Abstract
Transcription activator-like effector nucleases (TALENs) are versatile tools that enable the insertion of DNA into different organisms. Here, we confirmed TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna, a model organism for ecological and toxicological genomics. We tested two different TALENs, ey1 TALEN and ey2 TALEN, both of which target the eyeless locus. The donor DNA plasmid, harbouring the H2B-GFP reporter gene, was designed to contain both TALEN target sites and was co-injected with each TALEN mRNA into eggs. The ey1 TALEN and ey2 TALEN constructs both resulted in H2B-GFP expression in Daphnia with a germline transmission efficiency of 3%. Of the three transgenic animals generated, two had donor DNA at the targeted genomic site, which suggested concurrent cleavage of the injected plasmid DNA and genome DNA. The availability of such tools that are capable of targeted knock-in of foreign genes will be extremely useful for advancing the knowledge of gene function and contribute to an increased understanding of functional genomics in Daphnia.
Highlights
We developed a method to inject an exogenous solution into eggs of Daphnia magna[22] and injected TALEN mRNA to assess the corresponding effects[23]
We found that heterodimeric TALENs induced heritable mutations both in the transgene DsRed[2] and in the endogenous eyeless gene, and were associated with decreased lethality compared to homodimeric TALENs
Our findings show that we successfully integrated a H2B-GFP reporter gene into a targeted genomic locus of D. magna demonstrating that TALEN-mediated targeted knock-in via NHEJ can be used in Daphnia, and that the availability of this approach will facilitate future efforts towards characterizing and understanding gene function and genomics in this important model organism
Summary
We developed a method to inject an exogenous solution into eggs of Daphnia magna[22] and injected TALEN mRNA to assess the corresponding effects[23]. In the current study, using D. magna, we tested TALEN-mediated knock-in of plasmid DNA via NHEJ, a method which has been successfully reported in studies using Chinese hamster ovary (CHO) cells[26], human cell lines[27], and zebrafish[28]. This knock-in method requires concurrent cleavage of injected plasmid DNA and genome DNA at the targeted site, resulting in the knock-in of exogenous DNA fragments that are much longer than that possible with other approaches, and has the added benefits of simplicity, flexibility, and high efficiency. Our findings show that we successfully integrated a H2B-GFP reporter gene into a targeted genomic locus of D. magna demonstrating that TALEN-mediated targeted knock-in via NHEJ can be used in Daphnia, and that the availability of this approach will facilitate future efforts towards characterizing and understanding gene function and genomics in this important model organism
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