Mammalian germ-line transgenesis by transposition

Abstract

Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic screens. We tested a functional transposon called Sleeping Beauty in the one-cell mouse embryo. In this report, we describe experiments in which transposon vectors were injected into one-cell mouse embryos with mRNA expressing the SB10 transposase enzyme. Molecular evidence of transposition was obtained by cloning of insertion sites from multiple transgenic mice produced by SB10 mRNA/transposon coinjection. We also demonstrate germ-line transmission and expression from transposed elements. This technique has promise as a germ-line transgenesis method in other vertebrate species and for insertional mutagenesis in the mouse.

Figure 1

Verifying activity of SB10 mRNA. In vitro-transcribed SB10 mRNA was injected into zebrafish embryos homozygous for a transposon insertion (line E). Embryos are then collected after 24 h, and genomic DNA is extracted from them (a). PCR is then performed by using primers that flank the site of transposon insertion. Transposition can then be detected by appearance of the PCR product from the transposon insertion site after excision (b). Excision can be confirmed by sequencing the PCR product to detect the three nucleotide footprint (c). The sequence of the insertion site following transposon excision is indicated (*). Two different base-pair footprints are found in roughly equal proportion, CAG or CTG.

Figure 2

Structure and analysis of transposon integration sites. (a) The structure of the linear transposon that was used for pronuclear injection is shown. The transposon contains a GFP reporter (gray arrow) driven by the immediate early CMV promoter (black arrow) followed by the bovine GH polyadenylation site (black box) and flanked by the IR/DRs (white arrows). (b Upper) The structure of a Sleeping Beauty-mediated integration site including the expected fragments from a NcoI (N) digest. (b Lower) The structure of a random integration site and NcoI restriction map. The probes used for Southern blot analysis are also defined as black bars. (c) Examples of three embryonic DNAs analyzed by Southern blotting are shown. The results shown were achieved with the GFP probe (Left) and the plasmid probe (Right).

Figure 3

Molecular analysis of flanking genomic DNA sequences. The flanking genomic DNA sequences from transposon insertions were cloned by using either inverse PCR or splinkerette PCR. The last 10 nucleotides of the IR/DR (bold and underlined) next to the flanking genomic DNA is shown. Some clones were mapped by using the whole mouse genome database from Celera Coproration. The map position for some clones is listed. Chr., chromosome; cM, centimorgan.

Figure 4

Structure of pT/K14A transposon reporter and analysis of a T/K14A+ mouse line. (a) The structure of the linear pT/K14A transposon that was used for pronuclear injection is shown. The transposon contains an agouti reporter (Ag,gray arrow) driven by the keratin-14 gene promoter (K14, black arrow) followed by the human GH polyadenylation site (pA, black box) and flanked by the IR/DRs (white arrows). Also shown are the SspI restriction enzyme sites (S) and probes used for Southern blotting. The three different coat color phenotypes observed in the offspring and founder are also shown: B = normal C57BL/6, I = intermediate expression in the skin but not the coat, Y = yellow coat color in the skin and coat. (b) Southern blot analysis of one T/K14A+ founder (red box) and 14 offspring generated by backcrossing to C57BL/6 is shown. Four independent transposon insertion sites are indicated by arrows B–E at the left. Band A represents a junction fragment that so-segregates with bands C and D. The coat color phenotype is indicated above each lane corresponding to a mouse in the top panel.

Figure 5

PCR genotyping of founder and offspring genomic DNA. Offspring of an agouti founder were genotyped by using primers based on flanking sequences cloned from insertion site E (a) or insertion site B (b). In both cases, a second reaction was performed by using a transposon-specific primer and a primer in the flanking genomic DNA to detect the transposon insertion. (c) Mice heterozygous for the E insertion were intercrossed. Offspring were genotyped by PCR, and an example of a mouse with each genotype is shown.