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.