Targeted Deletion of Zebrafish lncRNAis18 with TALENs

Abstract

<p>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.</p>