Cloning and Activity determination of Enhancer-like sequence from Proboscis monkey (Nasalis larvatus)
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, pot...
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| Format: | Final Year Project Report |
| Language: | English English |
| Published: |
Universiti Malaysia Sarawak (UNIMAS)
2017
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| 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/ |
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| Summary: | 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 |
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