Differential Expression of Placental Genes in a Mouse Model of Infection-Associated Preterm Labor
Sandra E. Reznik, Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences
Chih-Hung Chen, Haoting Yen, Wei Wang
Preterm labor, defined as labor before 37 weeks’ gestation, occurs in 7 to 10% of pregnancies, but is the cause of over 85% of perinatal morbidity and mortality. Sadly, the existing therapeutic approaches for preterm labor are not effective and have not changed significantly over the past few decades. The identification of novel potential therapeutic targets in the control of preterm labor is the most promising approach to the prevention of birth defects and adverse perinatal outcome. As the single most common cause of preterm labor is infection, we have used genomics technology to identify the genes and molecular pathways that are differentially expressed in infection-associated preterm labor in a tightly controlled validated animal model.
Timed pregnant C57Bl/6 mice from Taconic Laboratories were used for all experiments. Twelve mice received an intraperitoneal (ip) injection of lipopolysaccharide at E15.5. Mice that delivered within 24 hours of the LPS injection (n=5) were included in the study. Control mice (n=6) received sham ip injections of phosphate buffered saline (PBS). Placentas were collected at E16.6 and stored in RNAlater. Total RNA extraction was accomplished with an RNeasy® Mini kit (Qiagen), following the manufacturer’s instructions. Messenger RNA was extracted from samples of total RNA, amplified and labeled by using an Amino Allyl MesageAmpTM II aRNA Amplification Kit (Ambion) and following the manufacturer’s instructions. RNA samples were quantified with both a traditional UV spectrophotometer and a nanodrop spectrophotometer. RNA integrity was confirmed with both RNA gel electrophoresis and a Bioanalyzer (Agilent). Microarray chips spotted with 32,000 oligonucleotides were first pre-hybridized with 35% formamide, 0.5%SDS, 2% ss salmon sperm DNA, in 4x SSPE-2.5x Denhardt’s at 50oC for one hour. For each hybridization, equimolar amounts of Cy3 and Cy5 labeled aRNA pre-incubated at 50oC for one hour with 50 µg/ml polydA, 0.5 mg/ml tRNA, 50 µg/ml human/mouse Cot1 DNA, 24% formamide, 0.3% SDS, 1.7x Denhardt’s, 2.7x SSPE were applied to a chip. Hybridization was carried out at 50oC for one hour. Chips were then washed in 2x SSC/0.1% SDS, 0.2x SSC/0.1% SDS, 0.2x SSC and promptly scanned at the Albert Einstein College of Medicine Microarray Facility. Hybridizations were performed with mRNA from each of the placentas from the delivering mice and reference mRNA pooled from all of the control placentas. Delivering mouse mRNA was tagged with the red fluorescent label and control RNA was tagged with the green fluorescent label. Chips were manually gridded and fluorescence was quantified with GenePix Pro 6.0 software. SAM and Pathway Architect Software (Stratagene) were used for analysis.
Differential gene expression and the regulation of specific placental molecular pathways are associated with endotoxin-triggered premature delivery in a mouse model.