Title page for ETD etd-02052010-153054


Type of Document Master's Thesis
Author Jenson, Lacey Jo
Author's Email Address ljenson@vt.edu
URN etd-02052010-153054
Title INDUCTION AND INHIBITION OF A NEURONAL PHENOTYPE IN SPODOPTERA FRUGIPERDA (SF21) INSECT CELLS
Degree Master of Science
Department Entomology
Advisory Committee
Advisor Name Title
Bloomquist, Jeffrey R. Committee Chair
Adelman, Zachary N. Committee Member
Paulson, Sally L. Committee Member
Keywords
  • Insecticides
  • Neuronal Phenotype
  • High-Throughput Screening
  • Insecticide Discovery
Date of Defense 2010-01-22
Availability restricted
Abstract
Due to the increasing resistance demonstrated by insects to conventional insecticides, the need for compounds with novel modes of action is becoming more urgent. Also, the discovery and production of new insecticides is vital as regulations and restrictions on conventional insecticides become increasingly stringent (Casida and Quistad 1998). Research in this area requires screening of many candidate compounds which is costly and time-consuming. The goal of this research was to produce in vitro insect neurons from Sf21 insect ovarian cell lines, which could lead to new high throughput screening methods and a way to mass produce insect material for basic research. This study used a culture of Sf21 cells and a mixture of differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, Sf21 cells began to express neuronal morphology, or the production of elongated, axon-like processes within 2-3 days. Maximal differentiation occurred when in the presence of 42 μM 20-HE or 10 μM insulin. Effects were maximal on day 2 for 20-E and day 3 for insulin. Insulin was more potent at day 2 for inducing differentiation (EC50 = 247 nM) than 20-HE (EC50 = 13 µM). In combination, 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system (CNS) stimulant, inhibited induction of elongated processes by 20-HE and/or insulin. Caffeine was a potent inhibitor of 42 µM 20-HE, with an IC50 of 9 nM, and the inhibition was incomplete, resulting in about one quarter of the differentiated cells remaining, even at high concentrations (up to 1 mM). The ability to induce a neural phenotype simplifies studies with of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined. If they are present, high throughput screening for new insecticides will be accelerated and made more economical by the utility of this method.
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