Expression of human amyloid precursor proteins in cultured neuronal cells through the use of HSV-1 defective vectors.
Postmortem investigations of Alzheimer's patients reveal senile plaques that contain, among other molecules, deposits of beta-amyloid protein. The role of the beta-amyloid deposits remains unclear but identification of mutations in the amyloid precursor protein (APP) gene within the beta-amyloid portion in hereditary forms of the disease provide evidence that these deposits are involved in the pathological state. To more fully investigate this hypothesis attempts have been made to create transgenic mice to overexpress the beta-amyloid protein but these models have not been successful in modeling the disease. We have chosen to utilize the HSV-1 defective vector system which allows the expression of experimental genes in neuronal cells to overexpress APPC100. We have cloned the rat tyrosine hydroxylase (TH) promoter into a defective HSV plasmid. Cloning the firefly luciferase gene under the control of the TH promoter (demonstrates that the promoter is active after infection of human SY5Y cells or rat PC12 cells. A synthetic APP cDNA which represents the last 100 amino acids of the carboxy terminus of APP including the beta-amyloid protein was synthesized and inserted under the control of the TH promoter. Infection and subsequent nuclease protection assays demonstrate expression of the synthetic gene in the infected cells. Current research focuses on detection of the expressed protein within the infected cells and determination of the time period for continued expression.
Biotechnology Department, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, MI 48105, USA.
R J
RJ
Wyborski
C A
CA
Goble
J
J
Scholten
D000682:Amyloid / Q000235:genetics*; D000818:Animals; D002522:Chlorocebus aethiops; D003673:Defective Viruses / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D016716:PC12 Cells; D010957:Plasmids; D000072002:Prion Proteins; D011328:Prions; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics*; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics
D000682:Amyloid; C512656:PRNP protein, human; D000072002:Prion Proteins; D011328:Prions; D011498:Protein Precursors; D008156:Luciferases; D014446:Tyrosine 3-Monooxygenase