I underwent an eight-week HotStart Studentship with the supervision of Dr Dawn Thompson, in a pharmacology research laboratory. Throughout my placement I developed invaluable laboratory experience, primarily through investigating GPCR signalling mechanisms. I carried out techniques such as Bacterial Transformation, Molecular Cloning, Mammalian Cell Cultures, Transfections, Drug Response Assays and Western Blotting.
The prostaglandin F receptor (PTGFR) is a vital G protein coupled receptor (GPCR). It can activate several downstream signalling pathways, with the outcome depending on which G protein it couples to. Examples of G Proteins include; stimulating Gas which stimulates Adenyl cyclase (AC), leading to an increase in Cyclic-Amp (cAMP) and protein kinase A (PKA). While inhibitory Gis decreases levels of AC thus leading to a decrease in cAMP and PKA. PKA is critical for the regulation of important cell process including metabolism, hormone activity and gene expression. Dysregulation of these cell process can cause infectious diseases in endocrine systems and the development of cancers. Gaq stimulates Phospholipase C (PLC), to produce IP3, leading to an increase in intracellular Ca2+ levels. Gaq also stimulates DAG which activates protein kinase C (PKC). PKC regulates physiological functions including the nervous, endocrine, inflammatory and immune system. It has protective properties during the development of cardiac ischemia and Alzheimer’s disease. Dysfunction of PKC has been linked to malignant tumour growth and diabetes development). Ca2+ is vital to normal cell homeostasis and the regulation of Cell development, metabolism, hormone secretion, gene transcription, muscle and neuronal function. The dysregulation of Ca2+ can be linked to several neuronal diseases.) β-Arrestin can also be recruited by receptor activation, to signal independently of G proteins. The investigation of these pathways is important for the understanding of different ligands producing selective cell responses and biased signalling.
My first few weeks primarily involved molecular cloning and plasmid preparations. My focus was to successfully develop a PTGFR based construct tagged with reporter luciferase. I used restriction enzymes EcoRV, HindIII and EcoRI to carry out a PCR and analysed the DNA with Agarose-gel electrophoresis. I also carried out a PCR clean up and used a NanoDrop Spectrophotometer to measure the DNA concentration and purity. I followed this by bacterial transformation, mini/midi preparations to further amplify the purified plasmid DNA. This enhanced my troubleshooting skills, by repeating unsuccessful transformations, to eventually obtain individual colonies and create a working PTGFR-luciferase tagged construct.
I also gained experience in genotyping, using DNA extracted from mouse ear clipping samples to analyse for the presence of GASP-1 and Cre proteins. I carried out a PCR to compare bands with controls and confirm the presence of proteins.
Another large part of my placement involved HEK293 mammalian cells used in culturing and transfections. I practised cell culture maintenance with aseptic techniques and cell confluency assessment. This allowed for a successful transfection with PEI, alongside P-Luc, with Gi, Gs, Gq and β-Arestin 3. I then calculated appropriate serial drug dilutions and added accordingly to a 96 well plate, before analysing with a CLARIOstar plate reader. Gi and Gs produced expected signals in response to drug treatment, while weak signalling β-Arestin 3 unexpectedly produced a small response and GQ produced anomalies. The data analysis highlighted the importance of controls and repeating experiments for more consistent and accurate results.
I also developed my western blotting skills to allow for the investigation of downstream signalling of drug PGF2a treated cells. I worked in step-by-step process; SDS-PAGE gel preparation, protein transfer, and antibody incubation with Phosphorylated ERK (PERK), total ERK (TERK) and GAPDH. After ECL detection, PERK, TERK and GAPDH were all analysed. Bands for PERK and TERK were shown strongest at 10 mins. This suggested the signal was produced in a short time then became desensitised for longer periods of time.
The Hotstart programme has helped me to gain invaluable research and laboratory experience. I have massively developed my confidence in molecular biology and pharmacology. I feel this opportunity has provided me with a strong foundation for future studies and further laboratory roles.
