Green fluorescent protein ( GFP ) gene was expressed transiently in 2-3 d old rice embryos by electroporation with the aid of a specially designed loading net. Under suitable conditions (500 μF capacitance, 300 V/c...Green fluorescent protein ( GFP ) gene was expressed transiently in 2-3 d old rice embryos by electroporation with the aid of a specially designed loading net. Under suitable conditions (500 μF capacitance, 300 V/cm Voltage, 100 μg/mL plasmid DNA), the percentage of embryos expressing GFP was up to 35%. The highest electroporation efficiency (40%) was obtained at pH 5.8 of the electroporation buffer. The GFP gene driven by the Ubi promoter produced the highest efficiency. Thus, on the basis of optimizing electroporation conditions, a transformation system has been developed for young embryos in rice. The electroporated 4-6 d old embryos regenerated plantlets under the controlled cultural conditions. Fluorescence microscopic observations indicated that GFP gene expressed in their calli and R0 plantlets.展开更多
Vertebrate Msx genes are unlinked,homeobox-containing genes that bear homology to the Drosophila muscle segment homeobox gene.These genes are expressed at multiple sites of tissue-tissue interactions during vertebrate...Vertebrate Msx genes are unlinked,homeobox-containing genes that bear homology to the Drosophila muscle segment homeobox gene.These genes are expressed at multiple sites of tissue-tissue interactions during vertebrate embryonic development.Inductive interactions mediated by the Msx genes are essential for normal craniofacial,limb and ectodermal organ morphogenesis,and are also essential to survival in mice,as manifested by the phenotypic abnormalities shown in knockout mice and in humans.This review summarizes studies on the expression,regulation,and functional analysis of Msx genes that bear relevance to craniofacial development in humans and mice.展开更多
Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been th...Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were Produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An 'all-fish' gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (goGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate ho- mogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century展开更多
The production of transgenic swine for xenotransplantation has been proposed as an optimal option to overcome the chronic shortage of human organ donors. Generation of genetically engineered swine has been elusive due...The production of transgenic swine for xenotransplantation has been proposed as an optimal option to overcome the chronic shortage of human organ donors. Generation of genetically engineered swine has been elusive due to the difficulties in gene transfer. In order to achieve effective gene delivery, a key step for the genetic modification, we applied electronic pulse delivery (EPD) technology to introduce HZKb-DC DNA construct into swine eggs. Using the developed EPD ProtocolsTM, we have achieved good viability of the EPD treated oocytes, satisfactory embryonic development of the EPD treated embryos, and stable DNA transfer into the swine embryos with high efficiency. Thus, application of the EPD technology promises to effectively facilitate the generation of large trangenic mammals.展开更多
In order to investigate glucose metabolism pathways and their changes in Kunming mouse preimplantation 1-,2-,4-,8-cell,and morula embryos,the mRNA level for the genes involved in glucose metabolism was tested by neste...In order to investigate glucose metabolism pathways and their changes in Kunming mouse preimplantation 1-,2-,4-,8-cell,and morula embryos,the mRNA level for the genes involved in glucose metabolism was tested by nested RT-PCR on embryos at different development stages in vivo.These genes were glucose 6-phosphate dehydrogenase(G6PDH),phospho-fructokinase(PFK),and phosphoglucomutase(PGM),representing pentose phosphate pathway(PPP),glycolysis,and glycogensis and glycogenolysis respectively.Three sets of inner and outer primers were designed and synthesized based on cDNA sequences of G6PDH,PFK and PGM.RT-PCR results revealed that G6PDH gene transcription was found in Kunming mouse 1-8 cell embryos,and not in morula embryos;it indicated that 1-8 cell embryos may metabolize glucose by pentose phosphate pathway,but morula embryos can not do so.PFK gene transcription was found in 1-8 cell and morula embryos;it is probable that there exists glycolysis in those embryos.PGM gene transcription was not found in 1-8 cell and morula embryos,so glycogenesis and glycogenolysis in these embryos were not present.展开更多
Date palm, like all other crops, is very sensitive to the injury by many insect pests, which may lead to the death of the affected plant and causes decrease in yield. In the present study, an efficient Agrobacterium f...Date palm, like all other crops, is very sensitive to the injury by many insect pests, which may lead to the death of the affected plant and causes decrease in yield. In the present study, an efficient Agrobacterium for genetic transformation was successfully achieved for well known date palm （Phoenix dactylifera L.） cv. Medjool and Khalas using callus as explant. Embryogenic callus were recorded 100% mortality when cultured on MS medium containing 100 mg/L kanamycin with different cultivars, thus it was chosen for the selection of transformed explants. Embryogenic callus of Medjool and Khalas were incubated with Agrobacterium tumefaciens strain LBA 4404 for 0.5, 1, 2, 4 and 24 h on LB medium. After the incubation periods, embryogenic callus was transferred to MS medium with 0.1 mg/L NAA, 0.05 mg/L BA, 250 mg/L carbenicillin and 100 mg/L kanamycin for detection of transgenic embryogenic callus. Polymerase chain reaction （PCR） was used for the rapid screening of Cry3Aa gene. For screening, total genomic DNA was isolated from transformants. Using primer specific to Cry3Aa gene （forward and reverse）, a PCR product with a size of about 2,000 bp was amplified when all nucleic acid from the transformants were utilized as templates. PCR analysis confirmed the appearance of the transgene of 2,000 bp in one individual plantlet. Presence and integration of foreign Cry3Aa gene in regenerated kanamycin resistant embryogenic callus was also confirmed by Southern blot hybridization. It was found that one transgenic embryogenic callus for both Medjool and Khalas showed a single copy of gene integration. These results signify the successful transfer of Cry3Aa gene into date palm plant.展开更多
In crab, embryogenesis is a complicated developmental program marked by a series of critical events. RNA-Sequencing technology offers developmental biologists a way to identify many more developmental genes than ever ...In crab, embryogenesis is a complicated developmental program marked by a series of critical events. RNA-Sequencing technology offers developmental biologists a way to identify many more developmental genes than ever before. Here, we present a comprehensive analysis of the transcriptomes of Eriocheir sinensis oosperms （Os） and embryos at the 2-4, cell stage （Cs）, which are separated by a cleavage event. Atotal of 18 923 unigenes were identified, and 403 genes matched with gene ontology （GO） terms related to developmental processes. In total, 432 differentially expressed genes （DEGs） were detected between the two stages. Nine DEGs were specifically expressed at only one stage. These DEGs may be relevant to stage-specific molecular events during development. A number of DEGs related to ＇hedgehog signaling pathway＇, ＇Wnt signaling pathway＇ ＇germplasm＇, ＇nervous system＇, ＇sensory perception＇ and ＇segment polarity＇ were identified as being up-regulated at the Cs stage. The results suggest that these embryonic developmental events begin before the early cleavage event in crabs, and that many of the genes expressed in the two transeriptomes might be maternal genes. Our study provides ample information for further research on the molecular mechanisms underlying crab development.展开更多
文摘Green fluorescent protein ( GFP ) gene was expressed transiently in 2-3 d old rice embryos by electroporation with the aid of a specially designed loading net. Under suitable conditions (500 μF capacitance, 300 V/cm Voltage, 100 μg/mL plasmid DNA), the percentage of embryos expressing GFP was up to 35%. The highest electroporation efficiency (40%) was obtained at pH 5.8 of the electroporation buffer. The GFP gene driven by the Ubi promoter produced the highest efficiency. Thus, on the basis of optimizing electroporation conditions, a transformation system has been developed for young embryos in rice. The electroporated 4-6 d old embryos regenerated plantlets under the controlled cultural conditions. Fluorescence microscopic observations indicated that GFP gene expressed in their calli and R0 plantlets.
基金supported by the NIH grants(R01DE12329,R01DE14044,P60DE13076)the National Science Foundation grant(IBN-9796321)the Millenium Trust Health Excellence Fund(HEF-2000-05-04)from the Louisiana Bpard of Regents
文摘Vertebrate Msx genes are unlinked,homeobox-containing genes that bear homology to the Drosophila muscle segment homeobox gene.These genes are expressed at multiple sites of tissue-tissue interactions during vertebrate embryonic development.Inductive interactions mediated by the Msx genes are essential for normal craniofacial,limb and ectodermal organ morphogenesis,and are also essential to survival in mice,as manifested by the phenotypic abnormalities shown in knockout mice and in humans.This review summarizes studies on the expression,regulation,and functional analysis of Msx genes that bear relevance to craniofacial development in humans and mice.
文摘Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were Produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An 'all-fish' gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (goGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate ho- mogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century
文摘The production of transgenic swine for xenotransplantation has been proposed as an optimal option to overcome the chronic shortage of human organ donors. Generation of genetically engineered swine has been elusive due to the difficulties in gene transfer. In order to achieve effective gene delivery, a key step for the genetic modification, we applied electronic pulse delivery (EPD) technology to introduce HZKb-DC DNA construct into swine eggs. Using the developed EPD ProtocolsTM, we have achieved good viability of the EPD treated oocytes, satisfactory embryonic development of the EPD treated embryos, and stable DNA transfer into the swine embryos with high efficiency. Thus, application of the EPD technology promises to effectively facilitate the generation of large trangenic mammals.
基金National Natural Science Foundation of China(39600106)
文摘In order to investigate glucose metabolism pathways and their changes in Kunming mouse preimplantation 1-,2-,4-,8-cell,and morula embryos,the mRNA level for the genes involved in glucose metabolism was tested by nested RT-PCR on embryos at different development stages in vivo.These genes were glucose 6-phosphate dehydrogenase(G6PDH),phospho-fructokinase(PFK),and phosphoglucomutase(PGM),representing pentose phosphate pathway(PPP),glycolysis,and glycogensis and glycogenolysis respectively.Three sets of inner and outer primers were designed and synthesized based on cDNA sequences of G6PDH,PFK and PGM.RT-PCR results revealed that G6PDH gene transcription was found in Kunming mouse 1-8 cell embryos,and not in morula embryos;it indicated that 1-8 cell embryos may metabolize glucose by pentose phosphate pathway,but morula embryos can not do so.PFK gene transcription was found in 1-8 cell and morula embryos;it is probable that there exists glycolysis in those embryos.PGM gene transcription was not found in 1-8 cell and morula embryos,so glycogenesis and glycogenolysis in these embryos were not present.
文摘Date palm, like all other crops, is very sensitive to the injury by many insect pests, which may lead to the death of the affected plant and causes decrease in yield. In the present study, an efficient Agrobacterium for genetic transformation was successfully achieved for well known date palm （Phoenix dactylifera L.） cv. Medjool and Khalas using callus as explant. Embryogenic callus were recorded 100% mortality when cultured on MS medium containing 100 mg/L kanamycin with different cultivars, thus it was chosen for the selection of transformed explants. Embryogenic callus of Medjool and Khalas were incubated with Agrobacterium tumefaciens strain LBA 4404 for 0.5, 1, 2, 4 and 24 h on LB medium. After the incubation periods, embryogenic callus was transferred to MS medium with 0.1 mg/L NAA, 0.05 mg/L BA, 250 mg/L carbenicillin and 100 mg/L kanamycin for detection of transgenic embryogenic callus. Polymerase chain reaction （PCR） was used for the rapid screening of Cry3Aa gene. For screening, total genomic DNA was isolated from transformants. Using primer specific to Cry3Aa gene （forward and reverse）, a PCR product with a size of about 2,000 bp was amplified when all nucleic acid from the transformants were utilized as templates. PCR analysis confirmed the appearance of the transgene of 2,000 bp in one individual plantlet. Presence and integration of foreign Cry3Aa gene in regenerated kanamycin resistant embryogenic callus was also confirmed by Southern blot hybridization. It was found that one transgenic embryogenic callus for both Medjool and Khalas showed a single copy of gene integration. These results signify the successful transfer of Cry3Aa gene into date palm plant.
基金Supported by the National High Technology Research and Development Program of China(863 Program)(No.2012AA10A409)the Scientific and Technological Innovation Project of Qingdao National Laboratory for Marine Science and Technology(No.2015ASKJ02)the National Natural Science Foundation of China(Nos.41276165 to Dr.CUI Zhaoxia,31302187 to Dr.HUI Min)
文摘In crab, embryogenesis is a complicated developmental program marked by a series of critical events. RNA-Sequencing technology offers developmental biologists a way to identify many more developmental genes than ever before. Here, we present a comprehensive analysis of the transcriptomes of Eriocheir sinensis oosperms （Os） and embryos at the 2-4, cell stage （Cs）, which are separated by a cleavage event. Atotal of 18 923 unigenes were identified, and 403 genes matched with gene ontology （GO） terms related to developmental processes. In total, 432 differentially expressed genes （DEGs） were detected between the two stages. Nine DEGs were specifically expressed at only one stage. These DEGs may be relevant to stage-specific molecular events during development. A number of DEGs related to ＇hedgehog signaling pathway＇, ＇Wnt signaling pathway＇ ＇germplasm＇, ＇nervous system＇, ＇sensory perception＇ and ＇segment polarity＇ were identified as being up-regulated at the Cs stage. The results suggest that these embryonic developmental events begin before the early cleavage event in crabs, and that many of the genes expressed in the two transeriptomes might be maternal genes. Our study provides ample information for further research on the molecular mechanisms underlying crab development.
