Cloning and Activity Determination of Enhancer-like Sequence from Proboscis Monkey (Nasalis larvatus)

Ch'ng, Vivien Jing Wen (2017) Cloning and Activity Determination of Enhancer-like Sequence from Proboscis Monkey (Nasalis larvatus). [Final Year Project Report] (Unpublished)

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Abstract

Enhancer elements are noncoding segments of DNA that are essential in spatiotemporal specificity of gene regulation pattern in animal development. Identification of enhancers is an important step for understanding the gene expression that regulate normal or undergo pathogenic cellular conditions. In this research, the study of enhancer in Proboscis monkey chromosome 18 is conducted as it has shared chromosome information with human chromosome in previous study. This experiment was initiated by identification of 644 bp enhancerlike sequence by using iEnhancer-2L software. A pair of primers were designed with addition of restriction sites BamHl in forward primer and Sall in reverse primer. An approximately 700 bp sequence was amplified by PCR. Subsequently, this putative enhancer was cloned into pGEM®-T Easy Vector with transformation efficiency of 5.53 x 104 transformants per pg. The plasmid was extracted and digested using BamHI and Sall along with pGL3.0 Basic Vector. This putative enhancer was cloned into pGL3.0 Basic vector with transformation efficiency of 6.18 x 104 transformants per pg and was sent for sequencing. The sequence verification of putative enhancer (654 bp) was conducted by comparing it to that of predicted, showing 99% similarities in pairwise alignment (BLASTn). Using MATCH TM software, this sequence was found to have binding sites for transcription factors such as NF-1, HNF-1 and HNF-3beta. This sequence will be used to test its enhancer activity by dual luciferase assay and to further explore the function of this sequence using in vivo assay. Through this analysis, this putative enhancer may play a role in regulating the gene expression in liver specific gene. The study of gene expression by putative enhancer can further contribute to the conservation of complex gene regulation to provide better information to the associate human disease for therapeutic purposes.

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