Hydrodynamics-Based Transfer of Human Apolipoprotein A-I Gene to Mice: Study of Factors Affecting the Efficiency and Duration of Gene Expression in the Mouse Liver

Human apolipoprotein A-I gene (apoA-I) inserted into a plasmid expression vector was transferred in vivo into C57Bl/6 mice using hydrodynamic injections into the tail vein. Two types of plasmid expression vectors were used: (1) pCMVcapoAI which contained cDNA of apoA-I driven by the human cytomegalovirus (CMV) early gene promoter and (2) pAlg, which contained a genomic locus of intron-containing apoA-I driven by its own extended 5-regulatory region (APOAI). Hydrodynamic intravenous injections of both expression vectors led to the appearance of human apoA-I mRNA in the liver and human ApoA-I protein in the serum of injected mice. The dynamics of human ApoA-I content in the sera of mice injected with pCMVcapoAI and pAlg were different. When pCMVcapoAI was used, the concentration of human ApoA-I in mouse serum was maximal one day after injection and decreased to zero within the next two weeks. In the case of pAlg, the content of human ApoA-I in serum was maximal (up to 20 g/ml) on days 5–7 after injection and then gradually decreased for several months (six months after injection, for example, it decreased to 25% of the maximal value). Experiments on saved pAlg plasmid isolated from the nuclei of hepatocytes 50 days after injection showed that the plasmid was retained for a long time in the form of an episome. A significant content of human ApoA-I in serum and its long-term persistence after injecting mice with pAlg may be accounted for by the properties of APOAI and/or the exon–intron structure of the apoA-I gene. Injecting mice with different variants of APOAI coupled with the luciferase gene did not lead to long-term expression of luciferase in the liver. It is concluded that the presence of introns in the apoA-I gene is required for its efficient and long-term expression after transfer to mice by means of hydrodynamic injections.     

Response of the afferent vagal fibers to lipopolysaccharides of gram-negative bacteria administered intrapulmonarily: An experimental model

Responses of the cervical afferent vagal fibers were studied in rats subjected to subdiaphragmatic vagotomy and administered intrapulmonarily with endotoxins of gram-negative bacteria. It was shown that 50-μg doses of lipopolysaccharides (LPSs) instilled into the rat lungs affected the activity of sensitive vagal fibers. The LPSs of Pseudomonas aeruginosa or Klebsiella pneumoniae produced a long-lasting increase in the frequency of afferent impulses, whereas the LPSs from Escherichia coli decreased it during the entire 100-min period of observation. Apyrogenic saline solution administered in control tests produced no considerable changes in the tonic activity of sensitive fibers. The endotoxins increased the respiratory rate and decreased the heart rate. The results experimentally support the idea that some components of the cell walls of gram-negative bacteria can affect both the activity of the afferent bronchopulmonary vagal fibers and intersystem interactions in the body.