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Efficient Transposition of Tol2 in the Mouse Germline

2009-12-01 14:54:22

Genetics; 2009 Dec; 183(4):1565-73


Vincent W. Keng, Barbara J. Ryan, Kirk J. Wangensteen, Darius Balciunas, Christian Schmedt, Stephen C. Ekker, and David A. Largaespada



Abstract



Insertional mutagenesis screens play an integral part in the annotating of functional data for all sequenced genes in the postgenomic era. Chemical mutagenesis screens are highly efficient but identifying the causative gene can be a laborious task. Other mutagenesis platforms, such as transposable elements, have been successfully applied for insertional mutagenesis screens in both the mouse and rat. However, relatively low transposition efficiency has hampered their use as a high-throughput forward genetic mutagenesis screen. Here we report the first evidence of germline activity in the mouse using a naturally active DNA transposon derived from the medaka fish called Tol2, as an alternative system for high-throughput forward genetic mutagenesis screening tool.



The Tol2 (transposable element of Oryzias latipes, number 2) element belongs to the hAT family (hobo of Drosophilia, Activator of maize and Tam3 of snapdragon) of transposons and was the first known autonomously active vertebrate type II transposable element (Koga et al. 1996; Kawakami et al. 1998). Unlike other DNA-type transposons like Sleeping Beauty (SB) (Ivics et al. 1997) or piggyBac (PB) (Fraser et al. 1996), Tol2 does not exhibit any known strong site specificity for integration nor does it exhibit any significant overexpression inhibition activity (Kawakami and Noda 2004; Balciunas et al. 2006) as seen in SB (Geurts et al. 2003). Recently, Tol2 was shown to effectively carry large DNA cargo of up to 10 kb in human and mouse cells without affecting its transposition efficiency (Balciunas et al. 2006). To date, Tol2 has also been demonstrated to transpose efficiently in zebrafish, frog, chicken, mouse cells, and human cells (Kawakami et al. 2000, 2004; Koga et al. 2003; Kawakami and Noda 2004; Balciunas et al. 2006; Hamlet et al. 2006; Sato et al. 2007).



Germline mutagenesis using the SB transposon system has been demonstrated in both the mouse (Dupuy et al. 2001; Horie et al. 2001) and rat (Kitada et al. 2007; Lu et al. 2007). In addition, PB germline mutagenesis in mice has also been demonstrated (Ding et al. 2005; Wu et al. 2007). However, the relatively low germline transposition efficiency of both transposon systems reported so far has hampered their use in a high-throughput forward genetic mutagenesis screen (Table 1). Using an SB gene-trap vector containing a GFP reporter poly(A) trap as an indication to the number of mutant mice generated per litter, 7 and 11% of newborn pups were GFP positive (GFP+) for mouse and rat, respectively (Keng et al. 2005; Kitada et al. 2007).



In search of an alternative tool for high-throughput forward germline mutagenesis screen in mice, a Tol2 transposon insertional mutagenesis system was generated on the basis of a similar strategy used for the SB transposon system (Horie et al. 2003; Keng et al. 2005). In the present study, we successfully demonstrate the novel use of the Tol2 transposon system for germline mutagenesis in mouse. Our results indicate the potential use of this transposon system for a high-throughput, large-scale forward mutagenesis screen in the mouse germline.



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