Kentucky State University

Poster Title

Cellular Effect of RNAi-Mediated Knockdown of the Metastasis Suppressor Gene,SSeCKS

Institution

Kentucky State University

Abstract

The purpose of this project is to observe cell behavior before and after the SSeCKS (Src Suppressed C Kinase Substrate) levels are knocked-down. The process of RNA interference (RNAi) is used to down regulate the levels of SSeCKS in normal mouse embryo fibroblast (MEF) or in MEF deficient for FAK /focal adhesion kinase). The latter cells are used, because they typically have 5-10 times more SSeCKS. Five evaluations are performed in order to obtain the various ways the cells may respond to SSeCKS RNAi. A proliferation assay is used in which the cells are grown over a four-day period and everyday an absorbance reading is taken on the plates. From the absorbance readings a growth curve is produced showing whether or not SSeCKS levels may have any affect on the rate of cell growth. Counting the cells after they are confluent for two days will produce a saturation density graph. The morphology of the cell is observed by using a process called immunostaining. Immunostaining is the technique used to stain for the proteins, in our case Vinculin and F-Actin, two cytoskeleton proteins. In addition to looking for the proteins, the shape changes in the cells are to be observed. The wound scratch assay is used to see if SSeCKS levels played a role in cell movement. Lastly, the clonogenic assay is used to test for cell survival after growing for eight days.

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Cellular Effect of RNAi-Mediated Knockdown of the Metastasis Suppressor Gene,SSeCKS

The purpose of this project is to observe cell behavior before and after the SSeCKS (Src Suppressed C Kinase Substrate) levels are knocked-down. The process of RNA interference (RNAi) is used to down regulate the levels of SSeCKS in normal mouse embryo fibroblast (MEF) or in MEF deficient for FAK /focal adhesion kinase). The latter cells are used, because they typically have 5-10 times more SSeCKS. Five evaluations are performed in order to obtain the various ways the cells may respond to SSeCKS RNAi. A proliferation assay is used in which the cells are grown over a four-day period and everyday an absorbance reading is taken on the plates. From the absorbance readings a growth curve is produced showing whether or not SSeCKS levels may have any affect on the rate of cell growth. Counting the cells after they are confluent for two days will produce a saturation density graph. The morphology of the cell is observed by using a process called immunostaining. Immunostaining is the technique used to stain for the proteins, in our case Vinculin and F-Actin, two cytoskeleton proteins. In addition to looking for the proteins, the shape changes in the cells are to be observed. The wound scratch assay is used to see if SSeCKS levels played a role in cell movement. Lastly, the clonogenic assay is used to test for cell survival after growing for eight days.