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Review
, 8 Suppl 1 (Suppl 1), S14

Germline Mutagenesis Mediated by Sleeping Beauty Transposon System in Mice

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Review

Germline Mutagenesis Mediated by Sleeping Beauty Transposon System in Mice

Junji Takeda et al. Genome Biol.

Abstract

Following the descovery of its transposition activity in mammalian culture systems, the Sleeping Beauty (SB) transposon has since been applied to achieve germline mutagenesis in mice. Initially, the transposition efficiency was found to be low in cultured systems, but its activity in germ cells was unexpectedly high. This difference in transposition efficiency was found to be largely dependent on chromosomal status of the host genomic DNA and transposon vector design. The SB transposon system has been found to be suitable for comprehensive mutagenesis in mice. Therefore, it is an effective tool as a forward genetics screen for tagged insertional mutagenesis in mice.

Figures

Figure 1
Figure 1
Constructs for generating the two transgenic mice. Sleeping Beauty (SB) transposon vector contains a green fluorescent protein (GFP) expression cassette flanked by SB transposon elements: inverted repeat/direct repeat (IR/DR)-L and IR/DR-R. SB transposase is driven by the CAG promoter. pA, poly(A) addition signal.
Figure 2
Figure 2
Generation and expression of GFP in double transgenic mice. (a) Transgenic mice bearing Sleeping Beauty (SB) transposon vector (green fluorescent protein [GFP] mice, left) were mated with transgenic mice expressing SB transposase (SB mice, right) to generate double transgenic mice. (b) No GFP signal was detected in peripheral blood by fluorescence activated cell sorting analysis. wt, wild-type.
Figure 3
Figure 3
Two possible schemes to account for the absence of GFP signal in double transgenic mice. In scheme I the integration step is extremely inefficient compared with the excision step. In scheme II both integration and excision steps are efficient, but the green fluorescent protein (GFP) signal is below the detection level, or suppression of the GFP gene is maintained at the new integration site.
Figure 4
Figure 4
GFP expression in progeny of double transgenic mice. Double transgenic mice were mated with wild-type mice to test transmission of Sleeping Beauty (SB) transposon insertions to progeny. If transmission of SB transposon insertions occur in progeny, then it would enable us to detect insertion with methods other than green fluorescent protein (GFP) expression, such as Southern blot analysis or fluorescence in situ hybridization analysis. Some of the progeny from the double transgenic mice are clearly GFP positive, and the intensity of GFP varied, suggesting that SB transposase mediated transpositions occurred and transposons integrated at various locations. wt, wild-type.
Figure 5
Figure 5
Possible explanation for why we could not detect GFP signal in double transgenic mice. Green fluorescent protein (GFP) expression at the donor site may be suppressed because of highly methylated transposon DNA (top panel). This suppression may be abrogated during germline transmission. However, the same suppression would be reintroduced at the donor site, probably because of the flanking sequence of the transposon DNA. The suppression may not be reintroduced into the transposed DNA (bottom panel).
Figure 6
Figure 6
A new version of transposon vector for gene trapping. Transcription of an endogenous gene is disrupted and β-galactosidase (β-gal) is expressed. Transcription by the CAG promoter generates a chimeric transcript of the green fluorescent protein (GFP) and the endogenous gene. IRES, internal ribosome entry site; pA, poly(A) addition signal; SA, splice acceptor; SD, splice donor.
Figure 7
Figure 7
Two possible strategies for comprehensive mutagenesis using SB transposition in mice. SB, Sleeping Beauty.

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