ESPE Abstracts (2014) 82 FC1.3

Genetic Engineering Using TALENs to Study the Redox Regulation of Steroidogenesis in vivo

Aliesha Griffina, Silvia Parajesa,b, Angela Taylora, Ferenc Muellera & Nils Kronea

aUniversity of Birmingham, Birmingham, UK; bMax Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

Background: Transcription activator-like effects nucleases (TALENs) have recently been developed as an efficient method for in vivo genome engineering. Zebrafish are becoming an increasingly popular model to study translational aspects in endocrinology. The redox cofactor ferredoxin (FDX1) is essential for mitochondrial cytochrome P450 (CYP) enzymes including those required for steroidogenesis. In vitro, FDX1 modifications influence the catalytic rate of steroidogenic CYP enzymes; however, to date mouse knockouts of Fdx1 have not been reported.

Objective and Hypotheses: To use TALENs to generate a zebrafish knockout of the mitochondrial redox cofactor ferredoxin (fdx1b) for investigating redox regulation of steroidogenesis in vivo.

Method: Genetic disruption was achieved by designing fdx1b specific TALENs using the golden gate assembly method. Genomic analysis for fdx1b knockouts was performed by high resolution melting curve analysis (HRM). RT-PCR of fdx1 and fdx1b was used to determine individual expression patterns. Transient knockdown studies were performed by microinjecting antisense morpholinos targeting fdx1b. Liquid chromatography tandem mass-spectrometry (LCMS/MS) was used to establish whole embryo steroid profiles.

Results: We identified duplicated zebrafish ferrdoxin genes; Fdx1 and Fdx1b. While Fdx1 was ubiquitously expressed, fdx1b was restricted to steroidogenic tissues; the interrenal gland (counterpart of the mammalian adrenal), gonads and brain. This suggests Fdx1b is the praralog required for steroidogenesis in zebrafish. Transient knockdown of fdx1b by antisense morpholino confirmed larvae fail to synthesize cortisol suggesting a key role in glucocorticoid production. Using TALENs we specifically targeted fdx1b to generate F1 knockout lines which was confirmed by HRM.

Conclusion: Developments in genome engineering make zebrafish an advantageous model to explore development and disease. Through transient knockdown studies we show Fdx1b is essential for cortisol synthesis. Using TALENs we generated the first vertebrate model of a ferredoxin knockout. This knockout line will provide a valuable tool for determining the mechanisms of how redox cofactors modulate steroidogenesis.

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