Characterization of Cre/loxP Inducible Transgenic Wnt5a Mouse Embryonic Stem Cell Line

Chong, Yoong Yi (2022) Characterization of Cre/loxP Inducible Transgenic Wnt5a Mouse Embryonic Stem Cell Line. [Project Paper] (Submitted)

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Abstract

Increasing studies have suggested the role of Wnt signalling family members in various aspects of neural development and function. One potential member is Wnt5a, which has been shown to promote neural differentiation of embryonic stem cells (ESCs), including into dopaminergic (DA) neurons. Our group has generated an inducible transgenic ESC line that carries a regulatable Wnt5a construct based on Cre/loxP binary system. The transgenic line allows us to study the effect of Wnt5a overexpression at specific time points during the neural differentiation process. However, the stability of the line before and after the induction of the Wnt5a transgene has not been properly evaluated. Objective: Our study aims to assess the stability of the Cre/loxP inducible transgenic Wnt5a ESC clone at the cellular and molecular levels. Methodology: A Wnt5a transgenic ESC line (NI-L7 line) was revived and propagated in a culture containing GMEM supplemented with 10% foetal bovine serum (FBS), other essential supplements and 20 ng/ml leukaemia inhibitory factor (LIF). The stability of the line was assessed for its pluripotency status and the inducibility of the Wnt5a transgene. The pluripotency status of the NI-L7 line was validated via RT-PCR for the expression of pluripotency-associated markers (Oct 4, Sox2 and Nanog) and the ability of the cells to form EBs using the cell suspension method. The cellular morphological assessment was carried out for the non- and tamoxifen-induced transgenic Wnt5a ESC clone (CE-L7). The Wnt5a expression of NI-L7 and CE-L7 lines were semi-quantified using ImageJ, an image analysis program upon RT-PCR. Results and Discussion: NI-L7 line successfully expressed all the pluripotency-associated markers and is capable of producing good qualities of EBs indicating that the Cre/loxP-Wnt5a construct does not compromise the pluripotency status of the transgenic Wnt5a ESC clone. Cellular morphology was indistinguishable between NI-L7 and CE-L7 lines revealing that the transgenic cells retained the stem cell properties even after tamoxifen induction. CE-L7 line showed higher Wnt5a transgene expression levels compared to NI-L7 line suggesting the functionality of the construct in overexpressing Wnt5a upon induction with tamoxifen. Conclusion: Findings of this study signify that genetic manipulation has not compromised the pluripotency status of ESCs and confirmed the functionality of the Cre/loxP-Wnt5a construct. Hence, the cells could be fully utilised to unravel the role of Wnt5a during the differentiation process of ESCs into the neural lineage including producing quality DA neurons.

Item Type:	Project Paper
Faculty:	Faculty of Medicine and Health Science
Depositing User:	Ms. Nor Safa'aton Saidin
Date Deposited:	22 Aug 2023 04:40
Last Modified:	22 Aug 2023 04:40
URI:	http://psaspb.upm.edu.my/id/eprint/1350

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