TY  - GEN
T1  - Codon optimized Tol2 transposase results in increased transient expression of a crystallin-GFP transgene in zebrafish
AU  - Mackey, Allison S
AU  - Redd, Priscilla S
AU  - DeLaurier, April
AU  - Hancock, C. Nathan
DO  - 10.17912/micropub.biology.000268
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000268/
AB  - Type II transposable elements (TEs) are segments of DNA that can be mobilized within the genome through the action of transposase (TPase) proteins (Craig 2002). In general, the rate at which TPase proteins bind the terminal sequences of the elements to form a functional transposition complex determines the relative mobility of the element (Mizuuchi et al. 1992; Zayed et al. 2004; Zhang et al. 2001). The rate of transposition complex formation is thus determined by the concentration of functional TPase protein, the number of TE sequences present, and the localization of the TPase proteins within the cell. Several studies suggest that transposition complex formation is regulated by access to the nucleus, as alteration of nuclear localization signals (NLS) and nuclear export signals (NES) influences transposition (Hancock et al. 2010; Payero et al. 2016; Ramakrishnan et al. 2019). Zebrafish studies often involve the integration of DNA sequences (i.e. overexpression cassettes) into embryos, leading to transient or germ line expression (Nusslein-Volhard and Dahm 2002). Inserting transgenes between TE sequences and providing a TPase protein source has been shown to increase transgenesis efficiency across multiple model organisms (Ding et al. 2005; Ivics and Izsvák 2004; Munoz-Lopez and Garcia-Perez 2010; Zayed et al. 2004). The Tol2 TE from Medaka fish (Oryzias latipes) has been developed as a means to improve zebrafish transgenesis, including both transient and germline integration (Kawakami 2007; Koga et al. 1996; Kwan et al. 2007; Ni et al. 2016). Tol2-mediated transgenesis is induced by co-injecting Tol2 TPase mRNA together with a Tol2 terminal sequence-flanked construct into 1-4 cell stage zebrafish embryos (Kawakami 2007; Kwan et al. 2007; Ni et al. 2016). Previous reports indicated that addition of the Tol2 TPase mRNA (expressed from pCS2FA) produced about a 3-fold increase in Tol2 flanked transgene expression compared to control (Kwan et al. 2007). Another study demonstrated that a zebrafish codon optimized version of Tol2 TPase led to successful germline transmission, but no direct comparison of efficiency was reported (Suster et al. 2011). The goal of this study was to explore the extent that efficiency of Tol2-mediated transgene expression could be improved by codon-optimizing the Tol2 TPase gene for zebrafish or altering a detected NES and NLS in the Tol2 TPase (Figure 1A). We hypothesized that increasing the translation efficiency (Gustafsson et al. 2004) and access to the nucleus would potentially improve transposition complex formation in transience. Analysis of the Tol2 TPase codon usage showed that it contained 19 codons (TTA, CTA, and TCG) that are rarely used by zebrafish (Nakamura et al. 2000). Codon optimization resulted in the development of a CO Tol2 TPase construct that more appropriately matched the zebrafish-specific codon bias, without altering the amino acid sequence (Zhou et al. 2016). In the CO Tol2 TPase N construct, the NES in the Tol2 TPase (Figure 1A) was removed by changing L641A and L643A and the existing NLS (PKRARLD, NLS score=8.5) was strengthened by changing it to PKKKRKV (NLS score=13) (Dingwall and Laskey 1991; Kosugi et al. 2009).
PY  - 2020
JO  - microPublication Biology
ER  -