Date of Graduation

Fall 2010

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Colette Witkowski

Abstract

The main purpose of this study was development of Caenorhabdtis. elegans axenic culture and RNAi reagents for testing single-walled carbon nanotubes (SWNTs) as a delivery system for RNA. C. elegans offers great potential for such a study due to some of its characteristics and the rapid identification of gene knockdowns generated by RNA interference using standard siRNAs such as unc-22.Unc-22 is required in muscle for regulation of the actinomyosin contraction-relaxation cycle and for maintenance of normal muscle morphology. Decrease in unc-22 activity produces severe twitching and loss of function resulting in impaired mobility of the worms. RNAi plasmids, pPD34.09 and pPD128.117, provided in a microtiter plate by Andrew Fire's lab, were transformed into DH5α competent cells using the heat shock method and isolated using alkaline lysis. Unc-22 inserts carried by these plasmids were PCR amplified using M13 forward and reverse primers. RNAi products were generated at a concentration of 5 mg/mL by in vitro transcription of the amplified inserts. RNAi products can be used to functionalize SWNTs (f-SWNTs) using standard protocols. For the uptake of f-SWNTS, C. elegans cultures were synchronized at the L1 stage using sodium hypochlorite. The synchronization procedure was established and optimized. Axenic cultures were established in CeHR medium for five days and technical problems were studied. Test groups to study the uptake of SWNTs-RNAi hybrids were designed.

Keywords

C. elegans, single walled carbon nanotubes, RNAi, unc-22, pPD34.09, synchronization, axenic, CeHR medium

Subject Categories

Medical Molecular Biology

Copyright

© Lala Shashmi Jaiswal

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