Parthenogenetic embryonic stem （pES） cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression p...Parthenogenetic embryonic stem （pES） cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression profiles of paternally expressed imprinted genes have not been fully explored. In this study, three mouse pES cell lines were established and the differentiation potential of these cells in extended culture was evaluated. The undifferentiated cells had a normal karyotype and homozygous genome, and expressed ES-cell-specific molecular markers. The cells remained undifferentiated after more than 50 passages and exhibited pluripotent differentiation capacity. All three lines of the established ES cells produced teratomas; two lines of ES cells produced chimeras and germline transmission. Furthermore, activation of the paternally expressed imprinted genes Snrpn, U2afl-rsl, Peg3, Impact, Zfp127, Dlkl and Mest in these cells was detected. Some paternally expressed imprinted genes were found to be expressed in the blastocyst stage of parthenogenetically activated embryos in vitro and their expression level increased with extended pES cell culture. Furthermore, our data show that the activation of these paternally expressed imprinted genes in pES cells was associated with a change in the methylation of the related differentially methylated regions. These findings provide direct evidence for the pluripotency of pES cells and demonstrate the association between the DNA methylation pattern and the activa- tion of paternally expressed imprinted genes in pES cells. Thus, the established ES cell lines provide a valuable model for studying epigenetic regulation in mammalian development.展开更多
目的检测常规精液冷冻技术中冷冻保护剂、冷冻过程及冷冻时间长短对精子父源印记基因H19及母源印记基因MEST印记控制区域(imprinting control region,ICR)的DNA甲基化的影响程度。方法以10例符合精子库捐精条件的正式志愿者为研究对象,...目的检测常规精液冷冻技术中冷冻保护剂、冷冻过程及冷冻时间长短对精子父源印记基因H19及母源印记基因MEST印记控制区域(imprinting control region,ICR)的DNA甲基化的影响程度。方法以10例符合精子库捐精条件的正式志愿者为研究对象,将每份精液样品平均分为4组:A组为新鲜精液,作为对照;B组加入同体积冷冻保护剂,不冷冻;C组加同体积冷冻保护剂,冷冻2d;D组加同等体积冷冻保护剂,冷冻两个月。通过亚硫酸氢盐克隆测序法分析H19及MEST ICR的DNA甲基化状态。结果四组(A、B、C、D组)H19基因ICR区的DNA甲基化率以克隆数计算时分别为58.70%、53.85%、49.46%和45.74%,以CpG岛数计算时分别为97.57%、97.33%、97.13%和96.56%,两者都有降低趋势,但组间差异均无统计学意义(P>0.05);四组MEST基因ICR区甲基化率以克隆数计算时分别为41.10%、45.33%、47.30%和50.68%,以CpG岛数计算甲基化率时分别为1.77%、1.74%、2.00%和2.26%,有升高趋势,但差异亦无统计学意义(P>0.05)。结论常规精液冷冻复苏技术对精子父源印记基因H19及母源印记基因MEST的ICR区甲基化程度未见明显影响。展开更多
美臀绵羊18号染色体端部的一个A-G的单碱基突变,产生了Callipyge表型,部分肌肉过度发育肥大,Callipyge基因属于印记基因家族,表现为父本极性超显性遗传,其作用机制可能是影响了1个顺式作用元件LRCE(long range control element)的功能,...美臀绵羊18号染色体端部的一个A-G的单碱基突变,产生了Callipyge表型,部分肌肉过度发育肥大,Callipyge基因属于印记基因家族,表现为父本极性超显性遗传,其作用机制可能是影响了1个顺式作用元件LRCE(long range control element)的功能,可能激活了1个增强子,也可能抑制了1个沉默子。Callipyge绵羊的瘦肉率和屠宰率较一般绵羊高,但是背最长肌的剪切力较高,影响了食用口感。育种工作可以利用Callipyge基因来提高绵羊的瘦肉率。展开更多
ARHI(aplasia ras homologue member I)/NOEY2是1999年新发现的一个母源性抑癌印迹基因,位于人染色体1p31,编码一个相对分子量为26kD的小GTP结合蛋白。ARHI属ras/rap超家族成员,与该家族成员有50%～60%的同源性并且两者具有相似的GTP/GD...ARHI(aplasia ras homologue member I)/NOEY2是1999年新发现的一个母源性抑癌印迹基因,位于人染色体1p31,编码一个相对分子量为26kD的小GTP结合蛋白。ARHI属ras/rap超家族成员,与该家族成员有50%～60%的同源性并且两者具有相似的GTP/GDP结合域,但与该家族其它成员不同,ARHI发挥抑癌基因作用,是该家族第一个被报道的肿瘤抑制基因。ARHI基因编码的蛋白在人类多种组织表达,而该基因在人卵巢癌、乳腺癌、胰腺癌、肝癌等多种肿瘤中表达下调或缺失,提示其与上述肿瘤的发生、发展密切相关。ARHI可能通过作用于cyclin D1,使其不能与CDK结合形成活性激酶,从而使细胞停止于G_1期来参与细胞周期调控;可能通过依赖caspase和calpain两条途径参与信号通路传导诱发细胞凋亡;另外,该基因可通过抑制STAT3的激活而发挥抑癌基因功能,也可以调节自体吞噬和肿瘤细胞休眠。目前研究显示,ARHI基因的表达缺失主要通过遗传事件和表观遗传学机制发生,包括DNA甲基化异常、杂合性丢失,乙酰化组蛋白的低水平表达及基因突变有关,但有待进一步深入研究。可以预见,ARHI基因的深入研究必将为早期肿瘤的基因诊治提供新的思路和理论依据。展开更多
Silver-Russell Syndrome(SRS) is clinically heterogeneous disorder characterized by low birth weight, postnatal growth restriction, and variable dysmorphic features. Current evidence strongly implicates imprinted genes...Silver-Russell Syndrome(SRS) is clinically heterogeneous disorder characterized by low birth weight, postnatal growth restriction, and variable dysmorphic features. Current evidence strongly implicates imprinted genes as an important etiology of SRS. Although almost half of the patients showed DNA hypomethylation at the H19/IGF2 imprinted domain, and approximately7%–10% of SRS patients have maternal uniparental disomy of chromosome 7(UPD(7) mat); the rest of the SRS patients shows unknown etiology. In this study, we investigate whether there are further DNA methylation defects in SRS patients. We measured DNA methylation in seven SRS patients and five controls at more than 485,000 CpG sites using DNA methylation microarrays. We analyzed methylation changes genome-wide and identified the differentially methylated regions(DMRs) using bisulfite sequencing and digital PCR. Our analysis identifies epimutations at the previously characterized domains of H19/IGF2,providing proof of principle that our methodology can detect the changes in DNA methylation at imprinted loci. In addition,our results showed a novel SRS associated imprinted gene OSBPL5 located on chromosome 11p14 with the probe cg25963939,which is hypomethylated in 4/7 patients(P=0.023, β=.0.243). We also report DMRs in other genes including TGFβ3, HSF1,GAP43, NOTCH4 and MYH14. These DMRs were found to be associated with SRS using GO pathway analysis. In this study,we identified the probe cg25963939, located at the 5′UTR of imprinted gene OSBPL5, as a novel DMR that is associated with SRS. This finding provides new insights into the mechanism of SRS etiology and aid the further stratification of SRS patients by molecular phenotypes.展开更多
Imprinted genes play significant roles in the regulation of fetal growth, development, function of the placenta and postnatal behavior in mammals, but little is known in pigs. In order to investigate the imprinting st...Imprinted genes play significant roles in the regulation of fetal growth, development, function of the placenta and postnatal behavior in mammals, but little is known in pigs. In order to investigate the imprinting status of porcine retro-transposon like 1 （RTL1） and type 3 iodothyronine deiodinase （DIO3） genes, DNA or RNA samples of the parents and F1 animals, generated with reciprocal crosses between Large White and Meishan breeds, were isolated, and analyzed by reverse transcription polymerase chain reaction restriction fragment length polymorphism （RT-PCR-RFLP）. The results demonstrated that the RTL1 gene was paternally expressed in 10 tissues, such as the skeletal muscle, heart, spleen, liver, kidney, lung, stomach, fat, small intestine and brain, and D103 gene exhibited paternal expression in the skeletal muscle, heart, spleen, lung, stomach, and brain, in 2-month-old pigs. The association of RTL1 and DI03 with carcass traits was further analyzed in the F2 population of Large White×Meishan pigs. The statistical results showed that the R TL1 A1101G polymorphism （EU781029） was significantly associated with lean meat percentage （LMP） and fat meat percentage （FMP） （P〈0.05）, while the D103 A744C polymorphism （AY533208） was not significantly associated with any carcass traits. These results indicate that the imprinting status of RTL1 and DIO3 is well kept across the mammalian species, and porcine RTL1 may have important roles in muscle growth and fat deposition.展开更多
文摘Parthenogenetic embryonic stem （pES） cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression profiles of paternally expressed imprinted genes have not been fully explored. In this study, three mouse pES cell lines were established and the differentiation potential of these cells in extended culture was evaluated. The undifferentiated cells had a normal karyotype and homozygous genome, and expressed ES-cell-specific molecular markers. The cells remained undifferentiated after more than 50 passages and exhibited pluripotent differentiation capacity. All three lines of the established ES cells produced teratomas; two lines of ES cells produced chimeras and germline transmission. Furthermore, activation of the paternally expressed imprinted genes Snrpn, U2afl-rsl, Peg3, Impact, Zfp127, Dlkl and Mest in these cells was detected. Some paternally expressed imprinted genes were found to be expressed in the blastocyst stage of parthenogenetically activated embryos in vitro and their expression level increased with extended pES cell culture. Furthermore, our data show that the activation of these paternally expressed imprinted genes in pES cells was associated with a change in the methylation of the related differentially methylated regions. These findings provide direct evidence for the pluripotency of pES cells and demonstrate the association between the DNA methylation pattern and the activa- tion of paternally expressed imprinted genes in pES cells. Thus, the established ES cell lines provide a valuable model for studying epigenetic regulation in mammalian development.
文摘目的检测常规精液冷冻技术中冷冻保护剂、冷冻过程及冷冻时间长短对精子父源印记基因H19及母源印记基因MEST印记控制区域(imprinting control region,ICR)的DNA甲基化的影响程度。方法以10例符合精子库捐精条件的正式志愿者为研究对象,将每份精液样品平均分为4组:A组为新鲜精液,作为对照;B组加入同体积冷冻保护剂,不冷冻;C组加同体积冷冻保护剂,冷冻2d;D组加同等体积冷冻保护剂,冷冻两个月。通过亚硫酸氢盐克隆测序法分析H19及MEST ICR的DNA甲基化状态。结果四组(A、B、C、D组)H19基因ICR区的DNA甲基化率以克隆数计算时分别为58.70%、53.85%、49.46%和45.74%,以CpG岛数计算时分别为97.57%、97.33%、97.13%和96.56%,两者都有降低趋势,但组间差异均无统计学意义(P>0.05);四组MEST基因ICR区甲基化率以克隆数计算时分别为41.10%、45.33%、47.30%和50.68%,以CpG岛数计算甲基化率时分别为1.77%、1.74%、2.00%和2.26%,有升高趋势,但差异亦无统计学意义(P>0.05)。结论常规精液冷冻复苏技术对精子父源印记基因H19及母源印记基因MEST的ICR区甲基化程度未见明显影响。
文摘美臀绵羊18号染色体端部的一个A-G的单碱基突变,产生了Callipyge表型,部分肌肉过度发育肥大,Callipyge基因属于印记基因家族,表现为父本极性超显性遗传,其作用机制可能是影响了1个顺式作用元件LRCE(long range control element)的功能,可能激活了1个增强子,也可能抑制了1个沉默子。Callipyge绵羊的瘦肉率和屠宰率较一般绵羊高,但是背最长肌的剪切力较高,影响了食用口感。育种工作可以利用Callipyge基因来提高绵羊的瘦肉率。
基金supported by Nature Science Foundation of China (81670713)The National Key Research and Development Program of China (2016YFC0901505)+1 种基金Basic and Clinical Medicine Cooperation Research Foundation of Capital Medical University (14JL75)Scientific Research Foundation for the Young and Middle-Aged Endocrinologists in GenSci's Department of Pediatrics
文摘Silver-Russell Syndrome(SRS) is clinically heterogeneous disorder characterized by low birth weight, postnatal growth restriction, and variable dysmorphic features. Current evidence strongly implicates imprinted genes as an important etiology of SRS. Although almost half of the patients showed DNA hypomethylation at the H19/IGF2 imprinted domain, and approximately7%–10% of SRS patients have maternal uniparental disomy of chromosome 7(UPD(7) mat); the rest of the SRS patients shows unknown etiology. In this study, we investigate whether there are further DNA methylation defects in SRS patients. We measured DNA methylation in seven SRS patients and five controls at more than 485,000 CpG sites using DNA methylation microarrays. We analyzed methylation changes genome-wide and identified the differentially methylated regions(DMRs) using bisulfite sequencing and digital PCR. Our analysis identifies epimutations at the previously characterized domains of H19/IGF2,providing proof of principle that our methodology can detect the changes in DNA methylation at imprinted loci. In addition,our results showed a novel SRS associated imprinted gene OSBPL5 located on chromosome 11p14 with the probe cg25963939,which is hypomethylated in 4/7 patients(P=0.023, β=.0.243). We also report DMRs in other genes including TGFβ3, HSF1,GAP43, NOTCH4 and MYH14. These DMRs were found to be associated with SRS using GO pathway analysis. In this study,we identified the probe cg25963939, located at the 5′UTR of imprinted gene OSBPL5, as a novel DMR that is associated with SRS. This finding provides new insights into the mechanism of SRS etiology and aid the further stratification of SRS patients by molecular phenotypes.
基金supported by the National Natural Sci-ence Foundation of China (30571331)the China Postdoctoral Science Foundation (2005038161)
文摘Imprinted genes play significant roles in the regulation of fetal growth, development, function of the placenta and postnatal behavior in mammals, but little is known in pigs. In order to investigate the imprinting status of porcine retro-transposon like 1 （RTL1） and type 3 iodothyronine deiodinase （DIO3） genes, DNA or RNA samples of the parents and F1 animals, generated with reciprocal crosses between Large White and Meishan breeds, were isolated, and analyzed by reverse transcription polymerase chain reaction restriction fragment length polymorphism （RT-PCR-RFLP）. The results demonstrated that the RTL1 gene was paternally expressed in 10 tissues, such as the skeletal muscle, heart, spleen, liver, kidney, lung, stomach, fat, small intestine and brain, and D103 gene exhibited paternal expression in the skeletal muscle, heart, spleen, lung, stomach, and brain, in 2-month-old pigs. The association of RTL1 and DI03 with carcass traits was further analyzed in the F2 population of Large White×Meishan pigs. The statistical results showed that the R TL1 A1101G polymorphism （EU781029） was significantly associated with lean meat percentage （LMP） and fat meat percentage （FMP） （P〈0.05）, while the D103 A744C polymorphism （AY533208） was not significantly associated with any carcass traits. These results indicate that the imprinting status of RTL1 and DIO3 is well kept across the mammalian species, and porcine RTL1 may have important roles in muscle growth and fat deposition.
