A transposon gene delivery system for the accelerated construction of HEK293S stable cell lines and its application for the study of rhodopsin retinitis pigmentosa mutants

Retinitis pigmentosa (RP) is a degenerative disease of the retina often leading to legal blindness. It is commonly the result of mutations in rhodopsin; a GPCR which initiates the visual signalling cascade. Characterisation of the large volume of such mutations is necessary due to their varied phenotypic outcomes. However, characterisation is often impeded by difficulties and time required to generate sufficient amounts of mutant rhodopsin. Here the PiggyBac transposon system, a rapid and facile method was used to overcome these limitations thus enabling the construction of several HEK293S inducible stable cell lines in under a month. Here, two groups of rhodopsin RP mutants were investigated. Group one comprised enigmatic mutants E122G, R252P, S298D, K311E. E122G had a λmax shifted to 481 nm and Meta II half-life twice WTs. S298D exhibited abnormal photobleaching and a Meta II half-life 4x shorter than WT. R252P and K311E remained enigmatic with photobleaching, Meta II decay and transducin activation unchanged. Group two comprised intradiscal domain mutants: P23A (N-terminus), G101V, V104F and G106W (all EL1). Each of the EL1 mutants were shown to have faster Meta II decay rates (5.96, 10.06, 9.97 mins respectively) than WT (17.68 mins). They also exhibited decreased transducin activation (35%, 64% and 18% relative to WT) and abnormal photobleaching was shown for G101V and V104F. Temperature-sensitivity was demonstrated for P23A, G101V and G106W with time-dependent restricted expression at 37°C which improved at 33°C. Overall, these results have showed the utility of the PiggyBac system for the expression and characterisation of rhodopsin mutants, particularly in high level expression for structural studies. Milligram amounts of E122G, E122Q and Y206F were purified for the collection of NMR data. This system will be valuable for future studies of rhodopsin RP mutants for which structural and functional information is needed for future clinical intervention in RP.