University of Twente Student Theses
Optimization of Nucleofection protocol for LX-2 cell line
Boelens, Boelo (2024) Optimization of Nucleofection protocol for LX-2 cell line.
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| Abstract: | From liver diseases, nonalcoholic steatohepatitis (NASH) is a disease that affects 5.3% of the people in the world. Patients with NASH have inflammation in the liver which is also known as fibrosis which can be chronic and lead to the development of hepatic cirrhosis. To treat patients with fibrosis, studies are ongoing to develop antifibrotic therapies to inhibit fibrosis. In these studies, the focus is on developing autotaxin inhibitors to reduce fibrosis. The enzyme autotaxin plays an important role in the production of LPA which is an extracellular signaling molecule. LPA can bind as an extracellular molecule to LPA1-3 receptors while LPA4-6 receptors preferably be activated by LPA with the guidance of ATX. Since the LPA receptors are involved in fibrosis, it is desirable to investigate the difference between these receptors in hepatic fibrosis using human hepatic stellate cells (HSCs) cell line LX-2 with knock-out of LPA receptors. To create this knock-out cell line, an optimized Nucleofection® protocol is needed to transfect the LX-2 cells with CRISPR/Cas9. Here the Nucleofector® programs EW-113 and CA-137 are compared to each other based on transfection efficiency and cell viability. For Nucleofection® experiment 150.000 cells were needed. To get the least cell loss after centrifugation, different centrifuge programs (90xg for 10 min, 240xg for 3 min, and 300xg for 5 min) were investigated. From the results of experiments, there were no significant differences between centrifuge programs in terms of least cell loss after centrifuge. From the results of transfection efficiency and cell viability, the program CA-137 is the most suitable program to have the highest cell viability combined with sufficient high transfection efficiency for Nucleofection® of LX-2 cells with CRISPR/Cas9. |
| Item Type: | Essay (Bachelor) |
| Faculty: | TNW: Science and Technology |
| Subject: | 42 biology, 50 technical science in general |
| Programme: | Biomedical Technology BSc (56226) |
| Link to this item: | https://purl.utwente.nl/essays/101767 |
| Export this item as: | BibTeX EndNote HTML Citation Reference Manager |
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