Parameters influencing ectopic gene expression in Aplysia neurons.
DNA microinjection for expressing exogenous genes in Aplysia neurons has been very useful for analyzing not only functions of encoded proteins, but also regulations of gene expression in the nervous system. Some of the factors that affect expression of foreign genes microinjected into Aplysia neurons are described. The effect of the DNA form (supercoiled or linear) and promoter modification in the expression vectors are analyzed as well as buffer composition and DNA concentration in the microinjection solution. The time course of reporter gene expression was also monitored. Reporter gene expression was first detected as early as 1 h and maintained at a high level even until 7 days after microinjection. The presence of AP-1 enhancer in the promoter region of the expression vectors was essential in driving a high-level constitutive expression of reporter genes. Particularly, a pNEX derivative containing eight copies of AP-1 enhancer drove constitutive overexpression more effectively than ones harboring either four or 12 copies of AP-1 enhancer. We also found that a prokaryotic promoter/operator from E. coli lacZ gene placed upstream from an eukaryotic enhancer/promoter repressed the expression of the downstream reporter gene in Aplysia neurons.
Kaang
B K
BK
Department of Biology, Seoul National University, South Korea. kaang@alliant.snu.ac.kr
D000818:Animals; D001048:Aplysia / Q000502:physiology*; D004247:DNA / Q000494:pharmacology; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D008156:Luciferases; D008845:Microinjections; D008969:Molecular Sequence Data; D009420:Nervous System / Q000166:cytology; D009474:Neurons / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000235:genetics*; D001616:beta-Galactosidase
D018808:Transcription Factor AP-1; D004247:DNA; D008156:Luciferases; D001616:beta-Galactosidase