Cell Biology and Biotechniques

Question: Discuss about theCell Biology and Biotechniques. Answer: Literature Review on Application of Flow Cytometry and Transfection Technique on Functional Analysis of Crohns Disease Risk Gene LACC1 The review of the literature by Assadi et al. (2016) highlights how biophysical technology has been efficiently utilized in addressing research questions and finding effective results in clinical investigation. This research study mainly utilized the method of flow cytometry and transfection technique apart from real time PCR and immunoflourescence microscopy to detect fatty acid metabolism-immune nexus (FAMIN) in Chrohns disease risk gene LACC1 (Laccase domain containing 1) encoded protein in human tissues. This method of fatty acid metabolism was decided because genetic variation in the LACC1 gene increased the risk of Chrohns disease. The main of this research was to elucidate the biological function of LACC1 gene and determine the characterization of the gene expression in human tissue and cells. The research question for the study was how characteristics expression of LACC1 gene can increase risk of Crohns disease (Assadi et al. 2016). The FAMIN protein encoded by the LACC1 gene control all the defense mechanism of human body and the research was important to gain additional insights into how FAMIN characterization and LACC1 gene expression may create risk of Crohns disease. The transfection technique was used by maintaining human cell line THP-1 in suitable medium and the cells were incubated wit phorbol-12-myristate 13-acetate (PMA) to induce macrophage differentiation. For transfection experiment the HeLa cell line was transfected with empty vectors and LACC1 vector constructs. Western blotting was done on this cell and expression level was calculated. As transfection technique lead to unexpected morphologies and abnormalities in target cells, it helped in studying the expression of gene. On the other hand, immunofluoroscence staining was used to calculate the number of peroxisomes and several antibodies were used both for immunofluorescence staining and western blot (Assadi et al. 2016).Hence, transfection tech nique was applied in this research to study macrophage differentiation and gene expression of LACC1 gene. A research which considers the efficiency and cytotoxic effect of tranfection methodology lead to better results, whereas highly cytotoxic approaches lead to unwanted effects and unknown changes in gene expression which may affect the results (Goehring et al. 2014). Flow cytometry is the biophysical technology done by means of laser and used to analyze the characteristics of particle in a fluid stream by passing through electronic detection device. . Flow cytometry technique is widely used in the cell tracking, cell viability and infection studies and to conduct functional analysis in cell signaling gene expression studies (Givan 2013). In the research study by Assadi et al. (2016), flow cytometry of human blood derived cell and granulocytes and erythrocytes were isolated from the cell and stained using monoclonal antibodies. The FAMIN cells labeled with antibodies and data analyzed with flow cytometer and flow analysis software. This technique helped in the characterization of FAMIN expression in THP-1 cells. The result indicated the FAMIN expression was not detected in untreated cells, however PMA induced differentiation was seen during THP-1 (positive) cell lines. Hence the result of the study finally showed that LACC1 gene codes for macropha ge immune-metabolic function and polymorphic variation influences risk of Crohns disease (Assadi et al. 2016). Considering the clinical use of flow cytometry in research studies, the main advantage of flow cytometry is its ability to characterize immunophenotyping deviation in cells and detect diagnosis of hematopoietic disorders like leukemia. It has led to new methods for assessing hematologic malignancy and MRD. It is cost-effective and quick method for disease detection and diagnosis. However the limitation of the method is that it cannot easily detect sarcoma and carcinomas as they tend to stick (Woo et al. 2014). The main principle of fluorescence and fluorescence microscopic techniques was used to analyze therapeutic effectiveness in Crohns disease. In patients with Crohns disease, the antibody to tumor necrosis factor (TNF) binds to the membrane bound TNF and suppresses immune response in person. Hence, the researcher used fluorescent antibodies for TNF imaging studies. Despite using TNF therapy, patient show little improvement in their clinical symptoms and it also increases the risk of side-effects like allergic reaction and skin disorders. The main limitation in treatment was that clinician cannot predict the response to anti-TNF therapy in patients. With this problem in treatment of patients with Crohns disease, the research question for the study was to see how fluorescence antibodies to TNF can help in studying response to anti-TNF therapy (Atreya et al. 2014). Therefore, the use of different techniques like transfection, fluorescence microscopy and flow cytometry helped in studying variation in expression of Crohns disease risk gene and increased understanding of Crohns disease risk gene. Higher LACC1 expression was seen in spleen and lymph nodes in this research study. Hence application of this technique helped in gaining better understanding of LACC1 function in regulating immune-mediated function and diseases. These tools can be exploited in the future for several diagnostic studies. Reference Assadi, G., Vesterlund, L., Bonfiglio, F., Mazzurana, L., Cordeddu, L., Schepis, D., Mjsberg, J., Ruhrmann, S., Fabbri, A., Vukojevic, V. and Percipalle, P., 2016. Functional Analyses of the Crohns Disease Risk Gene LACC1.PloS one,11(12), p.e0168276. Atreya, R., Neumann, H., Neufert, C., Waldner, M.J., Billmeier, U., Zopf, Y., Willma, M., App, C., Mnster, T., Kessler, H. and Maas, S., 2014. In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn's disease.Nature medicine,20(3), pp.313-318. Givan, A.L., 2013.Flow cytometry: first principles. John Wiley Sons. Goehring, A., Lee, C.H., Wang, K.H., Michel, J.C., Claxton, D.P., Baconguis, I., Althoff, T., Fischer, S., Garcia, K.C. and Gouaux, E., 2014. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.Nature protocols,9(11), pp.2574-2585. Woo, J., Baumann, A. and Arguello, V., 2014. Recent advancements of flow cytometry: new applications in hematology and oncology.Expert review of molecular diagnostics,14(1), pp.67-81.