Danielle Robinson Deletion of IFT20 in mammalian cells.

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Information about Danielle Robinson Deletion of IFT20 in mammalian cells.

Published on June 8, 2016

Author: DanielleRobinson34

Source: slideshare.net

1. The 2010 Summer Research Fellowship Program at UMass Medical School Website: http://www.umassmed.edu/summer Deletion of IFT20 in Cultured Mammalian Cells Robinson, Danielle., Keady, Brian., and Pazour, Greg. Abstract Primary cilia are located in many tissues throughout the body and are essential for converting an array of sensory stimuli , such as hormones, into signals for proper mammalian development. In order for cilia to function correctly, the receptors within the cilium must be localized appropriately. Defects in cilia lead to a broad spectrum of diseases such as polydactyly, obesity, and polycystic kidney disease. Intraflagellar transport protein 20 (IFT20), is a subunit of the IFT particle which is required for assembly and maintenance of cilia. IFT20 is normally localized to both the Golgi and cilia, and thought to be involved in ciliary trafficking of membrane proteins. To further study the function of IFT20, mice were previously generated that carried a floxed allele of IFT20 and a tamoxifen responsive Cre. Here we have developed a method to delete IFT20 in cell culture in order to better understand the role of IFT20 in ciliary membrane trafficking. To visualize the deletion of IFT20, we used immunofluorescence, western blot, and PCR. Within 48 hours of tamoxifen treatment we observe successful conversion of the floxed allele into the null allele and subsequent decrease in protein levels by western blot analyses and immunofluoresecence. This cell culture model will be used in the future to study trafficking of ciliary membrane proteins like polycystin. Experimental Design Results Conclusions Primary cilia play an important role in the development of mammals . Defects in the cilia or the machinery required to assemble them leads to a broad spectrum of human disease symptoms, including polycystic kidney disease, polydactyly, obesity and etc(Marshall et al., 2006). Intraflagellar transport (IFT), is the cellular process for assembling and maintaining cilia, and is carried out by a multi-subunit protein complex known as the IFT particle . Intraflagellar transport protein 20 (IFT20) , is a subunit of the IFT particle and is also involved in controlling Wnt signaling, cell proliferation , and is required for proper positioning of the centrosome in nondividing cells and correct orientation of the mitotic spindle in dividing cells (Jonassen et al., 2008). To further study the function of IFT20 , a “floxed IFT20 gene was created. By using a “floxed” IFT20 gene and Cre recombination we can cause a genetic rearrangement at Lox P sites so that the sequence between Lox P sites is deleted. Therefore, by adding the enzyme Cre recombinase to the floxed gene it deletes the IFT20 gene and then the two strands ligate back together with the IFT20 deleted. However, Cre has to be in the nucleus in order to work. Cre is located in the cytoplasm and goes into the nucleus after binding to the drug tamoxifin. This new Cre protein goes into the nucleus, via the tamoxifen, and this is where our project questions stem from. Our goal here is to 1) add tamoxifen to cells that have the floxed IFT20 gene to try and delete IFT20 and 2) determine the dosage and duration of tamoxifen treatment required to delete IFT20 in Mus Musculus kidney cells. Results •IFT20 levels decreased significantly suggesting that tamoxifen is working and deleting the IFT20. •PCR / agarose gel analysis proved that the floxed allele was present before tamoxifen treatment and the null allele afterwards , thus confirming that tamoxifen is deleteing IFT20 from the floxed allele. •Although traces of IFT20 proteins were seen on the western blot analyses, this could be due to the fact that although the gene has been deleted, the protein that was made before deletion is left over. •Number of cilia slightly decreased after tamoxifen treatment suggests that IFT20 may play a role in cilia maintenance. •Future research will use this tool to study the role of IFT20 in the trafficking of cilia membrane proteins Background References Acknowledgements I want to thank the Pazour lab for all their help and support. Special thanks to Brian Keady for teaching me cell biology techniques. •Fliegauf, M., Benzing T., and Omran H. (2007). When cilia go bad: cilia defects and ciliopathies. Molecular Cell Biology 8:880-893 •Jonassen, J.A., j. San Agustin, J.A. Follit, and G. Pazour (2008). Deletion of IFT20 in the mouse kidney causes misorientation of the mitotic spindle and cystic kidney disease. The Journal of Cell Biology 183: 377-384. •MarshalL W.F. and Nonaka S. (2006). Cilia:Tuning in to the Cell’s Antenna. Current Biology 16: R604-R614. Examples of Cilia and Diseases Caused by Cilia Defects (A) (B) (C) (D) Figure 1: Examples of Cilia and diseases caused by cilia defects. (A) Cilia are located all throughout the body and defects in the cilia results in an array of different diseases. (B) Gross morphology of a IFT20 mutant Mus Musculus experimental kidney (top pair) and control kidneys at p23. (C) Gross morphology of a kidney from a patient suffering from polycystic kidney disease (left) in comparison to healthy kidneys (right). (D) Image depicting opsin (green), a membrane protein found in the photoreceptor cilium, located in the retina of the eye. Conversion of Floxed IFT20 Allele into the Null Allele. Deletion of IFT20 in Cultured Mammalian Cells. Immunofluoresence of IFT20 and Cilia Merged DAPI IFT20 Cilia (A) 72hr untreated (B) 72hr tamoxifen (ciliated) (C) 72hr tamoxifen (unciliated) Figure 2: Deletion of IFT20 in mouse Fibroblasts. (A) IFT20(green) is localized in the golgi apparatus in the untreated mouse fibroblast cell.Cilia (red) were stained with acetylated tubulin. (B) After tamoxifen treatment for 72 hours, IFT20 is dramatically reduced (white arrow depicts cilia). (C) Mouse fibroblast cells after 72 hour tamoxifen treatment (unciliated). Control and experimental images at each of the time points were taken using a fluorescence microscope under identical conditions. Percentage of Ciliated Mouse Kidney Cells DNA Agarose Gel Analysis Floxed allele Wild type(+) Null allele Figure 4: DNA agarose gel analysis Agarose gel electrophoresis analysis depicting two conditions. Arrows indicate wild type (blue), floxed IFT20 allele (green), and the null allele after 72hr tamoxifen treatment (red). Thus, confirming tamoxifen is deleting IFT20. Figure 5: Percentage of ciliated mouse kidney cells versus concentration. Percentage of cilia present in the three experimental conditions (untreated, 10nM, and 100nM) and at three different time points (A) 24 hours (B) 48 hours, and (C) 72 hours. (A) (B) (C) Western Blot Analysis Figure 3. : Western blot analysis comparing IFT20 to actin protein levels. IFT20 protein levels are decreasing gradually over time with the most effective deletion being seen at 72hr. 100nM tamoxifen treatment. Actin , however is not affected by the treatment.

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