Regulated transposition of a fish transposon in the mouse germ line - PubMed (original) (raw)

Regulated transposition of a fish transposon in the mouse germ line

S E Fischer et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Tc1/mariner elements are able to transpose in species other than the host from which they were isolated. As potential vectors for insertional mutagenesis and transgenesis of the mouse, these cut-and-paste transposons were tested for their ability to transpose in the mouse germ line. First, the levels of activity of several Tc1/mariner elements in mammalian cells were compared; the reconstructed fish transposon Sleeping Beauty (SB) was found to be an order of magnitude more efficient than the other tested transposons. SB then was introduced into the mouse germ line as a two-component system: one transgene for the expression of the transposase in the male germ line and a second transgene carrying a modified transposon. In 20% of the progeny of double transgenic male mice the transposon had jumped from the original chromosomal position into another locus. Analysis of the integration sites shows that these jumps indeed occurred through the action of SB transposase, and that SB has a strong preference for intrachromosomal transposition. Analysis of the excision sites suggests that double-strand breaks in haploid spermatids are repaired via nonhomologous end joining. The SB system may be a powerful tool for transposon mutagenesis of the mouse germ line.

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Figures

Figure 1

Figure 1

Tc1/mariner transposons compared for their activity in HeLa cells. (A) Structure of the transposons tested and of the corresponding transposases (below the transposon). The black boxes represent the terminal inverted repeats. The diagonal lines indicate the positions of the SV40-G418-resistance cassette. The transposon sequence between these lines was deleted. The sizes of the wild-type and, parenthetically, disrupted transposons, are indicated. Each tagged transposon was cloned into the polycloning site in pUC18/19. The gray box in each the transposase protein represents the (predicted) DNA-binding domain, whereas the white part represents the catalytic domain containing the catalytic amino acids DD(34)E or DD(34)D(50). Relevant mutions are indicated beneath each transposon. All five ORFs have comparable numbers of optimal and nonoptimal human codons and are preceded and followed by identical sequences. (B) Activity of Tc1/mariner elements compared in HeLa cells. A vector with the transposon marked by an SV40-G418-resistance cassette was cotransfected with a CMV expression vector with either the ORF of the corresponding transposase or with the ORF of β-galactosidase. G418-resistant colonies were selected and counted. The transposition activity (indicated on the y axis) is the ratio of the number of resistant colonies obtained when the transposase expression vector is cotransfected over the number of resistant colonies when the β-galactosidase expression vector is cotransfected (1 indicates no transposition). The activity indicates an average of between three and nine independent transfections. The error bars indicate SEM.

Figure 2

Figure 2

Generation and analysis of the transgenic mice. (A) Structure of the transgenes. The_Prm1-SB_-β -globin transgene is a fragment of plasmid pRP1345. A fragment of the mouse protamine 1 (Prm1) promoter was cloned in front of the ORF encoding the SB transposase, followed by the rabbit β-globin polyadenylation signal. The SB/neo transgene is a fragment of plasmid pT/neo. It has an SV40-G418R cassette in between the inverted repeats of the transposon. The fragments were injected into different FVB/N oocytes. Five founders (designated as lines A–E) transmitting the Prm1_-SB transgene to their offspring were obtained, ranging in copy number from 1 to 5. One line carrying a single copy of the complete SB/neo transposon was obtained (line 1).(B)_ RT-PCR analysis of the expression of SB transposase mRNA in several tissues, including brain (B), liver (L), skeletal muscle (M), testis (T) and kidney (K) of wild-type and transgenic mice (shown are lines B and E) using primers indicated in A by arrows.

Figure 3

Figure 3

(A) Mating scheme to induce and detect SB/neo transposition in mouse spermatids. Transposition events that occur in the spermatids of double transgenic males can be detected in their progeny. The progeny are analyzed for the presence of the SB/neo transposon by PCR. SB/neo+ animals (boxed) are further analyzed by PCR for the presence of SB/neo at the original locus and by Southern blotting to detect possible transposition events (shown in_B_). Males with SB/neo at a new location were further crossed to wild-type mice. SB/neo+ progeny (boxed) were analyzed to detect possible transposition events (shown in_D_). As an example, the lineage of animal 1734 is indicated (black text boxes). The heterozygous mice with an SB insertion were not homozygosed and had no obvious phenotypes. (B) Southern blot analysis of_Eco_RI-digested tail tip DNA of_Prm1-_SB+, SB/neo+ fathers and their offspring. The Eco_RI site in SB/neo and the probe, a fragment of the neo-resistance gene, are indicated in Fig. 2_A. Animal 1047 is the SB/neo+ founder animal (line 1) and shows the SB/neo transgene at its original locus. This line was crossed to all five_Prm1-_SB lines, lines A to E. Double transgenic males of lines 1A, 1B, 1C, 1D, and 1E were obtained and crossed with wild-type females. Of their progeny, animal 1724 did not inherit the SB/neo transgene; animals 1726 and 1728 did inherit the SB/neo transgene at its original locus. The remaining progeny represent 11 of 20 animals in which the SB/neo transposon had moved from its original locus to a new site (as the change in fragment size indicates). PCR analysis using a primer flanking the transposon at its original locus and a primer in the transposon confirmed the presence or absence of the transposon at the original locus. (C) Map of mouse chromosome 5 indicating the SB insertion sites. The insertion sites were mapped between the markers indicated, using the T31 mouse/hamster radiation hybrid panel. The distance between the original site on distal chromosome 5 and the region on central chromosome 5 is ≈25 cM. (D) Southern blot analysis of_Eco_RI-digested tail tip DNA of a_Prm1-_SB+, SB/neo+ father and his offspring. Animal 1047 is the SB/neo+ founder animal (line 1). Animal 1734 is progeny of a double transgenic male (line 1A), which has SB/neo at a new site and inherited the_Prm1-_SB transgene. Six progeny of mouse 1734 crossed to a wild-type female are shown: animals 1866, 1872, and 1874 have the transposon at the location of their father, animal 1734. Animal 1869 did not inherit the transposon. In animals 1867 and 1868 new transposition events were detected. PCR analysis confirmed the presence or absence of the transposon at the 1734 locus in the progeny of animal 1734. The positions of the fragments containing SB at its original site in animal 1047, and at the site in animal 1734, are indicated by arrows.

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