Cloning and Activity determination of Enhancer-like sequence from Proboscis monkey (Nasalis larvatus)

• Main Author: Chung, Phey Lin
• Format: Final Year Project Report
• Language: English; English
• Published: Universiti Malaysia Sarawak (UNIMAS) 2017
• Subjects: Q Science (General); QH426 Genetics
• Online Access: http://ir.unimas.my/id/eprint/27802/3/CHUNG%20PHEY%20LIN%2024pgs.pdf; http://ir.unimas.my/id/eprint/27802/6/CHUNG%20PHEY%20LIN%20ft.pdf; http://ir.unimas.my/id/eprint/27802/

Enhancer is a distal cis-regulatory element that regulate spatiotemporal gene expression by integrating with transcription factors (TFs). Proboscis monkey (Nasalis larvatus) is endemic and potential to be a suitable study model due to its proximate chromosome karyotypess with human. Currently, potential in silico software for prediction of enhancer from vast DNA sequence is still in increasing trend of development. The aim of this study is to isolate enhancer sequence from N. larvatus and to determine its TFs bounded. In this research, a strong putative enhancer (683bp) with >90% confidence level was selected via iEnhancer-2L software for further PCR amplification. The PCR amplicon was purified and cloned into pGEM-'f® Easy vector followed by pGL 3.0 SV40 basic vector. Subsequently, transformed into E.coli XLI Blue with transformation efficiency of 2.1 XIO"4 transformantsiug. Double digested restriction enzymes BamHI and Sall was conducted to verify the propagation of plasmid with insert DNA. Sequencing result and BLASTn analysis indicated that the putative enhancer has 79% Query similarity to the expected one. Further transcription factors binding site search using MATCIfTM 1.0 public and Alibaba 2.1. This includes AP-I, CIEBP beta, NFl, HNF-l, ER receptor and SPl. This shows that the putative enhancer might playa role in regulating liver specific genes. Due to time and reagents constraint, the determination of enhancer activity through transienttransfection was not able to be performed. However, future validation on the usability of this approach can be supported by functional assays