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Optimazation of a genotyping protocol for Pink1 Knockout mice and cre mice

Celis, Eline
Professionele bachelor in de agro- en biotechnologie

Abstract :
Every year there are 1500 new cases of Parkinson’s disease (PD) in Flanders. This is the primary form of parkinsonism and the main symptoms are tremor, rigidity and bradykinesia. PD is progressive and some patients suffer more than others. The disease is a consequence of the loss of dopaminergic neurons in the substantia nigra. Due to the lack of dopamine, the nigrostriatal pathway changes which causes hyperactivity of the globus pallidus and subthalamic nucleus. This overactivity results in the hypoactivity of the frontal cortex. This makes it difficult for a person to plan movements so patients move slow. To diagnose PD is quite difficult for the symptoms could also represent other diseases. Usually a neurologist does some examinations and then prescribes a medicine for PD. If this works the person has PD, if not it could be another condition. Techniques that are used often are CAT scan, DaT-SPECT scan, MRI and an EEG.
PD still can’t be cured but there are some treatments that alleviate the symptoms and make the patient’s life easier. Examples of these treatments are medication, DBS-STN, pallidotomy and thalamotomy which are surgeries to remove some parts of the brain, kinesiotherapy, ergo therapy and speech therapy.
Researchers are still not sure what the actual cause of PD is. Most of them think it’s a combination of gene mutations and environmental factors. Also Lewy bodies and oxidative stress could trigger it. One of the genes in which a mutation can cause PD is the PINK1 gene. It codes for PTEN induced putative kinase 1 and plays a role in the pathway that gets rid of damaged mitochondria.
Research is necessary to better understand the mechanism of the brain and hence the mechanisms of Parkinson’s disease. At NERF, PINK1 knockout mice are used for this research. To create such knockout animals, labs use the cre-lox system that modulates the genomic DNA through homologous recombination. Before mice can be used for experiments the researchers have to be sure about the genotype. There are different techniques for genotyping like RFLP, sequence analysis, micro array,… In this case PCR is used to determine whether or not an animal contains the complete PINK1 gene and the cre gene. The protocol needs to work optimally to be sure that the results are right. An optimization is performed to determine which parameters need to change in a provided protocol to make sure that the results are clear.
First of all the provided protocols were tested to check if the results were clear. This was not the case in both the PINK1 and cre protocol. A touchdown PCR was executed with the PINK1 samples to check whether the primers worked. They worked but the results were rather confusing. Afterwards the right annealing temperature, primer concentration and DNA volume were determined. The optimal annealing temperature was 56,1°C, the right primer concentration was 0,125 µM and the right amount of DNA in a PCR sample of 40 µl was 0,5 µl.
On the cre samples only the optimization was performed not a touchdown because the primers worked. The protocol was used before but the results weren’t clear enough. Because it was used before, it wasn’t in the plan to determine the right annealing temperature but the results of the different primer concentrations showed a lot of nonspecific binding. So the annealing temperature was determined afterwards and this was 51°C. The right primer concentration is 0,25 µM. The right amount of DNA to be added is 0,25 µl.
The results of the optimized protocols are good. The bands are very clear so the researchers can be sure about the genotype of the animals used in their experiments.

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Celis, E. (2016). Optimazation of a genotyping protocol for Pink1 Knockout mice and cre mice. Unpublished thesis, Hogeschool PXL, PXL-Tech.
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