Generation A Transgenic Zebrafish Model Of Parkinson Disease

Abstract

The aim of this study was to clarify the mechanism of human Leucine- Rich Repeat Kinase 2 (hLRRK2) gene with point mutation of amino acid substitution from glycine to serine at residue 2019 (G2019S) in parkinson disease (PD). This is done by cloning the hLRRK2 G2019S gene into a vector with GFP (green fluorescence protein) under neuron-specific zebrafish Tyrosine Hydroxylase (zfTH) promoter. The promoter drives over-expression of a fusion protein between reporter gene-green fluorescent protein (GFP) - and LRRK2 gene holding a missense mutation (G2019S) driving in dopaminergic neuron. This research utilizing two methods of cloning, the first method was cloning the LRRK2 G2019S to pTGLwt by digestion and ligation. The second method was cloning of LRRK2 G2019S by site directed mutagenesis. This study also observing the behaviour of the wild type zebra fish using a locomotor activity test as a control for the transgenic zebrafish in further studies. Under the method of digestion and ligation, pTGLwt and pCGLG2019S were isolated (mini and maxi preparation). Subsequently, the sequencing process is applied to the whole construct of pTGLwt and pCGLG2019S prior to digestion of both constructs. Purification of pTGLwt and LRRK2G2019S is done using TAE agarose gels with Nucleospin Extract II Kit. Afterwards, transformation ligation mix is done from pTGL and LRRK2G2019S into E.coli competent cell, which the gene inserted is checked later using colony PCR before applying another sequencing. By site directed mutagenesis, PD- associated mutants (LRRK2 G2019S) were introduced in pTGLwt with primers that contain point mutation during PCR processing. This study is succeeded in resulting a construct of pTGLG2019S which will be useful in studying the transgenic zebrafish as the model of PD.