Construction and Evaluation of a Mammalian Expression Vector for Human REST Coding Sequence

Arrumugam, Crystal Arthini (2022) Construction and Evaluation of a Mammalian Expression Vector for Human REST Coding Sequence. [Project Paper] (Submitted)

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Abstract

Repressor Element-1 Silencing Transcription Factor (REST) is an epigenetic transcriptional repressor that regulates neurogenesis in undifferentiated neural progenitors and is involved in neuroprotection. Dysregulation of REST is implicated in many diseases, including Alzheimer’s disease and Down Syndrome. Thus, constructing a mammalian expression vector (AcGFP1) incorporated with REST is beneficial for a better understanding of REST regulation on human neuronal cells. It would be a promising fusion partner for future study of diseases, especially in the gain of function or rescue experiments. Objective: Thus, this study aims to construct a mammalian expression vector of human REST. Methodology: REST primers with restriction sites were designed, and REST coding sequences were amplified from human cell line-derived cDNA. BamH1 and Nhe1 were used to cleave the Ac-GFP1 plasmid to produce sticky ends compatible with the REST gene for ligation. The REST amplicon was cloned into the vector. The REST-AcGFP plasmid was transformed using the heat-shocked method into bacteria-competent cells (STBL3 and JM109). Subsequently, the transformant was spread on an LB agar plate with Kanamycin antibiotic (50ug/mL) and incubated at 37°C overnight. Later, cloning validation was confirmed by gel electrophoresis. Results: REST primer was successfully designed and amplified using gradient PCR. However, no transformed colonies were observed on the agar plates. Gel electrophoresis shows no ligated product with higher molecular weight, and only a plasmid band was observed. Discussion: Prior to ligation, there is a band based on gel electrophoresis indicating the p resence of amplified REST and digested plasmid. Nevertheless, no band was seen for both insert and ligated products. Large REST insert may occlude the ligation and form multiple polymers, leading to the failure to transform the plasmid into the STBL3 and JM109 competent cells. Conclusion: Although no colonies formed in these competent cells, more extensive research and alternative approach using DH5-α, competent cells with higher insert stability are encouraged.

Item Type:	Project Paper
Faculty:	Faculty of Medicine and Health Science
Depositing User:	Ms. Nor Safa'aton Saidin
Date Deposited:	22 Aug 2023 04:38
Last Modified:	22 Aug 2023 04:38
URI:	http://psaspb.upm.edu.my/id/eprint/1368

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