Assessment of Fetal Cell Chimerism in Transgenic Pig Lines Generated by <i>Sleeping Beauty</i> Transposition

<div><p>Human cells migrate between mother and fetus during pregnancy and persist in the respective host for long-term after birth. Fetal microchimerism occurs also in twins sharing a common placenta or chorion. Whether microchimerism occurs in multiparous mammals such as the domestic pig, where fetuses have separate placentas and chorions, is not well understood. Here, we assessed cell chimerism in litters of wild-type sows inseminated with semen of transposon transgenic boars. Segregation of three independent monomeric transposons ensured an excess of transgenic over non-transgenic offspring in every litter. Transgenic siblings (n = 35) showed robust ubiquitous expression of the reporter transposon encoding a fluorescent protein, and provided an unique resource to assess a potential cell trafficking to non-transgenic littermates (n = 7) or mothers (n = 4). Sensitive flow cytometry, fluorescence microscopy, and real-time PCR provided no evidence for microchimerism in porcine littermates, or piglets and their mothers in both blood and solid organs. These data indicate that the epitheliochorial structure of the porcine placenta effectively prevents cellular exchange during gestation.</p></div

Assessment of potential chimerism in non-transgenic littermates and sows.

<p>ID, unique identification number; Ct, threshhold cycle in a quantitative real time PCR;</p><p>∞, no threshold cycle reached during PCR;</p><p>n.d., not done;</p>§<p>, sow with two “transgenic” pregnancies.</p

Assessment of reporter-positive cells in solid organs.

<p>A, B) Cryosections of heart and testis of a Venus-transposon pig are depicted under specific excitation of Venus and brightfield conditions (insets). C) Heart, and D) testis sections of a non-transgenic littermate were recorded under identical camera settings. White bar = 50 µm.</p

Reporter transposon and potential routes of inter-fetal and feto-maternal cell trafficking in the pig.

<p>A) Schematic depiction of a monomeric reporter transposon in chromosomal context. The SB transposon consists of <i>CAGGS</i> promoter, <i>Venus</i> cDNA and polyadenylation sequence flanked by SB inverted terminal repeats (ITRs). Drawing not at scale. B) Fetal cells might traffic directly between fetuses (I.) or via the maternal circulation (II.). In both cases the non-transgenic fetuses should carry Venus-expressing cells. In case (II.), the mother should also show cell chimerism. For simplification only two fetuses are depicted and only the transgenic fetus is shown in green, however, cells of amnion and allantois are also Venus positive. C) Specific Venus fluorescence in embryonic allantois at day 25 of gestation. Cryosection of a <i>CAGGS-Venus</i> transgenic implantation in a wild-type sow. D) Overlay of fluorescence and brightfield views. E) Corresponding brightfield view. A dotted line indicates the border between embryonic and maternal tissue. Bar = 10 µm.</p

Flow cytometric measurement of porcine leucocytes for Venus-positive cells.

<p>A) Venus transposon transgenic pig (#515), B) Wild-type pig, C) Non-transgenic littermate, D) Sow #404 (delivered two litters of transposon piglets), E) Determination of the detection limit of flow cytometry. Leukocytes from a wild-type animal were spiked with decreasing amounts of Venus-positive leukocytes (n = 3 technical and biological replicates for each dilution). The dotted line indicates the theoretically expected cell counts. A detection limit of 1 Venus cell in 100 000 wild type cells was determined. F) RT-PCR of a dilution series of <i>Venus</i> gDNA in wildtype DNA.</p

<i>In vitro</i> and <i>in vivo</i> developmental rates of prepubertal and adult oocytes cultured pre- and during IVM with or without cAMP modulators.

<p><i>In vitro</i> and <i>in vivo</i> developmental rates of prepubertal and adult oocytes cultured pre- and during IVM with or without cAMP modulators.</p

Total number of OPU sessions, total number of follicles, total number of oocytes per donor and suitable for IVM obtained via ovum pick up in adult and prepubertal donors.

<p>Total number of OPU sessions, total number of follicles, total number of oocytes per donor and suitable for IVM obtained via ovum pick up in adult and prepubertal donors.</p

Gene expression profiles in expanded blastocysts produced from adult and prepubertal oocytes treated with or without cAMP modulators prior to and during IVM.

<p>Data are shown as the mean ± SEM (n = 12). Data were analyzed using two-way ANOVA followed by a Tukey's range test. The asterisk represents statistical significance among treatments for the same transcript; <i>P</i> < 0.05. <i>In vivo</i> produced expanded blastocysts were used for comparison. The mRNA relative abundance of the EGR1 gene was lower in all <i>in vitro</i> derived blastocysts compared to <i>in vivo</i> produced counterparts. No differences among treatments were found for <i>DNMT3b</i>, <i>BCL2L1</i>, <i>PRDX1</i>, <i>SLC2A8</i>.</p