Genome-wide target profiling of piggyBac and Tol2 in HEK 293: pros and cons for gene discovery and gene therapy

被引:57
作者
Meir, Yaa-Jyuhn J. [1 ]
Weirauch, Matthew T. [3 ,4 ]
Yang, Herng-Shing [5 ]
Chung, Pei-Cheng [2 ]
Yu, Robert K. [6 ,7 ]
Wu, Sareina C-Y [2 ]
机构
[1] Chang Gung Univ, Dept Biomed Sci, Tao Yuan 333, Taiwan
[2] Chang Gung Univ, Mol Med Res Ctr, Tao Yuan 333, Taiwan
[3] Univ Toronto, Banting & Best Dept Med Res, Toronto, ON M5S 3E1, Canada
[4] Univ Toronto, Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada
[5] YongKang Vet Hosp, Yongkang City 71051, Tainan, Taiwan
[6] Georgia Hlth Sci Univ, Inst Mol Med & Genet, Augusta, GA 30912 USA
[7] Georgia Hlth Sci Univ, Inst Neurosci, Augusta, GA 30912 USA
关键词
SLEEPING-BEAUTY; TRANSPOSABLE ELEMENT; GERM-LINE; TRANSPOSITION; ACTIVATION; GENERATION; SEQUENCES; CELLS;
D O I
10.1186/1472-6750-11-28
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background: DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery. Results: We have observed that (1) the Tol2 transposase (but not piggyBac) is highly sensitive to molecular engineering; (2) the piggyBac donor with only the 40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective transposition; and (3) a small amount of piggyBac transposases results in robust transposition suggesting the piggyBac transpospase is highly active. Performing genome-wide target profiling on data sets obtained by retrieving chromosomal targeting sequences from individual clones, we have identified several piggyBac and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear difference in targeting preferences in the human genome. Finally, we have observed that (5) only sites with a particular sequence context can be targeted by either piggyBac or Tol2. Conclusions: The non-overlapping targeting preference of piggyBac and Tol2 makes them complementary research tools for manipulating mammalian genomes. PiggyBac is the most promising transposon-based vector system for achieving site-specific targeting of therapeutic genes due to the flexibility of its transposase for being molecularly engineered. Insights from this study will provide a basis for engineering piggyBac transposases to achieve site-specific therapeutic gene targeting.
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页数:19
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