There are currently over 430 known SLC transporters, over 30% of which have an unknown function. Compared to other transporter gene families, the SLC genes are relatively understudied with many orphan genes. SLC transporters have a high disease relevance and can be associated with many different diseases like gout, type 2 diabetes and different forms of cancer. SLC transporters also appear to be very druggable, thus offering a rare opportunity of an underexplored gene family, that can be linked to many diseases and seem to have a general druggability with small organic molecules. This thesis is evaluating three specific SLC transporters of the SLC38 family to discover their different roles and purposes. In this project CRISPR-Cas9 is used to knockout three SLC38 transporters, called SNAT6, SNAT8 and SNAT10. The cell-line used is HEK293 cells, as they are easy to transfect and are thought to express the three genes, however it is not certain that they do express the three SNAT genes. The project aims to optimize the method for best possible transfection by trying different protocols. A literature study is done on what the future experiments of the knocked-out cells could be, including; ensuring the HEK293 cells express the three genes, controlling the effectiveness of the transfection and analyzing the result of such a transfection. To confirm that the HEK293 cells do express the three SNATs a western blot assay could be performed. RT-qPCR is found to be useful in evaluating whether the knockouts are successful, by measuring if the three SNAT transporter proteins are present or not in the knocked-out cells. A metabolic analysis study to determine the result of the knockouts is also described as a future experiment. The experimental finding was a CRISPR-Cas9 transfection method that yielded enough RNA, enabling future experiments such as RT-qPCR.

Transport across the membrane of a cell is of crucial importance for cellular functions. The solute carrier family,SLC38 is a family of membrane proteins that transports various substances through the membrane and thusperforms many physiologically important functions, for example, transport of glutamine from astrocyte toneurons in the central nervous system. In this paper, we demonstrate that one of the transporters in this familynamed SLC38A6 forms several protein complexes with a variety of proteins in the membrane and in synapticvesicles, suggesting that SLC38A6 is involved in the synaptic release of neurotransmitters in synapses. Weperformed sensitive protein interaction analysis between the protein of interest and a variety of proteinsexpressed at different sites in the neuronal cell. We showed that SLC38A6 interacts with proteins in the cellmembrane as well as in the membrane of synaptic vesicles. The current theory is that SLC38A6 interact withthese proteins when the synaptic vesicles are in close proximity with the cell membrane during the release of theneurotransmitters.