Figure 1.
Injection of ccc-plasmids into the cytoplasm of a zygote.
A) Schematic depiction of cytoplasmic plasmid injection (CPI) into an opaque zygote. B) Cytoplasmic injection into a porcine zygote. C) For comparative reasons a pronuclear injection in a porcine zygote is shown. To reveal the pronuclei, a high speed centrifugation at 12'000–15'000 g is necessary.
Table 1.
Injection parameters and rates of transgenesis.
Figure 2.
Venus–transposon expression in tissues of the three germ layers and extraembryonic membranes.
Images of selected organs of F0 transgenic porcine fetuses (d30) derived from cytoplasmic injection of transposon plasmids into zygotes. A–F, specific excitation of the Venus fluorophore; A′–F′, corresponding bright field images. A, limb bud; B, eye; C, heart; D, intestine; E, E′, amnion of a non-transgenic fetus, E″, amnion of a transgenic fetus; F, mesonephros. G) Flow cytometric determination of Venus-fluorescence. Black line represents wildtype fibroblasts, blue and red lines, non-transgenic fibroblasts from fetuses #37-1 and #40-1. H) Flow cytometry of Venus-transposon transgenic fibroblasts. Red, blue, green and purple lines, fibroblasts from fetuses #37-2, #37-3, #37-4 and #37-5, respectively. The difference of fluorescence intensities between wildtype (G) and transgenic fibroblasts was so great that the transgenic fibroblasts were measured with reduced gain settings. Fibroblasts of #37-3 (blue) were mosaic, approximately 50% of the cells displayed a reduced fluorescence. I) Southern blot of fetal fibroblasts. M, molecular size marker; wt, wild type DNA; #37-1 to #37-5, fetal IDs; c, positive control: Venus plasmid digested with NcoI. The NcoI digest produces a constant fragment of 1.4 kb and a variable fragment >1.4 kb.
Figure 3.
Persistent transgene expression in transposon-transgenic pigs.
A) Transgenic boar (#505) viewed under specific excitation from side and front at the age of 2 months playing with an auto-fluorescent toy ball (left). B) Wildtype boar (left) and transgenic boar (#503, right, 8 months of age) photographed side-by-side under a light source with specific excitation of the Venus fluorophore. The animals are separated by a fence visible in the middle of the image. Blue appearance of wildtype animal is due to reflected and scattered excitation light. C) PCR genotyping of ear biopsies of born piglets for the presence of Venus, plasmid backbone; SB100X transposase, and control amplicons [poly(A) polymerase, POL(A)]. M, size marker, lane 1 (#505), lane 2 (#503), lanes 3–11 littermates (no 3-11); wt, wildtype pig sample. Lane 7 and lane 8 correspond to deceased piglets with Venus-fluorescence. Right, genotyping of different organs from stillborn piglet (no 12) with Venus fluorescence: M, size marker; 1, ear; 2, heart, 3, muscle; 4, spleen; 5, kidney; 6, liver, 7, no template. D) Southern blot of transgenic piglets. Genomic DNA was isolated from ear biopsies, NcoI digested and blotted with the Venus probe. M, molecular size marker; wt, wildtype; 1–5, genomic DNA from transgenic piglets; c, Venus plasmid control. E) Analysis of cell chimerism; wild type (wt_e and wt_s) and transgenic boars (#503: 1_e and 1_s; #505: 2_e and 2_s) genomic DNA from ear biopsies (_e) and spermatozoa (_s) was blotted and hybridized with the Venus probe. Note the different fragment patterns between tissues of the founders. In addition genomic DNA from total fibroblasts of fetus #37-5 (#37-5) and of the cell fraction sorted for absence of Venus fluorescence was probed (#37-5n).
Figure 4.
Segregation of Venus-transposons in F1-fetuses.
Fetuses derived from insemination of a wildtype sow with semen of transgenic boar #503 were isolated at day 29 p.c., and fluorescence images were taken under normalized conditions. A) Typical image of a strongly fluorescent fetus (F1-2), B) Typical image of a weakly fluorescent fetus (F1-4). The fluorescence intensities correlated with the transposon copy numbers, as determined by Southern blotting. C) Flow cytometric measurements of Venus-fluorescence in fibroblasts derived from F1-fetuses with weak fluorescence intensity: F1-1 (red), F1-5 (purple), F1-11 (yellow) and strongly fluorescent fetuses: F1-5 (blue) and F1-9 (green). D) Expression of Venus in different tissues of d29 porcine fetus (F1-3, strongly fluorescent) as determined by Northern blotting with a Venus-specific probe (top): head (1); carcasse (2); mesonephros (3); liver (4); heart/lung (5) and control samples from wildtype pig: heart (6); lung (7); liver (8). In addition, RNAs from wildtype murine heart (9) and lung (10); and RNAs from Venus-transposon transgenic murine heart (11) and lung (12) were loaded. Bottom, reprobed blot with an actin-specific probe. Porcine tissues show organ-specific splice patterns of actin transcripts [51]. E) Segregation of Venus-transposons in F1-animals. Genomic DNA from F1-fetuses was analysed by Southern blot with the Venus-probe. M, size marker; 1–10, genomic DNA from ten F1 offspring. Black arrow, internal, constant band at ∼1.4 kb; blue arrow, external fragment of one integrant; red arrow, external fragment of the other integrant. 1× and 2× indicate the deduced transposon copy numbers.
Figure 5.
Recombinase-mediated cassette exchange in Venus-transposon transgenic fibroblasts.
Primary fibroblasts from fetus #37-5 carry a single Venus-transposon. The Venus-transposon includes heterologous loxP sites (see Figure S1). Five day after co-electroporation of a Cre expression plasmid and an mCherry exchange plasmid, the cells were screened under brightfield (A), Venus-optics (A′) and mCherry optics (A″). The dashed circle indicates a cell, which presumptively underwent Cre-mediated cassette exchange (mCherry positive and Venus-negative). The arrow points to a cell with an illegitimate recombination event (mCherry positive and Venus positive). Some round cells (most likely dead cells) are floating in the medium and are out of the focus plane and thus do not appear in A′or A″. B–B″) Clonal isolation and expansion of Cre-recombined cells. Importantly, the screening and clonal isolation procedures are based only on fluorescence criteria and no antibiotics selection was applied. C) PCR confirmation of specific cassette exchange. Batch fibroblasts were analysed 10 days after electroporation. In lanes 1-6, primers specific for the Venus-transposon (see Fig. 6A for primer positions) were employed (amplicon size 480 bp); in lanes 1′–6′, primers specific for a successful RMCE event (see Fig. 6A) were employed, which specifically amplify a 395-bp fragment. Lanes 1–5 correspond to fibroblasts (#37-5) electroporated with no plasmid (1), with mCherry exchange plasmid (2), with mCherry and Cre plasmids (3), or were untreated (4), or wildtype fibroblast (5). Lane 6 is a negative control with no template. In lane 3′ an amplicon of the expected size for a successful RMCE event is detectable.
Figure 6.
Recombination-mediated cassette exchange in the pig.
A) Schematic depiction of RMCE in transposon-tagged porcine cells. B–D) Fetus obtained from nuclear transfer of RMCE fibroblasts photographed under brightfield (B), mCherry fluorescence (C) and Venus fluorescence (D) conditions.
Figure 7.
Schematic depiction of targeted transgenes in the pig genome.
First, SB-catalyzed transgenesis was employed to tag genomic loci, which are suitable for expression. Founder animals with appropriate expression levels and only one transposon integration were selected and used to derive primary cell cultures. Then, the reporter construct (Venus) was specifically exchanged against a gene of interest (here mCherry) via Cre recombinase and heterospecific loxP sites (blue triangles). Cells which underwent successful RMCE events were isolated by screening for loss of Venus fluorescence and gain of mCherry fluorescence, and were used in somatic cell nuclear transfer to establish cloned piglets, with targeted mCherry integration into a pre-tested genomic locus.