Isolation and Characterisation of ABCB4 Promoter Region from Danio rerio

Mohamad Fikri Haikal, Salehuddin (2018) Isolation and Characterisation of ABCB4 Promoter Region from Danio rerio. [Final Year Project Report] (Unpublished)

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Abstract

ABC gene is a class of protein that have significant function in movement of molecule or transporting molecules across the cells. In human, ABCB4 gene is a multidrug resistance gene (MDR) which is involve in transport of biliary hosphatidylcholine (PC) across the cells. The purpose of this study is to isolate and characterize the ABCB4 gene promoter in zebrafish. The DNA of zebrafish was extracted and the fragment of the DNA was amplified by using specific primer. The gradient PCR was conducted to determine the optimum annealing temperature, which was 54.6 °C producing I single amplicon. The amplicon was then purified and cloned into pGEMO-T Easy Vector using T4 ligase. Then, transformation was conducted into the XL 1-Blue competent cells of Escherichia coli with the transformation efficiency of 0.30 x 105 transformants per μg. The colony PCR was conducted on 5 colony to determine the colony that carry the DNA of interest. Upon confirmation, the sample was sent for sequencing which produced a sequence with 1213 bp. Upon analysis, the sequence is 95% identical to the Zebrafish DNA sequence from clone DKEY-24124 in linkage group 16. Next, mini-preparation was conducted to isolate plasmid with insert before restriction enzyme digestion was done by using Sacl and EcoRI to cut the pGEMm-T Easy Vector before subsequently cloning it into pZsGreenI-1 vector. The transformation producing around 6 colony on the LB plate. Upon successful isolation of pZsGreenl -1 with insert, the plasmid was used for microinjection to transfer the plasmid and DNA into the 2 cell-stage zebrafish embryo. Upon successful microinjection, the injected embryo was viewed and autofluorescence was observed under the fluorescence microscope. Based on this study, the future characterization and analysis of ABCB4 gene promoter can be carried out especially in mutixenobiotic mechanisms studies.

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