Development of a PhiC31 system for functional characterisation of cis-regulatory elements in reporter transgenic zebrafish

Roberts, Jennifer Anne (2013). Development of a PhiC31 system for functional characterisation of cis-regulatory elements in reporter transgenic zebrafish. University of Birmingham. Ph.D.

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Abstract

Current methods to study enhancers in vivo involved integrating DNA sequences into the genome in a random position, in zebrafish the transposase Tol2 is utilised. However, this random genome integration can result in position effects which cause variations in reporter gene expression patterns preventing conclusions from being drawn about the enhancer being tested. To address the issue of variable position effects when using random integration techniques to study enhancers, this project aimed to establish a PhiC31 integrase system of targeted transgenesis in zebrafish. Firstly, we designed and tested a multicomponent system including a recipient vector, donor vector and PhiC31integrase mRNA. These components were co-injected into one cell stage embryos to test the integration function of PhiC31 integrase in zebrafish. The integration was detected by designing the recipient and donor vectors such that a switch from GFP to mCherry lens expression marks embryos with legitimate recombination. Over 97% of fluorescent embryos showed mCherry expression in the lens after coinjection of the three components indicating that integration of the two vectors by PhiC31 integrase is highly efficient. Legitimate recombination was shown to have occurred using PCR and sequencing techniques from total RNA extracted from embryos. A recipient transgenic line was made into which the donor vector and PhiC31 integrase RNA were co-injected. This resulted in around 70% of fluorescent embryos showing mCherry expression in the lens which suggests targeted genome integration is also efficient in zebrafish embryos. To establish whether variability of positional effects could be reduced using this system, an enhancer-promoter-reporter construct was modified to ii include donor vector sequences and injected into recipient line transgenic zebrafish embryos. Expression patterns observed in stable lines made with either Tol2 transposase or PhiC31 integrase suggest that variability in position effects is reduced when using the PhiC31 integrase system. Together, these results provide evidence that the PhiC31 integrase system I have tested is suitable for a number of uses in zebrafish, including enhancer screening.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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