NE102 Final Lab Write-Up

This was to ensure the plasmids were made correctly

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This was to ensure the plasmids were made correctly with the right components. Figure four displays our results from the restriction digests. The bands are roughly the same sizes as what we expected them to be by comparison to the DNA ladder on the left. Figures five and six prove our hypothesis that Ras induces neuronal differentiation. In figure five, we have two images: one with no sign of differentiation after transfection of pGFP, and the other has a clear sign of neurite outgrowth after transfection of pRas-G12V and pGFP. Right below that figure, figure six compares the relative percentage of cells that differentiated when the two different expression plasmids were transfected. Clearly, a larger percentage of cells underwent differentiation when the mutant form of Ras was introduced into the cells. This shows how Ras plays a role PC12 cell differentiation. Figure seven shows the relative amounts of β-actin and Ras in cells following transcription. β-actin was used as a control and the figure illustrates that its levels stay constant. For Ras, on the other hand, the band on the right is a lot thicker than on the left. This means that relatively there is more Ras present in the cells that were transfected with the pRas-G12V plasmids, or simply, Ras was overexpressed in that case. Through the series of experiments and the final investigation, we were able to conclude that Egr1 is necessary for neuronal differentiation. Figure eight simply shows how cells did not differentiate when they were transfected with the dominant-negative, regardless of treatment with NGF. It also shows how the cells transfected with the vector
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did undergo cell differentiation. Figure nine more accurately displays our results from the experiment. Image (a) is our immunoblot for our control, β-actin and image (b) shows the results for our cells that were transfected. β-actin levels did not change, which is what we expected. As our control, β-actin was used to show that our transfections and ICC worked on the cells. However, we get more interesting results from the picture on the right. As labeled beneath the image, lane one represents the cells transfected with ZnEgr1 and treated with NGF while lane two represents the cells transfected with ZnEgr1 and not treated with NGF. Lanes three and four represent the cells transfected with the vector with and without NGF respectively. We see that lanes three and four have distinct bands representing the presence of NFT L. The band in lane three is larger indicating there is more NFT L. This is expected because those cells were treated with NGF, which induces cell differentiation. In lanes one and two, there are no indications of NFT L being present in the cells transfected with ZnEgr1. We correctly predicted this because the dominant negative protein for Egr1 inhibits its function. If Egr1 does not initiate transcription of NFT L, then it will not be present. Despite the treatment of NGF, neuronal differentiation did not occur in the cells that were transfected with ZnEgr1. Because the dominant negative protein inhibited Egr1, which stops the transcription of NFT L, we can conclude that Egr1 does play a necessary role in neuronal differentiation of PC12 cells.
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