Targeted Expression of Cre Recombinase Provokes Placental-Specific DNA Recombination in Transgenic Mice

Background: Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth. Principal Findings: By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein a (Tpbpa) and adenosine deaminase (Ada), we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus. Significance: In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of thi

The detrimental role of angiotensin receptor agonistic autoantibodies in intrauterine growth restriction seen in preeclampsia

Growth-restricted fetuses are at risk for a variety of lifelong medical conditions. Preeclampsia, a life-threatening hypertensive disorder of pregnancy, is associated with fetuses who suffer from intrauterine growth restriction (IUGR). Recently, emerging evidence indicates that preeclamptic women harbor AT1 receptor agonistic autoantibodies (AT1-AAs) that contribute to the disease features. However, the exact role of AT1-AAs in IUGR and the underlying mechanisms have not been identified. We report that these autoantibodies are present in the cord blood of women with preeclampsia and retain the ability to activate AT1 receptors. Using an autoantibody-induced animal model of preeclampsia, we show that AT1-AAs cross the mouse placenta, enter fetal circulation, and lead to small fetuses with organ growth retardation. AT1-AAs also induce apoptosis in the placentas of pregnant mice, human villous explants, and human trophoblast cells. Finally, autoantibody-induced IUGR and placental apoptosis are diminished by either losartan or an autoantibody-neutralizing peptide. Thus, these studies identify AT1-AA as a novel causative factor of preeclampsia-associated IUGR and offer two possible underlying mechanisms: a direct detrimental effect on fetal development by crossing the placenta and entering fetal circulation, and indirectly through AT1-AA–induced placental damage. Our findings highlight AT1-AAs as important therapeutic targets

Angiotensin II induces soluble fms-Like tyrosine kinase-1 release via calcineurin signaling pathway in pregnancy

Maternal endothelial dysfunction in preeclampsia is associated with increased soluble fms-like tyrosine kinase-1 (sFlt-1), a circulating antagonist of vascular endothelial growth factor and placental growth factor. Angiotensin II (Ang II) is a potent vasoconstrictor that increases concomitant with sFlt-1 during pregnancy. Therefore, we speculated that Ang II may promote the expression of sFlt-1 in pregnancy. Here we report that infusion of Ang II significantly increases circulating levels of sFlt-1 in pregnant mice, thereby demonstrating that Ang II is a regulator of sFlt-1 secretion in vivo. Furthermore, Ang II stimulated sFlt-1 production in a dose- and time-dependent manner from human villous explants and cultured trophoblasts but not from endothelial cells, suggesting that trophoblasts are the primary source of sFlt-1 during pregnancy. As expected, Ang II-induced sFlt-1 secretion resulted in the inhibition of endothelial cell migration and in vitro tube formation. In vitro and in vivo studies with losartan, small interfering RNA specific for calcineurin and FK506 demonstrated that Ang II-mediated sFlt-1 release was via Ang II type 1 receptor activation and calcineurin signaling, respectively. These findings reveal a previously unrecognized regulatory role for Ang II on sFlt-1 expression in murine and human pregnancy and suggest that elevated sFlt-1 levels in preeclampsia may be caused by a dysregulation of the local renin/angiotensin system

Generation of <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> chimeric expression vector and <i>in vitro</i> analysis of its Cre recombinase activity in human trophoblast cells.

<p>(<b>A</b>) Structure of p<i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> construct. <i>Tpbpa</i>r/<i>Ada</i>f chimeric enhancer and <i>Ada</i> basal promoter (<i>Ada</i>P) were ligated to the sequence encoding Cre cDNA containing a nuclear localization signal (NLS). (<b>B</b>) Schematic representation of pCAG-CATZ vector. The PCR primers, primer pair 1 (AG and Z3) were used to monitor Cre-mediated loxP-dependent DNA recombination (2100 bp for parental DNA, 690 bp for the recombined DNA). Primer pair 2 (CAT2 and CAT3) were internal primers used to detect pCAG-CATZ. (<b>C</b>) PCR analysis: pCAG-CATZ was transfected alone or together with CMV-Cre or different amounts of <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> into human trophoblast cells (HTR). DNA was isolated 48 h after transfection and assayed for the presence of the recombination-dependent 690 bp fragment. In the absence of Cre, only the 2100 bp precursor PCR fragment was observed. However, in the presence of Cre, both the 2100 bp precursorand the 690 bp product PCR fragments were detected. The amount of 690 bp PCR fragment observed increased with additional <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> transfected to the cells. The 320 bp PCR fragment was used to determine that pCAG-CATZ was transfected into the cells. (<b>D</b>) Schematic representation of AdMA19 vector. Spacer interposed between the <i>loxP</i> sites precludes efficient lucifearse expression in the absence of the Cre recombinase. (<b>E</b>) Luciferase analysis. AdMA19 vector was transfected with CMV-<i>Cre (CMV-Cre/AdMA19</i>,1∶1), different amounts of <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> (<i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre/AdMA19,1∶1 or 5∶1</i>) or alone. Cellular extracts were isolated 48 h after transfection and luciferase activity was measured. All data are expressed as mean ± SEM. n = 6. * <i>P</i><0.05 versus cells transfected with AdMA19 construct only. **<i>P<0.05 versus Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre/AdMA19,1∶1</i>.</p

Random assembly of placental specific enhancers and <i>in vitro</i> analysis of their ability to activate the basal promoter of the <i>Ada</i> gene in multiple cell types.

<p>(<b>A</b>) Schematic illustrations of the constructs. (<b>B</b>) Luciferase activity in human trophoblast (HTR) cells transfected with each construct. (<b>C</b>) Luciferase activity driven by <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P chimeric enhancer in multiple cell lines. Data are expressed as mean ± SEM. n = 4–6. * <i>P</i><0.05 versus cells transfected with <i>Ada</i>f-<i>Ada</i>P construct.</p

Placental-restricted DNA recombination in female <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> transgenic mice mated with male Z/EG double-reporter transgenic mice.

<p>(<b>A</b>) Schematic representation of <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> female mating with Z/EG transgenic male, double reporter mice. (<b>B</b>) <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> female transgenic mice were mated with Z/EG transgenic mice. On gestation E16.5, pregnant <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> mice were sacrificed and embryos and placentas were isolated. To define the genotype of each placenta, embryonic DNA was analyzed for the presence of <i>Tpbpa</i>r<i>/Ada</i>f-<i>Ada</i>P-<i>Cre</i> and <i>Z/EG</i> by PCR. β-actin was used as an internal control. (<b>C</b>, <b>D</b>) Expression patterns of <i>Cre</i> mRNA and GFP mRNA analyzed by RT-qPCR. nd, not detectable. (<b>E</b>) X-gal staining of multiple placentas from <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> pregnant transgenic mice. Nuclear fast red was used for counterstaining. Large arrows indicate the junctional zone and small arrows indicate giant cells. Scale bar: 1 mm. (<b>F</b>) Quantification of LacZ staining in spongiotrophoblast cell layer (sp layer). de, decidual cells; sp, spongiotrophoblast cells; gi, giant cells, la, labyrinth zone.</p

Placenta-restricted gene expression in <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> transgenic mice.

<p>Pregnant mice were sacrificed on gestation day 16.5 and placentas, multiple organs and embryos were collected. (<b>A</b>) Copy number of transgenes was determined by qPCR analysis in <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> transgenic founders. Genotyping analysis (<b>B</b>, <b>D</b>) of <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-<i>Cre</i> transgenes by PCR and expression pattern of <i>Cre</i> mRNA (<b>C</b>, <b>E</b>) analyzed by RT-qPCR in placenta, fetus and multiple maternal organs from female transgenic mice (derived from Tg 5 founder) mated with wild type FVB male mice. β-actin was used as an internal control. Tg placental RNA is used as positive control. nd, not detectable; P, Positive control; N, Negative control. (<b>F</b>) Immunochemistry staining of Cre recombinase using anti-Cre antibody in the placentas of pregnant <i>Tpbpa</i>r<i>/Ada</i>f<i>-Ada</i>P<i>-Cre</i> females mated with wild type FVB males. Placentas with <i>Tpbpa</i>r<i>/Ada</i>f-<i>Ada</i>P-<i>Cre</i> transgenes (Cr⁺(Tg)) expressed Cre protein in giant cells (indicated by long arrow), spongiotrophoblast cells (indicated by short arrow) and cells in the labyrinthine zone (indiated by arrow head)of placentas, with highest expression in the spongiotrophoblast zone. Panel <b>F</b> (inset) showed nuclear localization of Cre in trophoblast cells. Placentas lacking <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-Cre transgenes (Cre⁻, panel <b>F</b>) and multiple organs from pregnant transgenic dams (<b>G</b>) showed no Cre immunostaining. Endogenous ADA immunostaining was performed in placentas with or without <i>Tpbpa</i>r/<i>Ada</i>f-<i>Ada</i>P-Cre transgenes using anti-ADA antibody (panel <b>F</b>). Scale bar, 100 µm (placenta) or 50 µm (inset) and 500 µm for maternal organs.</p