The so-called transgenes, by definition, is an exogenous gene integration into the host genome, present in mammalian systems most commonly used transgenic technology including Sleeping beauty (SB), PiggyBac ( PB) and Tol2 transposon family, however, one of the "star turn The seat is "Tol2!"
1, Tol2 transposition mechanism
The Tol2 transposon is a reverse repeat of the hAT transposon family, belonging to the hAT transposon family, with a full length of 4682 bp and 17 bp and 19 bp, respectively, containing four exons (Fig. 1). The encoded protein contains 649 amino acid residues.
As an autonomous transposable element, Tol2 itself encodes a transposase and, by far, is the only transposon found in vertebrates with autonomous transposable activity [1] . The partial deletion of the transposase coding region sequence, but retaining the transposase recognition and binding region sequences, Tol2 could not undergo autonomous transposition (Fig. 1). 
Figure 1 Tol2 transposon sequence [1]
Using this property, the foreign gene can be substituted for the transposase coding sequence, and the Tol2 transposon vector can be constructed and transferred into the host cell together with the transposase cDNA vector plasmid, and the foreign gene will be integrated in a "cut-and-paste" manner. Into the genome and does not cause rearrangement and modification of any target sites [2] , as shown in Figure 2.
Figure 2 Tol2 transposition mechanism [2]
2, Tol2 application
The Tol2 transposon is non-directional, ie it is randomly inserted in the genome, and there is no sequence specificity. Since Tol2 is so capricious, what is the use?
Initially, scientists used this feature as a tool for gene capture and enhancer capture to perform mutant screening to identify mutants of important functional genes during the body's development. However, with the continuous advancement of science and technology, automated high-throughput screening and identification of genes has long been a problem, thinking that tol2 will be eliminated. nonexistent! Today's tol2 transposon still plays a unique technical advantage in establishing stable gene expression cell lines and preparation of transgenic mice and mice.
Of course, practice is the only criterion for testing truth. In 2009, Zoltán Ivics et al. [3] elaborated on the application of the Tol2 transposition mechanism in rats. The specific process is shown in Figure 3. Through continuous phenotypic screening, the Tol2 system can Used to construct transgenic mice and stable gene expression cell lines.
Figure 3 Construction of transgenic mice and gene expression cell lines using Tol2 [3]
3, Tol2 transposition advantage
Compared with the traditional transgenic method, the Tol2 transposon method has obvious advantages, mainly in the following aspects:
Relatively higher transgenic efficiency
Different strains of mice, the same differences in the efficiency of transposition of the transposon, the same strains of mice, compared with the Tol2 SB, PB transposition higher efficiency germ cells [4], as shown in Table 1.
Table 1 Transposition efficiency of different transposons in germ cells 
Site-specific
Compared with PB and SB, the Tol2 transposon target has higher selectivity, no obvious site specificity (8-bp hybrid base sequence), and tends to be integrated on the chromosome at the 5' end of the gene. Area [3] , as shown in Table 2 .
Table 2 Genomic site-specific comparison of SB, PB and Tol2 
Abasic propensity
Tol2 transposon, the base sequence of the insertion site can be different, and there is no obvious base tendency [5] . As shown in Table 3.
Table 3 Base propensity of Tol2 insertion site 
No excessive inhibition effect
As shown in Table 4 below, as the ratio of transposase to transposon increased, the efficiency of PB transposition gradually became saturated or even decreased, indicating that overexpressed transposase inhibited the transposition reaction of PB, ie, excessive inhibition effect. However, Tol2 is not affected by the transposition efficiency, and there is no inhibition effect [6] .
Table 4 Comparison of PB and Tol2 transposition inhibition effects 
In addition, tol2 transposition does not require insulators and can carry large fragments of exogenous DNA. At present, Biotech has successfully transferred 13kb exogenous DNA fragments into mice using this system!
Ok, the space is limited. Today, science is over, but I will be back soon. ! Boss who need tol2 transgenic mice, the Olympics is waiting for you~~
[ Reference ]
[1] Koga A. and Hori H. The Tol2 transposable element of the medaka fish: an active DNA-based element naturally occurring in a vertebrate genome [J]. Genes & Genetic Systems, 2001, 76:1-8.
[2] DNA transposon-based gene vehicles - scenes from an evolutionary drive. J Biomed Sci. 2013 Dec 9;20:92.
[3] Transposon-mediated Genome Manipulations in Vertebrates. Nat Methods. 2009 June ; 6(6): 415–422.
[4] Efficient transposition of Tol2 in the soap germline. Genetics. 2009 Dec;183(4):1565-73.
[5] A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection. Genomics. 2010 May;95(5):306-11.
[6]. Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons. Proc Natl Acad Sci US A. 2012 Jun 5;109(23):E1466-72.
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