Comparison of Shotgun Fragment Library Approaches for Functional Enhancer Screen of the Six3 Gene

Jones, Carmen (2022) Comparison of Shotgun Fragment Library Approaches for Functional Enhancer Screen of the Six3 Gene. Masters thesis, University of East Anglia.

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Cyclopia is defined as the failure of the eye field to successfully divide into two. This is encompassed by the broader brain defect, Holoprosencephaly (HPE), where the brain lacks any ventral identity, and the forebrain fails to divide.

Key signalling pathways such as Sonic Hedgehog (Shh) and Nodal have been identified as critical for the ventralization of the neural plate and prosencephalic regions of the forebrain during embryo development. Specific mutations within key genes of these pathways have identified as them as causative genes for the Cyclopic/HPE phenotype.

Despite extensive knowledge surrounding these causative genes, little is known about the regulatory elements, enhancers, that control the spatial-temporal expression of these genes. In recent years many different techniques have been developed to identify and characterise potential regulatory elements.

Using the Cyclopia gene network as a disease model, different methods for detecting regulatory enhancer elements were tested. This project aimed to construct a 3D shotgun fragment reporter library for the causative Holoprosencephaly gene, Six3. Utilising the use of BAC clones, our approach focused on optimising the random amplification of fragments followed by a modified Golden Gate cloning strategy to circumvent the loss of material associated with traditional methods for creating BAC clone fragment libraries.

Here we successfully edited our reporter vector to contain 5 unique barcode sequences for rapid detection of enhancer activity using RT-PCR. We successfully modified our Golden Gate Cloning strategy to produce hundreds of colonies ready for library construction. Using a candidate approach, a putative enhancer sequence was identified for the cMyc gene. Using phylogenetic foot printing, ECR2 was identified and using RT-PCR this sequence was amplified. This ECR was then cloned into the pTK-Citrine reporter vector to validate the enhancer’s spatio-temporal activity in vivo using the model organism, Gallus gallus (chick).

Item Type: Thesis (Masters)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 27 Mar 2023 10:44
Last Modified: 27 Mar 2023 10:44

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