a clean tube and stored at a temperature of +4 °C. The presence of the transgene was checked by polymerase chain reaction (PCR) using primers specific to the insertion causing the expression of the transgenic (human) genes PSEN1 and APP in individual brain neurons. We used the following primers: The primer pair for determining the PSEN1 transgene and(flanking the DNA region with the length of 600 nucleotide base pairs); The primer pair for determining the APP transgene and (flanking the DNA region with the length of 350 nucleotide base pairs); The control primer pair and (flanking the DNA region with the length of 324 nucleotide base pairs). Preparation of hippocampal lysates. Mice aged 4 and 6 months were transcardially perfused with cold phosphate buffer at a 3 ml/min rate for 10 min. Prior to this, the mouse was anesthetized with 200 µl of urethane solution (Sigma, U2500-250G) in normal saline (0.9% NaCl), with a concentration of 300 mg/ml. The hippocampus was removed, homogenized and lysed in a buffer with the following chemical composition, mM: NaCl 137, KCl 2.7, Na2HPO4 4.3, KH2PO4 1.4, 5.0 EDTA, EGTA 5.0, 1.0 PMSF, NaF 50, Na3VO4 1.0 (pH 7.2); 1% CHAPS, inhibitors of proteases (1 tablet per 100 ml of lysis buffer, SIGMAFAST Protease Inhibitor tablets, Sigma, S8820-20TAB) and phosphatases (200 µl per 100 ml of lysis buffer, Phosphatase Inhibitor Cocktail, Sigma, P0044-5ML).