A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of ...A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of primary and secondary needles,cotyledons,and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens.Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy.In fluorometric assays,GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles,while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly.Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons,all tested WWP tissues using the protocol had high levels of transient GUS expression.Thus,heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach.The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.展开更多
DNA expression constructs containing hGH gene directed by bovine αsl-casein gene regulatory sequences were injected into mouse heart or mammary glands in vivo. hGH expression was readily detected by radioimmunoassay ...DNA expression constructs containing hGH gene directed by bovine αsl-casein gene regulatory sequences were injected into mouse heart or mammary glands in vivo. hGH expression was readily detected by radioimmunoassay in milk of the nursing mice throughout lactation. Our results demonstrated the value of this simple and rapid approach in screening gene constructs and identifying transcription regulation sequences in vivo prior to the generation of transgenic animals. For the First time we report the successful expression in mammary gland via intracardial injection.展开更多
The key factors for genome-editing in plants using CRISPR/Cas9,such as the Cas9 nuclease and guide RNA(gRNA)are typically expressed from a construct that is integrated into the plant genome.However,the presence of for...The key factors for genome-editing in plants using CRISPR/Cas9,such as the Cas9 nuclease and guide RNA(gRNA)are typically expressed from a construct that is integrated into the plant genome.However,the presence of foreign DNA in the host genome causes genetic and regulatory concerns,particularly for commercialization.To address this issue,we developed an accelerated pipeline for generating transgene-free genome-edited sorghum(Sorghum bicolor)in the T0 generation.For proof-of-concept,we selected the Phytoene desaturase(PDS)gene as the target due to its visible phenotype(albinism)upon mutation.Following microprojectile-mediated co-transformation with a maize(Zea mays)-optimized Cas9 vector and a guide RNA(gRNA)cassette with a geneticin(G418)resistance gene,we divided tissue derived from immature embryos into two groups(with and without antibiotic selection)and cultured them separately as parallel experiments.In regenerated plants cultured on medium containing MS basal nutrition(to allow albino plants to survive),we detected higher rates of albinism in the non-selection group,achieving editing rates of 11.1–14.3%compared with 4.2–8.3%in the antibiotic selection group.In the T0 generation,22.2–38.1%of albino plants from the non-selection group were identified as transgene-free,whereas only 0–5.9%from the selection group were transgene-free.Therefore,our strategy efficiently produced transgene-free genome-edited plants without the need for self-crossing or outcrossing,demonstrating the feasibility of achieving transgene-free genome-edited sorghum plants within a single generation.These findings pave the way for commercializing transgene-free genome-edited lines,particularly for vegetatively propagated crops like pineapple,sugarcane,and banana.展开更多
AIM： To explore the possibility of using the Noninvasive Micro-test Technique （NMT） to investigate the role of Transient Receptor Potential Canonical 1 （TRPC1） in regulating Ca^2＋ influxes in HL-7702 cells, a no...AIM： To explore the possibility of using the Noninvasive Micro-test Technique （NMT） to investigate the role of Transient Receptor Potential Canonical 1 （TRPC1） in regulating Ca^2＋ influxes in HL-7702 cells, a normal human liver cell line.METHODS： Net Ca^2＋ fluxes were measured with NMT, a technology that can obtain dynamic information of specific/selective ionic/molecular activities on material surfaces, non-invasively. The expression levels of TRPCl were increased by liposomal transfection, whose effectiveness was evaluated by Western-blotting and single cell reverse transcription-polymerase chain reaction.RESULTS： Ca^2＋ influxes could be elicited by adding 1 mmol/L CaCl2 to the test solution of HL-7702 cells. They were enhanced by addition of 20 μmol/L noradrenalin and inhibited by 100 μmol/L LaCl3 （a non-selective Ca^2＋ channel blocker）; 5 μmol/L nifedipine did not induce any change. Overexpression of TRPCl caused increased Ca^2＋ influx. Five micromoles per liter nifedipine did not inhibit this elevation, whereas 100 μmol/L LaCI3 did.CONCLUSION： In HL-7702 cells, there is a type of TRPCl-dependent Ca^2＋ channel, which could be detected v/a NMT and inhibited by La^3＋.展开更多
文摘A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of primary and secondary needles,cotyledons,and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens.Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy.In fluorometric assays,GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles,while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly.Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons,all tested WWP tissues using the protocol had high levels of transient GUS expression.Thus,heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach.The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.
文摘DNA expression constructs containing hGH gene directed by bovine αsl-casein gene regulatory sequences were injected into mouse heart or mammary glands in vivo. hGH expression was readily detected by radioimmunoassay in milk of the nursing mice throughout lactation. Our results demonstrated the value of this simple and rapid approach in screening gene constructs and identifying transcription regulation sequences in vivo prior to the generation of transgenic animals. For the First time we report the successful expression in mammary gland via intracardial injection.
基金GRDC(Grains Research&Development Corporation)for funding the GRDC Better Sorghum with Larger Grain project(2018–2022).
文摘The key factors for genome-editing in plants using CRISPR/Cas9,such as the Cas9 nuclease and guide RNA(gRNA)are typically expressed from a construct that is integrated into the plant genome.However,the presence of foreign DNA in the host genome causes genetic and regulatory concerns,particularly for commercialization.To address this issue,we developed an accelerated pipeline for generating transgene-free genome-edited sorghum(Sorghum bicolor)in the T0 generation.For proof-of-concept,we selected the Phytoene desaturase(PDS)gene as the target due to its visible phenotype(albinism)upon mutation.Following microprojectile-mediated co-transformation with a maize(Zea mays)-optimized Cas9 vector and a guide RNA(gRNA)cassette with a geneticin(G418)resistance gene,we divided tissue derived from immature embryos into two groups(with and without antibiotic selection)and cultured them separately as parallel experiments.In regenerated plants cultured on medium containing MS basal nutrition(to allow albino plants to survive),we detected higher rates of albinism in the non-selection group,achieving editing rates of 11.1–14.3%compared with 4.2–8.3%in the antibiotic selection group.In the T0 generation,22.2–38.1%of albino plants from the non-selection group were identified as transgene-free,whereas only 0–5.9%from the selection group were transgene-free.Therefore,our strategy efficiently produced transgene-free genome-edited plants without the need for self-crossing or outcrossing,demonstrating the feasibility of achieving transgene-free genome-edited sorghum plants within a single generation.These findings pave the way for commercializing transgene-free genome-edited lines,particularly for vegetatively propagated crops like pineapple,sugarcane,and banana.
基金Supported by The National Natural Science Foundation of China,No.30270532 and No.30670774Tsinghua-Yue-Yuen Medical Science Foundation,No.20240000531 and No.20240000547
文摘AIM： To explore the possibility of using the Noninvasive Micro-test Technique （NMT） to investigate the role of Transient Receptor Potential Canonical 1 （TRPC1） in regulating Ca^2＋ influxes in HL-7702 cells, a normal human liver cell line.METHODS： Net Ca^2＋ fluxes were measured with NMT, a technology that can obtain dynamic information of specific/selective ionic/molecular activities on material surfaces, non-invasively. The expression levels of TRPCl were increased by liposomal transfection, whose effectiveness was evaluated by Western-blotting and single cell reverse transcription-polymerase chain reaction.RESULTS： Ca^2＋ influxes could be elicited by adding 1 mmol/L CaCl2 to the test solution of HL-7702 cells. They were enhanced by addition of 20 μmol/L noradrenalin and inhibited by 100 μmol/L LaCl3 （a non-selective Ca^2＋ channel blocker）; 5 μmol/L nifedipine did not induce any change. Overexpression of TRPCl caused increased Ca^2＋ influx. Five micromoles per liter nifedipine did not inhibit this elevation, whereas 100 μmol/L LaCI3 did.CONCLUSION： In HL-7702 cells, there is a type of TRPCl-dependent Ca^2＋ channel, which could be detected v/a NMT and inhibited by La^3＋.
