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
AIM:To examine the imprinted Dlk1-Dio3 locus in pluripotent embryonic stem(ES)cell/fibroblast hybrid cells.METHODS:Gtl2,Rian,and Mirg mRNA expression in mouse pluripotent ES cell/fibroblast hybrid cells was examined b...AIM:To examine the imprinted Dlk1-Dio3 locus in pluripotent embryonic stem(ES)cell/fibroblast hybrid cells.METHODS:Gtl2,Rian,and Mirg mRNA expression in mouse pluripotent ES cell/fibroblast hybrid cells was examined by real-time reverse transcription-polymerase chain reaction.Pyrosequencing and bisulfate sequencing were used to determine the DNA methylation level of the Dlk1-Dio3 locus imprinting control region. RESULTS:The selected hybrid clones had a near-tetraploid karyotype and were highly pluripotent judging from their capacity to generate chimeric embryos and adult chimeras.Our data clearly demonstrate that Gtl2,Rian,and Mirg,which are imprinted genes within the Dlk1-Dio3 locus,are active in all examined ES cell/fibroblast hybrid clones.In spite of interclonal variability,the expression of the imprinted genes is comparable to that of ES cells and fibroblasts.Quantitative analysis of the DNA methylation status of the intergenic differentially methylated region(IG DMR)within the Dlk1-Dio3 locus by pyrosequencing and bisulfite sequencing clearly showed that the DNA methylation status of the imprinted region in the tested hybrid clones was comparable to that of both ES cells and fibroblasts.CONCLUSION:Reprogramming process in a hybrid cell system is achieved without marked alteration of the imprinted Dlk1-Dio3 locus.展开更多
Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially ...Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially DNA-methylated region)and IG(intergenic)-DMR deletions that partially mimic paternal imprinting of H19-Igf2 and Dlk1-Dio3 can be employed as sperm replacement to efficiently support full-term embryonic development.However,how H19-DMR and IG-DMR act together to regulate embryonic development is still largely unknown.Here,using androgenetic ha ESC(AG-ha ESC)-mediated semi-cloned(SC)technology,we showed that paternal H19-DMR and IG-DMR are not essential for pre-implantation development of SC embryos generated through injection of AG-ha ESCs into oocytes.H19-DMR plays critical roles before 12.5 days of gestation while IG-DMR is essential for late-gestation of SC embryos.Interestingly,we found that combined deletions of H19 and H19-DMR can further improve the efficiency of normal development of SC embryos at mid-gestation compared to DKO SC embryos.Transcriptome and histology analyses revealed that H19 and H19-DMR combined deletions rescue the placental defects.Furthermore,we showed that H19,H19-DMR and IG-DMR deletions(TKO)give rise to better prenatal and postnatal embryonic development of SC embryos compared to DKO.Together,our results indicate the temporal regulation of paternal imprinted loci during embryonic development.展开更多
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.
基金Supported by Grant from the Russian Academy of Sciences,Siberian Branch N48
文摘AIM:To examine the imprinted Dlk1-Dio3 locus in pluripotent embryonic stem(ES)cell/fibroblast hybrid cells.METHODS:Gtl2,Rian,and Mirg mRNA expression in mouse pluripotent ES cell/fibroblast hybrid cells was examined by real-time reverse transcription-polymerase chain reaction.Pyrosequencing and bisulfate sequencing were used to determine the DNA methylation level of the Dlk1-Dio3 locus imprinting control region. RESULTS:The selected hybrid clones had a near-tetraploid karyotype and were highly pluripotent judging from their capacity to generate chimeric embryos and adult chimeras.Our data clearly demonstrate that Gtl2,Rian,and Mirg,which are imprinted genes within the Dlk1-Dio3 locus,are active in all examined ES cell/fibroblast hybrid clones.In spite of interclonal variability,the expression of the imprinted genes is comparable to that of ES cells and fibroblasts.Quantitative analysis of the DNA methylation status of the intergenic differentially methylated region(IG DMR)within the Dlk1-Dio3 locus by pyrosequencing and bisulfite sequencing clearly showed that the DNA methylation status of the imprinted region in the tested hybrid clones was comparable to that of both ES cells and fibroblasts.CONCLUSION:Reprogramming process in a hybrid cell system is achieved without marked alteration of the imprinted Dlk1-Dio3 locus.
基金partly supported by the Genome Tagging Project,Fountain-Valley Life Sciences Fund of University of Chinese Academy of Sciences Education Foundation and grants from the Chinese Academy of Sciences(XDB19010204,OYZDJ-SSW-SMC023 and Facility-based Open Research Program)the National Natural Science Foundation of China(31530048,81672117,31730062,31821004,and31601163)+1 种基金the Ministry of Science and Technology of China(2019YFA0109900)Shanghai Municipal Commission for Science and Technology(16JC1420500,17JC1420102,17JC1400900,and17411954900)
文摘Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially DNA-methylated region)and IG(intergenic)-DMR deletions that partially mimic paternal imprinting of H19-Igf2 and Dlk1-Dio3 can be employed as sperm replacement to efficiently support full-term embryonic development.However,how H19-DMR and IG-DMR act together to regulate embryonic development is still largely unknown.Here,using androgenetic ha ESC(AG-ha ESC)-mediated semi-cloned(SC)technology,we showed that paternal H19-DMR and IG-DMR are not essential for pre-implantation development of SC embryos generated through injection of AG-ha ESCs into oocytes.H19-DMR plays critical roles before 12.5 days of gestation while IG-DMR is essential for late-gestation of SC embryos.Interestingly,we found that combined deletions of H19 and H19-DMR can further improve the efficiency of normal development of SC embryos at mid-gestation compared to DKO SC embryos.Transcriptome and histology analyses revealed that H19 and H19-DMR combined deletions rescue the placental defects.Furthermore,we showed that H19,H19-DMR and IG-DMR deletions(TKO)give rise to better prenatal and postnatal embryonic development of SC embryos compared to DKO.Together,our results indicate the temporal regulation of paternal imprinted loci during embryonic development.
