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Assessing the generalizability of FRAP by comparing results from two confocal microscopes

Bree, Fabian de (2021) Assessing the generalizability of FRAP by comparing results from two confocal microscopes.

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Abstract:SOX9 is the master transcription factor for cartilage formation. SOX9 is, just like other DNA binding transcription factors, highly mobile when it is not bound to DNA. However, this mobility will decrease when a transcription factor binds to DNA, thereby being able to regulate gene expression. SOX9 activity may be regulated via various pathways and cytokines. For example, BMP7 is an anabolic cytokine that can stimulate cartilage formation and upregulate SOX9 gene expression, and previous work in the DBE group has shown that BMP7 also regulates SOX9 activity at the protein activity level. In one of those previous studies it has been shown with a Nikon A1 confocal microscope that the changes in SOX9 mobility can be measured with Fluorescence Recovery After Photobleaching (FRAP) and can be related to SOX9 activity. It has also been shown that BMP7 promotes SOX9 activity. However, there are multiple variables that can influence the FRAP results. In this research, C20/A4 cells were treated with BMP7 and the mobility of SOX9 was studied. The purpose of this study was to compare the results from the Nikon with a Zeiss confocal microscope to study whether FRAP is a generally applicable method for studying protein dynamics, and to develop a protocol for FRAP experiments on the Zeiss microscope. The results show significant differences between the data obtained on the Zeiss and Nikon microscopes. Based on the Zeiss data, BMP7 has an insignificant inhibiting effect on SOX9. There are also differences between data processing with MATLAB and the Zeiss Zen software. It was expected that the FRAP rates between the Nikon and Zeiss would be similar, but differences in these rates were observed. Other challenges include a relatively low transfection efficiency and changes in the MATLAB scripts. Due to the large number of variables in this study it is inconclusive if FRAP is a generalizable method.
Item Type:Essay (Bachelor)
Faculty:TNW: Science and Technology
Subject:42 biology
Programme:Biomedical Technology BSc (56226)
Link to this item:http://purl.utwente.nl/essays/88555
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