After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resist...After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.展开更多
The preparation, regeneration and mutagenesis of the taxol-producing fungus UV40-19 protoplasts were discussed in the experiment. Totally 42 strains displayed hygromycin resistance. Six strains were found to be positi...The preparation, regeneration and mutagenesis of the taxol-producing fungus UV40-19 protoplasts were discussed in the experiment. Totally 42 strains displayed hygromycin resistance. Six strains were found to be positive mutants when screened on plate containing 90μg/mL hygromycin. One hereditarily stable strain UN05-6 was obtained, which raised the taxol yield from （376.38 ± 8.41）μg/L to （493.12 ±11.36） μg/L. The optimal conditions for the preparation, regeneration and mutagenesis of the taxol producing fungus UV40-19 were as follows： 1 ）enzymolysis in a solution containing 3% lywallzyme, 4% snailase, 1% lysozyme and 3% cellulose at 30~C water bath, pH5.5 - 6.0 for 5h; 2） The prepared protoplasts were regenerated by using bilayer plate culturing method; 3）To mutagenize the fungus UV40-19, the protoplast suspension was treated with 0.8mg/mL NTG for 15min, followed by UV irradiation （30W, 30cm distance）for 40s under magnetic stirring. The purified products of the fungus UN05-6 fermented extracts have significant inhibitive effects on SMMC-7721 cell.展开更多
Glial cell derived neurotrophic factor （GDNF） holds promises for treating neurodegenerative diseases such as Parkinson＇s dis- ease. Human neural stem cells （hNSCs） have proved to be a suitable cell delivery vehic...Glial cell derived neurotrophic factor （GDNF） holds promises for treating neurodegenerative diseases such as Parkinson＇s dis- ease. Human neural stem cells （hNSCs） have proved to be a suitable cell delivery vehicle for the safe and efficient introduction of GDNF into the brain. In this study, we used hNSCs-infected with a lentivirus encoding GDNF and the hygromycin re- sistance gene as such vehicles. A modified tetracycline operator 7 （tetO7） was inserted into a region upstream of the EFI-α promoter to drive GDNF expression. After hygromycin selection, hNSCs were infected with a lentivirus encoding a KRAB-tetracycline repressor fusion protein （TTS）. TTS bound to tetO7 and suppressed the expression of GDNF in hNSCs. Upon administration of doxycycline （Dox） the TTS-tetO7 complex separated and the expression of GDNF resumed. The hNSCs infected with GDNF expressed the neural stem cell specific markers, nestin and sox2, and exhibited no significant change in proliferation rate. However, the rate of apoptosis in hNSCs expressing GDNF was lower compared with normal NSCs in response to actinomycin treatment. Furthermore, a higher percentage of Tuj-I positive cells were obtained from GDNF-producing NSCs under conditions that induced differentiation compared to control NSCs. The inducible expression of GDNF in hNSCs may provide a system for the controllable delivery of GDNF in patients with neurodegenerative diseases.展开更多
基金the Natinnal Biotechnology Reseaxch Project of 863 High Technology, contract No. 101-01-01-02.
文摘After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.
基金Supported by the National Science Foundation of China (30570025)the Fifteen Important Items of Heilongjiang (GA02C101)+3 种基金Key Scientific Program of Harbin (011421126)Research Program for Scholars Overseas by Heilongjiang Education Bureau ( 1152HZ06)Harbin Youth Science Foundation (2005AFOXJ063)Outstanding Young Scientist Foundation of Heilongjiang University
文摘The preparation, regeneration and mutagenesis of the taxol-producing fungus UV40-19 protoplasts were discussed in the experiment. Totally 42 strains displayed hygromycin resistance. Six strains were found to be positive mutants when screened on plate containing 90μg/mL hygromycin. One hereditarily stable strain UN05-6 was obtained, which raised the taxol yield from （376.38 ± 8.41）μg/L to （493.12 ±11.36） μg/L. The optimal conditions for the preparation, regeneration and mutagenesis of the taxol producing fungus UV40-19 were as follows： 1 ）enzymolysis in a solution containing 3% lywallzyme, 4% snailase, 1% lysozyme and 3% cellulose at 30~C water bath, pH5.5 - 6.0 for 5h; 2） The prepared protoplasts were regenerated by using bilayer plate culturing method; 3）To mutagenize the fungus UV40-19, the protoplast suspension was treated with 0.8mg/mL NTG for 15min, followed by UV irradiation （30W, 30cm distance）for 40s under magnetic stirring. The purified products of the fungus UN05-6 fermented extracts have significant inhibitive effects on SMMC-7721 cell.
基金supported by the National Basic Research Program of China (2007CB947704)the High-level Technical Personnel Training of Health Plan of Beijing
文摘Glial cell derived neurotrophic factor （GDNF） holds promises for treating neurodegenerative diseases such as Parkinson＇s dis- ease. Human neural stem cells （hNSCs） have proved to be a suitable cell delivery vehicle for the safe and efficient introduction of GDNF into the brain. In this study, we used hNSCs-infected with a lentivirus encoding GDNF and the hygromycin re- sistance gene as such vehicles. A modified tetracycline operator 7 （tetO7） was inserted into a region upstream of the EFI-α promoter to drive GDNF expression. After hygromycin selection, hNSCs were infected with a lentivirus encoding a KRAB-tetracycline repressor fusion protein （TTS）. TTS bound to tetO7 and suppressed the expression of GDNF in hNSCs. Upon administration of doxycycline （Dox） the TTS-tetO7 complex separated and the expression of GDNF resumed. The hNSCs infected with GDNF expressed the neural stem cell specific markers, nestin and sox2, and exhibited no significant change in proliferation rate. However, the rate of apoptosis in hNSCs expressing GDNF was lower compared with normal NSCs in response to actinomycin treatment. Furthermore, a higher percentage of Tuj-I positive cells were obtained from GDNF-producing NSCs under conditions that induced differentiation compared to control NSCs. The inducible expression of GDNF in hNSCs may provide a system for the controllable delivery of GDNF in patients with neurodegenerative diseases.
