Isolation and Characterization of ABCG2 Promoter from Danio rerio.

ABC gene is a group of protein that functions in the transport and movement of molecules into and out of the cells. ABC genes have seven distinct subfamilies and ABCG2 protein is a member of White subfamily. It is also known as breast cancer esistance protein (BCRP) which functions as xenobiotic transporter that play important role in multi-drug resistance (MDR). This study was done to isolate and characterize the promoter of ABCG2 gene in zebrafish. Since there are multiple variants encoding different isoforms were found, the promoter of ABCG2a gene was selected to be studied. The genomic DNA of zebrafish was extracted and amplified by using the designed primers that is specific to the promoter region. The optimum temperature obtained from the gradient PCR is 54.5 °C in producing a single amplicon. The amplicon was purified and cloned into pGEM"-T Easy vector. Then it was transformed into XL- I Blue competent cells of Escherichia coli with efficiency of 0.30 x 105 transformants per kg. Colony PCR was conducted to determine the colony that carry the DNA of interest. For confirmation, the sample was sent for sequencing that resulted in 1, 239 bp. Analysis has showed that the sequence is 98% identical to the zebrafish DNA sequence from clone CH73-67C22 in linkage group 23. After that, the plasmid with the insert was isolated and continued by restriction digestion using Sacl and EcoRI before subsequently clone it into an expression vector, pZsGreen 1. 1. The cloning and confirmation of colonies by PCR was repeated for pZsGreen 1. 1. Next, the isolated pZsGreen 1. 1 vector with insert was used for microinjection into the zebrafish embryos. The injected embryos were left to hatch in the incubator at 27 °C. The hatchlings was viewed under fluorescence microscope and resulted with an auto fluorescence. Besides that, the characterization and analysis of the promoter region of ABCG2 gene was carried out to find the transcription factors involved in activating the promoter by using MatInspector software. The transcription factors that was chosen to be discussed are O$YTBP, V$CEBP and V$CREB. These particular transcription factors were compared with human's ABCG2 promoter region. Results show that both organisms have the same transcription factors in regulating the promoter's activity of ABCG2 gene