Animals

This study was conducted with female imprinting control region strain mice, 7-8 weeks old (SLC, Shizuoka, Japan). The mice were housed in an animal care facility with a controlled environment, and females were kept with males of the same strain overnight. When a vaginal plug was detected the next morning, that was considered postcoital day 0.5 of the pregnancy. All animal experiments were approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine.

Preparation of the hemagglutinating virus Japan envelope (HVJ-E) vector–mediated siRNA for in vivo transfer

Ten thousand hemagglutinating activity units of an inactivated column-purified HVJ-E vector, Genome-ONE NEO (Ishihara Sangyo Co Ltd, Fukuoka, Japan) initially were centrifuged at 13,500 g for 15 minutes at 4°C. The pellet was mixed with 20 μL of 2% Triton-X/TE and centrifuged at 13,500 g for 15 minutes at 4°C. After removal of the supernatant, the tube was tapped to collapse the pellet. The pellet was dissolved in 5 nm of siRNA solution for 5 mice and mixed well. Finally, human tubal medium (Nippon Medical and Chemical Instrument Co Ltd, Osaka, Japan) was added to yield a final volume of 250 μL. RCAS1 siRNA (5′-CAACUAUACGGAAAACUCA-3′; n = 21) and the scrambled siRNA (5′-ACCUAACUAAUCGGAAAAC-3′; n = 14) as a negative control were used (Sigma-Genosys Ltd, Hokkaido, Japan). SiRNA target sequence was defined with the use of a web-based online software system (siDirect sequences) for computing highly effective siRNA sequences with maximum target specificity for mammalian RNA interference ( http://genomics.jp/sidirect/index.php?type=fc ).

In vivo siRNA transfer experiments

Mice on postcoital day 7.5 were anesthetized with Nembutal (Dainippon Sumitomo Pharma, Osaka, Japan) that was diluted in phosphate-buffered saline solution at a concentration of 1.25 mg/mL (1.5 mL/mouse introduced intraperitoneally) and subjected to laparotomy to expose the pregnant uterus. Twenty-five microliters of vector-mediated siRNA suspension was injected into the upper (ovarian) side of the uterine cavity of each horn with a 30G needle. The needle was inserted into the uterine cavity between the amniotic sacs, outside the amniotic cavity. The cervix was clamped for 10 minutes. All the procedures were performed slowly, taking care not to rupture the amniotic membranes or not to damage the cervix. Finally, the incision was closed to allow the recovery of the mice.

Sample preparation for Western blotting and immunohistochemical analysis

We collected uteri that contained fetuses from pregnant mice on postcoital day 8.5 at 24 hours after siRNA transfer. Samples from 5 mice were obtained from each group at the designated day of gestation. A portion of the collected tissues was paraffin embedded; the remaining tissues were frozen in liquid nitrogen and stored at –80°C until use.

Western blotting analysis of whole uteri from pregnant mice on postcoital day 8.5 at 24 hours after siRNA transfer

The specimens of uteri with placentae and fetuses (each approximately 100 mg in weight, frozen at –80°C) were homogenized with 0.5 mL of lysis buffer that consisted of distilled water, 0.5 mol/L Tris-HCl (pH 6.8), 10% sodium dodecylsulfate, glycerol, 6% β-mercaptoethanol and 1% bromophenol blue. Homogenates were centrifuged at 4°C for 20 minutes at 1200 g . An Immunomini NJ-2300 apparatus (InterMed, Tokyo, Japan) was used to determine the protein concentration in the lysates. Fifty micrograms of protein was subjected to electrophoresis on 10% sodium dodecylsulfate-polyacrylamide gel with a Bio-Rad Power-Pac 200 apparatus (Bio-Rad Laboratories Inc, Hercules, CA) then were transferred to a nitrocellulose membrane (0.45 μm) with a Bio-Rad Mini-Transblotter apparatus (Bio-Rad Laboratories Inc). Commercially available mouse lung tissue extract was used as a positive control (Santa Cruz Biotechnology Inc, Santa Cruz, CA). The membrane was incubated with 5% dry milk (Amersham Biosciences, Arlington, IL) in Tris-tween buffered saline (TTBS) buffer (pH 8.0) that contained 0.005 mol/L Tris-HCl, 0.138 mol/L NaCl, 0.0027 mol/L KCl, and 0.1% Tween x 20, (Nacalai Tesque Inc, Kyoto, Japan) followed by incubation with the primary goat polyclonal antibody to RCAS1/EBAG9 (Abcam Inc, Cambridge, MA) in TTBS buffer at a dilution of 1:500. The primary polyclonal goat anti–β-actin antibody (Abcam Inc) at a dilution of 1:500 and a normal goat immunoglobulin G (Santa Cruz Biotechnology Inc) were used as loading and negative controls, respectively. After overnight incubation of the membrane at 4°C, we used anti-goat immunoglobulin, horseradish peroxidase–linked antibody (from donkey; Abcam Inc) in TTBS buffer at a dilution of 1:5000 as the secondary antibody. RCAS1 immunoreactivity was visualized with an enhanced chemiluminescence Western blotting detection kit (Amersham Biosciences). Western blotting experiment for each specimen from 5 mice was performed 3 times. Blot densitometry analysis was performed with the National Institutes of Health Image J software. The results were normalized by the expression of β-actin in each sample.

Immunohistochemical staining of RCAS1 in whole uteri from pregnant mice on postcoital day 8.5 at 24 hours after siRNA transfer

To determine the gene silencing efficiency of the transferred siRNA against RCAS1 protein in the uterus, whole uteri specimens that contained placenta and fetuses from 5 mice from each group on postcoital day 8.5 at 24 hours after siRNA transfer were examined immunohistochemically. Immunohistochemical staining was performed with paraffin-embedded sections. The sections were deparaffinized and rehydrated in Xylene, and 100%, 95%, 70% ethanol. A heat-induced antigene retrieval method with sodium citrate buffer (pH 6.0) was used, followed by incubation with 10% rabbit serum in PBS buffer (pH 7.4; Nacalai Tesque Inc, Kyoto, Japan) for 1 hour at room temperature. Next, the sections were incubated overnight with goat polyclonal antibody to RCAS1/EBAG9 in PBS buffer at a dilution of 1:50 (Abcam Inc). Normal goat immunoglobulin G at a dilution of 1:50 was used as a negative control (Santa Cruz Biotechnology Inc). After being washed, the sections were incubated with Alexa Fluor 555 rabbit anti-goat immunoglobulin G (H+L) secondary antibody in PBS buffer at a dilution of 1:1000 (Invitogen, Eugene, OR). The slides were counterstained with Bis-benzimides (Hoechst 33258; Nacalai Tesque Inc, Kyoto Japan), washed with water, and dehydrated.

For calculation of the positively stained placental cell number, the quantification system (field = 1 mm ² at ×100 magnification) was used. Two examiners counted randomly selected 8 fields from each section. The number of RCAS1 positively stained placental cells was quantified as the percentage of the sum of unstained and stained cells in the placenta.

Urine ACR measurement

Urine was collected at designated gestational days in the morning at 8:00-9:00 before blood pressure measurements were taken. Urine samples were centrifuged at 11,100 g for 10 minutes at room temperature and stored at –20°C until used. Albumin and creatinine concentrations were evaluated with the Albuwell M ELISA kit (Exocell Inc, Philadelphia, PA) and Creatinine Companion chemical assay kit (Exocell Inc), respectively. Intra- and interassay precision of samples within the useful range have a coefficient of variation of <10% of the mean for both assays. The ACR was calculated in the following manner: albumin (mg/dL)/creatinine (mg/dL) × 1000 = ACR (mg/g). Urine ACR measurements were performed in the RCAS1 siRNA group (n = 16) and the scrambled siRNA group (n = 9). ACR was measured on postcoital days 3.5, 5.5, 7.5, 8.5, 9.5, 10.5, 11.5, 13.5, 15.5, 17.5, and 19.5 before labor.

Blood pressure measurement

Blood pressure was measured in awake mice with the use of an automated tail cuff system (Softron BP-98; Softron, Tokyo, Japan) according to the manufacturer’s instructions at designated days of pregnancy. Results from the first 5 measurements were discarded; the average value that was obtained from the next 5 measurements was counted. The mice were in the restrainer for 15-20 minutes. Measurements were performed at each designated gestational day in the morning from 11:00-12:30. Blood pressure was measured in RCAS1 siRNA group (n = 16) and the scramble siRNA (negative control) group (n = 9). Blood pressure measurements were performed on postcoital days 3.5, 5.5, 7.5, 8.5, 9.5, 10.5, 11.5, 13.5, 15.5, 17.5, and 19.5 before labor.

Evaluation of course of pregnancy after in vivo siRNA transfer

To evaluate the placental size and fetal weight after siRNA transfer, 5 mice from each group were killed on postcoital day 18.5; the remaining mice in each group were monitored until delivery.

Statistical analysis

Data that were obtained from study groups on each designated day of gestation were subjected to the Mann-Whitney U test; Western blotting and immunohistochemistry data were subjected to 1-way analysis of variance with the use of the Statview statistics package (Abacus Concepts, Inc, Berkeley, CA); a probability value of < .05 was considered to indicate significance.