ESR4: Functional assessment of a cis-regulatory element of ABCA4 in a frog model
Partner
Ghent University, Belgium (www.ugent.be/en; www.debaerelab.com) Supervisor Prof. Dr. K. Vleminckx |
Munevver Burcu Cicekdal
My name is Munevver Burcu Cicekdal and I work at UGent as a part of the StarT project. I was born and raised in Istanbul where I was exposed to both European and Asian culture. I graduated from Molecular Biology and Genetics department at Istanbul University. Then, I followed my passion towards science and joined a research group at Yeditepe University where I completed my Master’s degree in Biotechnology and worked as a Research assistant for 4 years. When I saw the opportunity for StarT project, I wanted to step out of my comfort zone and try to be a part of such an important project where several perspectives of science came together to fight against a hereditary disease, Stargardt’s disease . Now, here I am challenging myself as ESR4 at UGent and I am grateful for the opportunity to work with state-of-the-art-technology under the top scientists’ supervision! During my StarT adventure, I will be working on cis-regulatory elements and generating a stable knock-out model of a cis-regulatory element of ABCA4 gene in Xenopus tropicalis. I am a self-motivated and a very excited young scientist with full of passion for science. I am looking forward to see what is coming for us as ESRs and I am eager to contribute with my knowledge and experience to do better in every mission for StarT project. |
Abstract
Xenopus tropicalis contains the major cell types of the human eye and unlike the rod-dominated rodent retina, Xenopus (X.) has equal numbers of cones and rods, which is more similar to humans. In addition, abca4 is not duplicated in X. and is located in a region with high synteny, facilitating the identification and characterisation of possible CREs. Previous CRISPR/Cas9 based knock out a CRE of Shh (i.e. ZRS) in X. tropicalis resulted in a phenocopy of the human limb phenotype. This work demonstrated that X. can be used as a model organism to study the non-coding genome. To generate a stable knockout of a previously characterised CRE of ABCA4, ESR4 will use CRISPR/Cas9-mediated genome editing to disrupt this element in X. ESR4 will screen F0 or F1 animals for a STGD1 phenotype by histology, immunofluorescence and TUNEL assays. To study the effect of variations in CREs in F0 X. and to add to the stable transgenesis experiments of ESR2, ESR4 will use a recently described direct plasmid-injection method generating robust, promoter-typical expression in tadpoles. A reference gene present on the plasmid controls for variations in the injections. The plasmids further contain phiC31 AttB sites to favour integration and chromosomal insulators to reduce possible chromosomal position effects.
Xenopus tropicalis contains the major cell types of the human eye and unlike the rod-dominated rodent retina, Xenopus (X.) has equal numbers of cones and rods, which is more similar to humans. In addition, abca4 is not duplicated in X. and is located in a region with high synteny, facilitating the identification and characterisation of possible CREs. Previous CRISPR/Cas9 based knock out a CRE of Shh (i.e. ZRS) in X. tropicalis resulted in a phenocopy of the human limb phenotype. This work demonstrated that X. can be used as a model organism to study the non-coding genome. To generate a stable knockout of a previously characterised CRE of ABCA4, ESR4 will use CRISPR/Cas9-mediated genome editing to disrupt this element in X. ESR4 will screen F0 or F1 animals for a STGD1 phenotype by histology, immunofluorescence and TUNEL assays. To study the effect of variations in CREs in F0 X. and to add to the stable transgenesis experiments of ESR2, ESR4 will use a recently described direct plasmid-injection method generating robust, promoter-typical expression in tadpoles. A reference gene present on the plasmid controls for variations in the injections. The plasmids further contain phiC31 AttB sites to favour integration and chromosomal insulators to reduce possible chromosomal position effects.
