Publication: Construction of plasmid vector for expression of preliminary microrna-367 in mammalian cell
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Date
2025-01
Authors
Yee, Angelyn Lee Chui
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Abstract
This thesis focuses on the construction of plasmid vector for the expression of preliminary microRNA-367, with a particular emphasis on its connection to miRNA-367-3p and its potential role in regulating key biological processes. Pre-miRNAs serve as essential intermediates in the biogenesis of mature miRNAs, which regulate gene expression by binding to complementary mRNA targets, leading to their degradation or translational inhibition. MiRNA-367-3p, in particular, has been implicated in various cellular functions such as apoptosis, cell proliferation, and differentiation, suggesting its potential therapeutic applications in diseases linked to miRNA dysregulation. The primary aim of this study was to construct a plasmid capable of expressing pre-miRNA 367 in mammalian cells, thereby facilitating the investigation of its functional roles and therapeutic potential. The methodology employed involved designing a synthetic pre-miRNA 367 sequence with necessary restriction sites for cloning into the pCMV-MIR plasmid vector. Following digestion with XhoI and BamHI, the pre-miRNA insert was ligated into the vector and subsequently transformed into E. coli XL1-Blue cells. Despite optimisation efforts, including adjustments to the ligation conditions and insert-to-vector molar ratio, no colonies were obtained, suggesting that potential issues during the ligation or transformation process may have contributed to the lack of successful plasmid incorporation. The discussion provides a detailed analysis of the experimental results, highlighting the steps taken to enhance the ligation process and the challenges encountered. While the cloning was unsuccessful, this study provides valuable insights into the challenges associated with the construction of pre-miRNA expression vectors and offers recommendations for overcoming these challenges to achieve successful expression of pre-miRNA 367 in mammalian cells
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