Generation of mutated expression plasmid KRT1 and comparison of HaCaT cells transfected with expression plasmid KRT1 or KRT10 concerning keratin aggregates

• Main Author: Eriksson, Jennifer
• Format: Others
• Language: Swedish
• Published: Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi 2012
• Subjects: Epidermolytic ichtyosis; keratinocytes; keratinfilaments; keratin genen mutation; 4-PBA; Cell and Molecular Biology; Cell- och molekylärbiologi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-176426

Introduction The genetic skin disease epidermolytic ichtyosis is caused by mutations in either keratin gene 1 or 10 and leads to blisters and hyperkeratosis of the epidermis. At cellular level the disease is seen as aggregates in the keratin filaments. Since medicines are hard to investigate and produce mainly due to lack of reproducible model systems, there is no good treatment available for this disease today. In this article we describe how an in vitro model consisting of cells from a stable cell line transfected with expression plasmids to mimic patient cells, may be a possible alternative for screening compounds for therapies. The first step was to generate an expression plasmid required to complete the in vitro model. The model was tested by a preliminary experiment with 4-phenylbuturate (4-PBA) to see if the substance had an effect on the amount of cells with keratin aggregates. Methods PCR and primers containing the desired mutation were used to incorporate a deletion in wild type keratin gene1 plasmid to generate the expression plasmid. HaCaT cells were transfected with the plasmid for expression of keratin. The percentage of cells with keratin aggregates was assessed by fluorescence microscopy. Results/Conclusion Cells containing mutated plasmid had a higher percentage of keratin aggregates compared to cells transfected with wild type plasmid. 4-PBA was found not to affect the amount of cells with keratin aggregates. According to this project the model might be a useful tool for screening compounds, but it needs to be more developed and tested.