Targeted Deletion of Zebrafish lncRNAis18 with TALENs

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Jones, Crystal
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Symposium on Undergraduate Research and Creative Expression
Iowa State University Conferences and Symposia

The Symposium provides undergraduates from all academic disciplines with an opportunity to share their research with the university community and other guests through conference-style oral presentations. The Symposium represents part of a larger effort of Iowa State University to enhance, support, and celebrate undergraduate research activity.

Though coordinated by the University Honors Program, all undergraduate students are eligible and encouraged to participate in the Symposium. Undergraduates conducting research but not yet ready to present their work are encouraged to attend the Symposium to learn about the presentation process and students not currently involved in research are encouraged to attend the Symposium to learn about the broad range of undergraduate research activities that are taking place at ISU.

The first Symposium was held in April 2007. The 39 students who presented research and their mentors collectively represented all of ISU's Colleges: Agriculture and Life Sciences, Business, Design, Engineering, Human Sciences, Liberal Arts and Sciences, Veterinary Medicine, and the Graduate College. The event has grown to regularly include more than 100 students presenting on topics that span the broad range of disciplines studied at ISU.

Genetics, Development and Cell Biology

Long non-coding RNAs (lncRNAs) are important players in epigenetic regulation of gene expression during development and disease (Niland et al, 2012). A number of mechanisms have been proposed for lncRNA action, however, few functional studies of lncRNAs have been described. We are using Transcription Activator-Like Effector Nucleases (TALENs), engineered site-specific nucleases, to create targeted mutations in a novel zebrafish lncRNA. We previously mapped a highly penetrant retinal tumor model to transgene disruption of the zebrafish lncRNAis18 gene. The objective of this project is to isolate a second zebrafish lncRNAis18 allele that contains a deletion of part of the lncRNAis18 gene. Two TALEN pairs were designed to simultaneously target double-strand breaks to exons 2 and 5 of lncRNAis18. Injection of 25-40pg of the TALENs targeting individual exons into zebrafish embryos resulted in efficient mutagenesis of the target sites. To isolate the lncRNAis18 deletion allele we co-injected embryos with the TALEN pairs targeting both exons 2 and exon 5. We predicted co-injection of TALEN pairs targeting exons 2 and 5 of lncRNAis18 would create a 147kb deletion after loss of the intervening sequence and repair by the non-homologous enjoining pathway. PCR products spanning the fusion of exons 2 to 5 were amplified from somatic tissue in 9 out of 14 co-injected embryos. We verified the deletion allele by sequencing PCR products from 3 embryos. We have identified one founder that transmits the deletion allele to the F1 generation. F1 embryos are being raised to establish a new lncRNAis18del line. The lncRNAis18 deletion allele will provide a new genetic tool to study the function of lncRNAis18 in zebrafish development and cancer.

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