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. 2001 Jul 31;98(16):9191-6.
doi: 10.1073/pnas.161071798.

Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice

K Horie  1 , A Kuroiwa, M Ikawa, M Okabe, G Kondoh, Y Matsuda, J Takeda
Affiliations

Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice

K Horie et al. Proc Natl Acad Sci U S A. .

Abstract

The presence of mouse embryonic stem (ES) cells makes the mouse a powerful model organism for reverse genetics, gene function study through mutagenesis of specific genes. In contrast, forward genetics, identification of mutated genes responsible for specific phenotypes, has an advantage to uncover novel pathways and unknown genes because no a priori assumptions are made about the mutated genes. However, it has been hampered in mice because of the lack of a system in which a large-scale mutagenesis and subsequent isolation of mutated genes can be performed efficiently. Here, we demonstrate the efficient chromosomal transposition of a Tc1/mariner-like transposon, Sleeping Beauty, in mice. This system allows germ-line mutagenesis in vivo and will facilitate certain aspects of phenotype-driven genetic screening in mice.

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Figures

Figure 1
Figure 1
An overview of the experimental system. (a) Strategy to identify transposition events. The multiply integrated GFP genes flanked by transposon elements (gray arrows) are epigenetically repressed at the original integration site. Excision of transposons by SB transposase releases GFP genes from repression status, and transposition events are detected by GFP activity at the novel site. (b) Constructs used to create transgenic mice. pTransCX-GFP:Neo contains a GFP expression cassette flanked by IR/DR-R and IR/DR-L, constituting an SB transposon element. Two fragments used for generating transgenic mice are described in the text. pCX-SB is the expression vector of SB transposase. Black arrowheads, loxP site; gray arrows, IR/DR-L and IR/DR-R; open bar, pBluescript II; CAG, CAG promoter; pA, polyA addition signal. The GFP probe used for Southern blot analysis in c is shown as a black bar. (c) Estimation of transposon copy number by Southern blot analysis. Genomic DNA from the lTRA-GFP-B transgenic mice was digested by KpnI, and indicated amounts of DNAs were loaded on a gel. As a control for copy number estimation, 5 μg of KpnI–XhoI digested genomic DNA from an ES cell line (ES/GFP) containing a single copy of GFP gene was included. The band sizes are shown on the left side.
Figure 2
Figure 2
Transposon excision in lTRA-GFP-B:SB doubly transgenic mice. (a) Structure of vector DNA at the original integration site. PCR primers are located 4.8 kb apart before excision, which is beyond the PCR capacity. Transposon excision by SB transposase brings two primers close enough for PCR amplification. PCR primers are shown by thin arrows. Other symbols and abbreviations are as shown in the legend to Fig. 1. (b) Transposon excision specific for lTRA-GFP-B:SB doubly transgenic mice. Tail DNAs from two mice carrying both transposon and SB transposase gene (GFP+SB+), four mice carrying only transposon (GFP+SB−), and two mice without both transgenes (GFP−SB−) were analyzed. Size marker is a 100-bp ladder. (c) Sequence of the PCR product. Three nucleotides (cag or ctg) and TA dinucleotides repeat shown in bold are the unique footprints typically left at the excision sites (11). Both “a” and “t” were identified in the middle of the footprints by direct sequencing of the PCR product. The rest of the sequence completely matched the vector sequence. (d) High frequency of excision events in lTRA-GFP-B:SB doubly transgenic mice. Genomic DNAs from tail (Upper) and blood (Lower) of lTRA-GFP-B:SB doubly transgenic mice were serially diluted and analyzed for excision events by PCR. Amount of DNAs used per reaction is shown at the top of the panels.
Figure 3
Figure 3
Activation of GFP expression in the progeny from lTRA-GFP:SB doubly transgenic mice. lTRA-GFP:SB doubly transgenic mice were obtained by the mating of lTRA-GFP mice and SB transgenic mice. Neither lTRA-GFP transgenic mice carrying GFP gene only or lTRA-GFP:SB doubly transgenic mice showed GFP signal. However, some of the progeny from lTRA-GFP:SB doubly transgenic mice were clearly GFP positive.
Figure 4
Figure 4
Molecular characterization of transposition. (a) Restriction map at the parental and novel integration sites. Location of GFP probe used in b–d is shown by a black bar. Other symbols and abbreviations are as described in the legend to Fig. 1. (b) Southern blot analysis of the progeny from a male lTRA-GFP-B:SB doubly positive mouse by AseI–KpnI digestion. GFP+SB− is the lTRA-GFP-B mouse carrying the transposon only, GFP+SB+ is the lTRA-GFP-B:SB doubly positive mouse, and the rest of the mice are the progeny from the mating of a male lTRA-GFP-B:SB doubly positive mouse and a wild-type mouse (Fig. 3). Presence or absence of GFP signals is shown under the panel. Location of the strong 4-kb band coming from multiple copies of transposon at the original integration site is shown on the left side. Arrowheads indicate the extra bands. Seven micrograms of DNAs were loaded per lane. (c) Southern blot analysis of the same progeny by PstI–KpnI digestion. Twenty micrograms of DNAs were loaded per lane except in GFP+SB−, in which 3 μg were loaded. Location of the 2.9-kb band resulting from the original integration site is shown on the left side. A faint band in mouse 1-8 is indicated by an asterisk. Presence or absence of the SB transposase gene determined by PCR is shown under the panel. (d) Southern blot analysis of the progeny from a female lTRA-GFP-B:SB doubly positive mouse by AseI–KpnI digestion. Seven micrograms of DNAs were loaded per lane. (e) Flanking sequences at the novel integration sites in mice 1-8 and 1-9. The TA dinucleotides flanking the SB transposon are shown in bold uppercase.
Figure 5
Figure 5
Chromosomal localization of the parental integration site (a and b), the SB transposase gene (c), and the novel integration sites in the progeny from the lTRA-GFP-B:SB doubly transgenic mouse (d–h) by FISH analysis. The names of the mice examined are shown at the right bottom corner of each panel, and the chromosomal localizations of the signals are presented by arrows. In d–h, the parental integration site and the SB transposase gene transmitted from their parent are indicated by lTRA-GFP-B and SB in parentheses, respectively. In d, the signals are shown at high magnification at the upper right corner. G-banded and R-banded patterns are demonstrated in a and b–h, respectively.
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