Pranav Ganapathy

Determining CLC5 Genotype in Mice for Crohns Biomarker Analysis

Research Plan
Rationale:
The CLC5 gene has been known to create proteins and ion channels involving the
exchange of hydrogen and chlorine. It plays an integral role in Dents disease, obesity, diabetes,
diarrhea, and especially Crohns disease. In order to establish crucial biomarkers involved in
Crohns disease, qPCR and electrophoresis methods were used to distinguish CLC5 genotypes
from Wild Type genotypes associated with breeding. The results of this experiment contribute to
the further research of Crohns biomarker analysis.
Procedures:
Samples of the tails of the labs mice are soaked in lysis buffer and centrifuged over
night. After heating at 37 degrees Celsius for 12 hours, the RNA is centrifuged and chilled in a
refrigerator, followed by 3 hours of qPCR. Gel is made by heating water with agarose powder
and pouring over an electrophoresis reservoir. After the gel is chilled, pipette the post-qPCR
RNA into the wells. Run the electrophoresis at 110 volts for 30 min. Follow up with running the
gel under a scanner, where bands for CLC5 and WT genes will be visible and can determine
genotype of the mice.
Risk and Safety:
Animal tissue will be used, so we will work under a hood with appropriate waste receptacles,
gloves, goggles, etc.
Bibliography:
Amarapurkar DN, Patel ND, Amarapurkar AD, Agal S, Baigal R, Gupte P. Tissue polymerase
chain reaction in diagnosis of intestinal tuberculosis and Crohn's disease. J Assoc Physicians
India. 2004 Nov; 52:863-7.
Weiwei Jiang, Xuhang Li. Molecular Analysis of Inflammatory Bowel Disease: Clinically Useful
Tools for Diagnosis, Response Prediction, and Monitoring of Targeted Therapy. Mol Diagn Ther.
2015.
Alex, P, Li, X. Applications of Proteomics in the Study of Inflammatory Bowel Diseases: Current
Status and Future Directions with Available Technologies. Inflamm Bowel Dis. 2009 Apr; 15:4.

Pranav Ganapathy

Xie G., Cheng K., Shant J., Raufman J.P. Acetylcholine-induced activation of m3 muscarinic
receptors stimulates robust matrix metalloproteinase gene expression in human colon cancer
cells. Am. J. Physiol. Gastrointest. Liver Physiol. 2009;296:G755G763.
Van der Jagt M.F., Wobbes T., Strobbe L.J., Sweep F.C., Span P.N. Metalloproteinases and their
regulators in colorectal cancer. J. Surg. Oncol. 2010;101:259269.

PRECAUTIONS INVOLVING ANIMAL TISSUE

I was not directly involved in procuring or using animal tissue. I was simply given samples in
test tubes, but regardless, I was still trained in the IACUC Animal Protocol at the JHU Ross
Research Building. No animals were killed in the procurement of tissue. Small snippets of tails
or ears were cut off (with pain killer dosage given to the animals) to alleviate the pain. All
IACUC and university protocols were followed, and no mice were directly killed for the
experiment.