Figure 1.
Random assembly of placental specific enhancers and in vitro analysis of their ability to activate the basal promoter of the Ada gene in multiple cell types.
(A) Schematic illustrations of the constructs. (B) Luciferase activity in human trophoblast (HTR) cells transfected with each construct. (C) Luciferase activity driven by Tpbpar/Adaf-AdaP chimeric enhancer in multiple cell lines. Data are expressed as mean ± SEM. n = 4–6. * P<0.05 versus cells transfected with Adaf-AdaP construct.
Figure 2.
Generation of Tpbpar/Adaf-AdaP-Cre chimeric expression vector and in vitro analysis of its Cre recombinase activity in human trophoblast cells.
(A) Structure of pTpbpar/Adaf-AdaP-Cre construct. Tpbpar/Adaf chimeric enhancer and Ada basal promoter (AdaP) were ligated to the sequence encoding Cre cDNA containing a nuclear localization signal (NLS). (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. (C) PCR analysis: pCAG-CATZ was transfected alone or together with CMV-Cre or different amounts of Tpbpar/Adaf-AdaP-Cre 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 Tpbpar/Adaf-AdaP-Cre transfected to the cells. The 320 bp PCR fragment was used to determine that pCAG-CATZ was transfected into the cells. (D) Schematic representation of AdMA19 vector. Spacer interposed between the loxP sites precludes efficient lucifearse expression in the absence of the Cre recombinase. (E) Luciferase analysis. AdMA19 vector was transfected with CMV-Cre (CMV-Cre/AdMA19,1∶1), different amounts of Tpbpar/Adaf-AdaP-Cre (Tpbpar/Adaf-AdaP-Cre/AdMA19,1∶1 or 5∶1) or alone. Cellular extracts were isolated 48 h after transfection and luciferase activity was measured. All data are expressed as mean ± SEM. n = 6. * P<0.05 versus cells transfected with AdMA19 construct only. **P<0.05 versus Tpbpar/Adaf-AdaP-Cre/AdMA19,1∶1.
Figure 3.
Placenta-restricted gene expression in Tpbpar/Adaf-AdaP-Cre transgenic mice.
Pregnant mice were sacrificed on gestation day 16.5 and placentas, multiple organs and embryos were collected. (A) Copy number of transgenes was determined by qPCR analysis in Tpbpar/Adaf-AdaP-Cre transgenic founders. Genotyping analysis (B, D) of Tpbpar/Adaf-AdaP-Cre transgenes by PCR and expression pattern of Cre mRNA (C, E) 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. (F) Immunochemistry staining of Cre recombinase using anti-Cre antibody in the placentas of pregnant Tpbpar/Adaf-AdaP-Cre females mated with wild type FVB males. Placentas with Tpbpar/Adaf-AdaP-Cre 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 F (inset) showed nuclear localization of Cre in trophoblast cells. Placentas lacking Tpbpar/Adaf-AdaP-Cre transgenes (Cre−, panel F) and multiple organs from pregnant transgenic dams (G) showed no Cre immunostaining. Endogenous ADA immunostaining was performed in placentas with or without Tpbpar/Adaf-AdaP-Cre transgenes using anti-ADA antibody (panel F). Scale bar, 100 µm (placenta) or 50 µm (inset) and 500 µm for maternal organs.
Figure 4.
Characterization of Tpbpar/Adaf-AdaP-Cre transgene expression at different times during pregnancy.
Pregnant mice were sacrificed on days E9.5, E14.5 and E16.5 and placentas were collected. (A) RT-PCR was used to analyze the expression levels of Cre and β-actin mRNA in the placentas. Representative expression patterns of Cre and β-actin mRNA in placentas (lane 1 and 2) are shown. N, negative control placenta without transgenic Cre mRNA. (B) The presence of the Cre transgene was assessed by PCR analysis of Tpbpar/Adaf-AdaP-Cre DNA in placentas of pregnant mice. β-actin DNA was used as an internal control.
Figure 5.
Placental-restricted DNA recombination in female Tpbpar/Adaf-AdaP-Cre transgenic mice mated with male Z/EG double-reporter transgenic mice.
(A) Schematic representation of Tpbpar/Adaf-AdaP-Cre female mating with Z/EG transgenic male, double reporter mice. (B) Tpbpar/Adaf-AdaP-Cre female transgenic mice were mated with Z/EG transgenic mice. On gestation E16.5, pregnant Tpbpar/Adaf-AdaP-Cre mice were sacrificed and embryos and placentas were isolated. To define the genotype of each placenta, embryonic DNA was analyzed for the presence of Tpbpar/Adaf-AdaP-Cre and Z/EG by PCR. β-actin was used as an internal control. (C, D) Expression patterns of Cre mRNA and GFP mRNA analyzed by RT-qPCR. nd, not detectable. (E) X-gal staining of multiple placentas from Tpbpar/Adaf-AdaP-Cre 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. (F) Quantification of LacZ staining in spongiotrophoblast cell layer (sp layer). de, decidual cells; sp, spongiotrophoblast cells; gi, giant cells, la, labyrinth zone.