The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r）...The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.展开更多
[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter seq...[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter sequence and the coding region of EGFP were amplified by polymerase chain reactions （PCR） with vector pCambia 1303 as the template.Using reverse transcript PCR technology,with total RNAs of cucumber as template,the coding region of acid α-galactosidase Ⅰ in cucumber was amplified.The above three fragments were inserted into the multiple cloning sites of expression vector pCambia 1381c.The fusion expression vector of α-galactosidase-EGFP located at the C-terminal of the target genes was constructed.[Result] After enzyme digestion and sequencing,the fusion expression of α-galactosidase-EGFP in cucumber was constructed successfully.[Conclusion] The research laid the experimental basis for further study on the subcellular localization of α-galactosidase in cucumber.展开更多
Plasmid pVK1001 which carried the gfp gene of GFPmut2, a mutant of GFP, was introduced into Azospirillum brasilense Yu62 by electroporation. Maize seedlings were inoculated with the GFP-labelled baeteria and grown gno...Plasmid pVK1001 which carried the gfp gene of GFPmut2, a mutant of GFP, was introduced into Azospirillum brasilense Yu62 by electroporation. Maize seedlings were inoculated with the GFP-labelled baeteria and grown gnotobiotically in flask with semi-solid agar medium. Observations were performed with confocal laser scanning microscopy (CLSM) and electron microscopy, respectively, at 8 d and 12 d after inoculation. Confocal laser scanning microscopy showed that A. brasilense Yu62 could penetrate into the cortex tissue, colonizing in the intercellular spaces of the parenchyma cells of the cortex tissue. Transmission and scanning electron microscopy (TEM) showed that the majority of the bacteria colonized on the root surface and only a minority of them resided in the root interior.展开更多
Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the fil...Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the filamentous bacteria indicated that the NtFtsZs could recognize the potential division sites in E. coli and be polymerized with heterogeneous FtsZ from bacteria. The overexpression of NtFtsZs ∶ gfp inhibited the division of host strain cells and resulted in the long filamentous bacterial morphology. These results suggested that eukaryotic ftsZs have similar function to their prokaryotic homologs. Meanwhile, the different deletions of motifs of NtFtsZs are also employed to investigate the functions of these proteins in E. coli . The results showed that the C_terminal domains of NtFtsZs were related to the correct localization of NtFtsZs in E. coli and the N_terminal domains of NtFtsZs were responsible for the polymerization of homogeneous and heterogeneous FtsZ proteins. The significance of these results in understanding the functions of NtFtsZs in plastid division were discussed.展开更多
To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a pro...To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a probe.The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid.The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21 （DE3 ）cells,the GFP-conjugated MIM-I-BAR （MIM-I-BAR-GFP ） exhibits strong visible fluorescence,and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃,but not at the culture temperature of 37 ℃.This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.展开更多
Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, ...Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, A005-G-T-1-2. Observations were made on pollen at four major developmental stages, viz. I. uni-nucleate microspore stage; II. early bi-cellular pollen stage; III. late bi-cellular pollen stage; and IV. tri-cellular pollen stage. At each of these developmental stages vegetative nucleus, generative nucleus/ cell, and sperm cells were seen undergoing continuous and coordinated motion and migration. These movements seemed to be influenced by associated microfilament networks existing in the pollen. Based on these observations we propose that it is the interaction between the microfilament networks (usually one existing in the central cytoplasm and another in the cortex) that controls the dynamic movement of the vegetative nucleus, generative nucleus/cell and sperm cells. Furthermore, we have also observed that there is an array of microfilaments (oriented mostly parallel to the long axis of the cell) existing in the generative and sperm cells. As far as we are aware, this is the first report showing the existence of microfilaments in living generative and sperm cells of rice pollen. The implication and significance of the existence of microfilaments in generative and sperm cells in rendering self-propelled motion of these cells in relation to their passage and movement in the pollen tube and embryo sac for fertilization were discussed.展开更多
Green fluorescent protein (GFP) fused to the F_actin binding domain of mouse talin labels the actin cytoskeleton in the immature pollen of stable transformed rice (Oryza sativa L.) plants. Actin microfilaments could b...Green fluorescent protein (GFP) fused to the F_actin binding domain of mouse talin labels the actin cytoskeleton in the immature pollen of stable transformed rice (Oryza sativa L.) plants. Actin microfilaments could be visualized only in the late_developmental stage of the immature pollen. During this developmental stage, microfilaments, initially composed of very short fibrils, develop into a very complex and novel network that sometimes totally and sometimes partially encloses the vegetative nucleus and the spherical shaped generative cell in the central cytoplasm of the immature pollen. The behavior of the actin microfilamentous structure throughout the late_developmental stage of the immature pollen is extremely dynamic, and the likelihood of this structure in generating forces for vegetative nucleus and generative cell movement in the immature pollen has been discussed. No actin filaments were visualized in the spherical generative cells.展开更多
AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluor...AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.展开更多
Primordial germ cells (PGCs), as precursors of mam-malian germ lineage, have been gaining more attention as anew resource of pluripotent stem cells, which bring a greatpossibility to study developmental events of germ...Primordial germ cells (PGCs), as precursors of mam-malian germ lineage, have been gaining more attention as anew resource of pluripotent stem cells, which bring a greatpossibility to study developmental events of germ cell invitro and at animal level. EG4 cells derived from 10.5 dayspost coitum (dpc) PGCs of l29/svJ strain mouse wereestablished and maintained in an undifferentiated state.With an attempt to study the differentiation capability ofEG4 cells with a reporter protein: green fluorescence pro-tein, and the possible application of EG4 cells in the re-search of germ cell development, we have generated severalEG4-GFP cell lines expressing enhanced green fluorescenceprotein (EGFP) and still maintaining typicaI characteris-tics of pluripotent stem cells. Then, the differentiation ofEG4-GFP cells in vitro as well as their developmental fatein chimeric embryos which were produced by aggregatingEG4-GFP cells to 8-cell stage embryos were studied. Theresults showed that EG4 cells carrying green fluorescencehave a potential use in the research of germ cell develop-ment and other related studies.展开更多
To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the con...To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the control of promoters： RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae： RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ. In addition, a threefold increase of GFP in Tran-Ⅱ was induced by heat shock at 40℃. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.展开更多
文摘The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.
基金Supported by National Basic Research Program of China( 2009CB119000)National Natural Science Foundation(30871721)~~
文摘[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter sequence and the coding region of EGFP were amplified by polymerase chain reactions （PCR） with vector pCambia 1303 as the template.Using reverse transcript PCR technology,with total RNAs of cucumber as template,the coding region of acid α-galactosidase Ⅰ in cucumber was amplified.The above three fragments were inserted into the multiple cloning sites of expression vector pCambia 1381c.The fusion expression vector of α-galactosidase-EGFP located at the C-terminal of the target genes was constructed.[Result] After enzyme digestion and sequencing,the fusion expression of α-galactosidase-EGFP in cucumber was constructed successfully.[Conclusion] The research laid the experimental basis for further study on the subcellular localization of α-galactosidase in cucumber.
文摘Plasmid pVK1001 which carried the gfp gene of GFPmut2, a mutant of GFP, was introduced into Azospirillum brasilense Yu62 by electroporation. Maize seedlings were inoculated with the GFP-labelled baeteria and grown gnotobiotically in flask with semi-solid agar medium. Observations were performed with confocal laser scanning microscopy (CLSM) and electron microscopy, respectively, at 8 d and 12 d after inoculation. Confocal laser scanning microscopy showed that A. brasilense Yu62 could penetrate into the cortex tissue, colonizing in the intercellular spaces of the parenchyma cells of the cortex tissue. Transmission and scanning electron microscopy (TEM) showed that the majority of the bacteria colonized on the root surface and only a minority of them resided in the root interior.
文摘Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the filamentous bacteria indicated that the NtFtsZs could recognize the potential division sites in E. coli and be polymerized with heterogeneous FtsZ from bacteria. The overexpression of NtFtsZs ∶ gfp inhibited the division of host strain cells and resulted in the long filamentous bacterial morphology. These results suggested that eukaryotic ftsZs have similar function to their prokaryotic homologs. Meanwhile, the different deletions of motifs of NtFtsZs are also employed to investigate the functions of these proteins in E. coli . The results showed that the C_terminal domains of NtFtsZs were related to the correct localization of NtFtsZs in E. coli and the N_terminal domains of NtFtsZs were responsible for the polymerization of homogeneous and heterogeneous FtsZ proteins. The significance of these results in understanding the functions of NtFtsZs in plastid division were discussed.
基金The National Basic Research Program of China(973Program)(No.2011CB933503)the National Natural Science Foundation of China for Key Project of International Cooperation(No.61420106012)China Postdoctoral Science Foundation(No.2013M541592)
文摘To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a probe.The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid.The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21 （DE3 ）cells,the GFP-conjugated MIM-I-BAR （MIM-I-BAR-GFP ） exhibits strong visible fluorescence,and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃,but not at the culture temperature of 37 ℃.This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.
文摘Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, A005-G-T-1-2. Observations were made on pollen at four major developmental stages, viz. I. uni-nucleate microspore stage; II. early bi-cellular pollen stage; III. late bi-cellular pollen stage; and IV. tri-cellular pollen stage. At each of these developmental stages vegetative nucleus, generative nucleus/ cell, and sperm cells were seen undergoing continuous and coordinated motion and migration. These movements seemed to be influenced by associated microfilament networks existing in the pollen. Based on these observations we propose that it is the interaction between the microfilament networks (usually one existing in the central cytoplasm and another in the cortex) that controls the dynamic movement of the vegetative nucleus, generative nucleus/cell and sperm cells. Furthermore, we have also observed that there is an array of microfilaments (oriented mostly parallel to the long axis of the cell) existing in the generative and sperm cells. As far as we are aware, this is the first report showing the existence of microfilaments in living generative and sperm cells of rice pollen. The implication and significance of the existence of microfilaments in generative and sperm cells in rendering self-propelled motion of these cells in relation to their passage and movement in the pollen tube and embryo sac for fertilization were discussed.
文摘Green fluorescent protein (GFP) fused to the F_actin binding domain of mouse talin labels the actin cytoskeleton in the immature pollen of stable transformed rice (Oryza sativa L.) plants. Actin microfilaments could be visualized only in the late_developmental stage of the immature pollen. During this developmental stage, microfilaments, initially composed of very short fibrils, develop into a very complex and novel network that sometimes totally and sometimes partially encloses the vegetative nucleus and the spherical shaped generative cell in the central cytoplasm of the immature pollen. The behavior of the actin microfilamentous structure throughout the late_developmental stage of the immature pollen is extremely dynamic, and the likelihood of this structure in generating forces for vegetative nucleus and generative cell movement in the immature pollen has been discussed. No actin filaments were visualized in the spherical generative cells.
基金Supported by grants from the Nationl Natural Scientific Foundation of China, No.30300082, 30470467, and Scientific Foundation Committee of Guangdong Province, China, No.04009360
文摘AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.
文摘Primordial germ cells (PGCs), as precursors of mam-malian germ lineage, have been gaining more attention as anew resource of pluripotent stem cells, which bring a greatpossibility to study developmental events of germ cell invitro and at animal level. EG4 cells derived from 10.5 dayspost coitum (dpc) PGCs of l29/svJ strain mouse wereestablished and maintained in an undifferentiated state.With an attempt to study the differentiation capability ofEG4 cells with a reporter protein: green fluorescence pro-tein, and the possible application of EG4 cells in the re-search of germ cell development, we have generated severalEG4-GFP cell lines expressing enhanced green fluorescenceprotein (EGFP) and still maintaining typicaI characteris-tics of pluripotent stem cells. Then, the differentiation ofEG4-GFP cells in vitro as well as their developmental fatein chimeric embryos which were produced by aggregatingEG4-GFP cells to 8-cell stage embryos were studied. Theresults showed that EG4 cells carrying green fluorescencehave a potential use in the research of germ cell develop-ment and other related studies.
基金Supported by the High Technology Research and Development Program of China (863 Program) (No. 2005AA601010-05)the Natural Science Foundation of Guangdong Province (No.5010492)the Technology Project of Shenzhen City
文摘To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the control of promoters： RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae： RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ. In addition, a threefold increase of GFP in Tran-Ⅱ was induced by heat shock at 40℃. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.
