Next-Generation Sequencing Transcriptome Quantitative Analysis of Hearts of Male Fetuses From Obese Female Mice

Obesity during pregnancy increase the risk for cardiac in the offspring. We used RNA next-generation sequencing analysis to have a snapshot of the heart transcriptome in male offspring exposed to maternal obesity and weaned onto an obesogenic diet. Female C57BL/6J mice received either commercial standard (RM1) or a high-fat diet [20% lipids (Special Dietary Services)] plus sweetened condensed milk [55% simple carbohydrates/8% lipids (Nestle, UK)] from weaning. After around 6 weeks on their respective diets, mice were mated with male counterparts and went through a first pregnancy and lactation to ensure their breeding competence. Mice were mated a second time, and first day of pregnancy was marked by the detection of a vaginal plug. On day 18.5 of gestation, pregnant mice were killed by raising CO2 concentrations or cervical dislocation. Fetuses were surgically removed and immediately killed by laying in cold buffer. Fetal heart ventricles were dissected, weighed, snap frozen and stored at −80 °C. Cardiac RNA from male offspring was extracted using a Qiazol/miRNeasy mini kit protocol (Qiagen, Hilden, Germany). Library preparation for mRNA sequencing followed manufacturer protocol of TruSeq RNA Library Preparation kit (Illumina, Cambridge, UK).

妊娠期肥胖可升高子代罹患心脏疾病的风险。本研究采用RNA下一代测序（RNA next-generation sequencing）技术，对暴露于母体肥胖环境且断奶后饲喂致肥胖饮食的雄性子代小鼠的心脏转录组（transcriptome）进行分析，获取其瞬时表达全貌。我们选用雌性C57BL/6J小鼠，自断奶起分别饲喂商品化标准饲料（RM1）或高脂饲料[20%脂肪（Special Dietary Services供应）]加甜炼乳[55%简单碳水化合物/8%脂肪（英国雀巢公司出品）]。饲喂对应饲料约6周后，将雌性小鼠与雄性小鼠合笼交配，完成首次妊娠与哺乳以验证其具备正常繁育能力。随后进行第二次合笼交配，以检测到阴道栓的当日作为妊娠第1天。妊娠第18.5天时，采用二氧化碳窒息法或颈椎脱臼法处死孕鼠。通过外科手术取出胎鼠，随即置于低温缓冲液中处死。分离胎鼠心脏心室，称重后快速冷冻并保存于-80℃环境中。采用Qiazol/miRNeasy迷你试剂盒（德国凯杰公司，希尔德市）的操作规程提取雄性子代小鼠的心脏总RNA。mRNA测序的文库构建严格遵循TruSeq RNA文库制备试剂盒（英国Illumina公司，剑桥）的厂商说明书进行。