A total of 105 transgenic dce lines independently transformed with a pea ferritin gene （Fer） were previously obtained. After seven generations of selfing and β-glucuronidase （GUS） assisted selection, 82 transgeni...A total of 105 transgenic dce lines independently transformed with a pea ferritin gene （Fer） were previously obtained. After seven generations of selfing and β-glucuronidase （GUS） assisted selection, 82 transgenic lines with stable agronomic traits were got. Among the 82 transgenic lines, two high-iron transgenic rice lines Fer34 and Fer65, with the iron contents in the milled rice being 4.82 and 3.46 times of that of the wild type Xiushui 11, respectively were identified. In the two transgenic lines, the exogenous Fer gene was highly expressed, and inherited as a single locus. The transgene had no negative effect on the agronomic traits of rice plant, other mineral nutritional components, appearance quality and eating quality of the milled rice, indicating that these two lines were elite high-iron breeding lines. Furthermore, the practical application and further studies facilitating utilization of the two elite breeding lines were discussed.展开更多
To understand the use of real-time reverse transcription-polymerase chain reaction （real-time RT-PCR） for detecting the relative abundance of mRNA, the expression of a tobacco ferrltin gene （NtFer1） was detected b...To understand the use of real-time reverse transcription-polymerase chain reaction （real-time RT-PCR） for detecting the relative abundance of mRNA, the expression of a tobacco ferrltin gene （NtFer1） was detected by Northern blot and real-time RT-PCR. The results indicated that both of the two methods were able to detect mRNA expression of NtFer1 cleady and similady, namely NtFer1 expression was responsive to iron-ovedoad, and the abundance of NtFer1 mRNA was greatly increased after iron loaded for 6 h. To compare the effect and sensitivity of two methods, results revealed that Northern blot need 30 μg of total RNA and at least 3 days for the total protocol performance, whereas real-time RT-PCR only need 2 μg of total RNA and 1.5 h. The real-time RT-PCR is rather sensitive and effective than Northern blot. Real-time RT-PCR analysis can be used to rapidly detect the relative abundance of mRNA expression instead of Northern blot analysis.展开更多
The neuronal differentiation of mesenchymal stem cells offers a new strategy for the treatment of neurological disorders.Thus,there is a need to identify a noninvasive and sensitive in vivo imaging approach for real-t...The neuronal differentiation of mesenchymal stem cells offers a new strategy for the treatment of neurological disorders.Thus,there is a need to identify a noninvasive and sensitive in vivo imaging approach for real-time monitoring of transplanted stem cells.Our previous study confirmed that magnetic resonance imaging,with a focus on the ferritin heavy chain 1 reporter gene,could track the proliferation and differentiation of bone marrow mesenchymal stem cells that had been transduced with lentivirus carrying the ferritin heavy chain 1 reporter gene.However,we could not determine whether or when bone marrow mesenchymal stem cells had undergone neuronal differentiation based on changes in the magnetic resonance imaging signal.To solve this problem,we identified a neuron-specific enolase that can be differentially expressed before and after neuronal differentiation in stem cells.In this study,we successfully constructed a lentivirus carrying the neuron-specific enolase promoter and expressing the ferritin heavy chain 1 reporter gene;we used this lentivirus to transduce bone marrow mesenchymal stem cells.Cellular and animal studies showed that the neuron-specific enolase promoter effectively drove the expression of ferritin heavy chain 1 after neuronal differentiation of bone marrow mesenchymal stem cells;this led to intracellular accumulation of iron and corresponding changes in the magnetic resonance imaging signal.In summary,we established an innovative magnetic resonance imaging approach focused on the induction of reporter gene expression by a neuron-specific promoter.This imaging method can be used to noninvasively and sensitively detect neuronal differentiation in stem cells,which may be useful in stem cell-based therapies.展开更多
目的利用昆虫细胞/杆状病毒系统表达细粒棘球蚴Eg95-Eg.ferritin融合蛋白,用于开发包虫病新型疫苗以及建立相关血清学诊断方法等研究。方法从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA为模板,通过RT-PCR扩增细粒棘球蚴Eg95和Eg.f...目的利用昆虫细胞/杆状病毒系统表达细粒棘球蚴Eg95-Eg.ferritin融合蛋白,用于开发包虫病新型疫苗以及建立相关血清学诊断方法等研究。方法从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA为模板,通过RT-PCR扩增细粒棘球蚴Eg95和Eg.ferritin基因,采用基因拼接法将Eg95和Eg.ferritin融合,将该融合基因Eg95-Eg.ferritin插入到p Fast Bac DUAL载体中,构建重组转座载体后转化DH10Bac感受态细胞,获得重组Bacmid质粒后转染Sf-9昆虫细胞,传毒3代,对表达蛋白进行Western blot鉴定。结果成功克隆了Eg95和Eg.ferritin基因,通过柔性氨基酸linker成功获得了融合基因Eg95-Eg.ferritin,经PCR和酶切鉴定成功构建了重组质粒p Fast Bac DUAL-Eg95-Eg.ferritin,Western blot结果证实表达蛋白能够被包虫病人标准阳性血清识别。结论在Bac-to-Bac杆状病毒表达系统中成功表达了细粒棘球蚴Eg95-Eg.ferritin融合蛋白,与包虫病人标准阳性血清具有良好的反应性。展开更多
Congenital cataract is a crystallin severe blinding disease and genetic factors in disease development are important. Crystallin growth is under a combination of genes and their products in time and space to complete ...Congenital cataract is a crystallin severe blinding disease and genetic factors in disease development are important. Crystallin growth is under a combination of genes and their products in time and space to complete the coordination role of the guidance. Congenital cataract-related genes, included crystallin protein gene (CRYAA, CRYAB, CRYBA1/A3, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD, CRYGS), gap junction channel protein gene (GJA1, GJA3, GJA8), membrane protein gene (GJA3, GJA8, MIP, LIM2), cytoskeletal protein gene (BF-SP2), transcription factor genes (HSF4, MAF, PITX3, PAX6), ferritin light chain gene (FTL), fibroblast growth factor (FGF) and so on. Currently, there are about 39 genetic loci isolated to which primary cataracts have been mapped, although the number is constantly increasing and depends to some extent on definition. We summarized the recent advances on epidemiology and genetic locations of congenital cataract in this review.展开更多
文摘A total of 105 transgenic dce lines independently transformed with a pea ferritin gene （Fer） were previously obtained. After seven generations of selfing and β-glucuronidase （GUS） assisted selection, 82 transgenic lines with stable agronomic traits were got. Among the 82 transgenic lines, two high-iron transgenic rice lines Fer34 and Fer65, with the iron contents in the milled rice being 4.82 and 3.46 times of that of the wild type Xiushui 11, respectively were identified. In the two transgenic lines, the exogenous Fer gene was highly expressed, and inherited as a single locus. The transgene had no negative effect on the agronomic traits of rice plant, other mineral nutritional components, appearance quality and eating quality of the milled rice, indicating that these two lines were elite high-iron breeding lines. Furthermore, the practical application and further studies facilitating utilization of the two elite breeding lines were discussed.
基金Supported in Part by the Key Project of Chinese Ministry of Education (106065) Heilongjiang Provincial Natural ScienceFoundation (C200533)
文摘To understand the use of real-time reverse transcription-polymerase chain reaction （real-time RT-PCR） for detecting the relative abundance of mRNA, the expression of a tobacco ferrltin gene （NtFer1） was detected by Northern blot and real-time RT-PCR. The results indicated that both of the two methods were able to detect mRNA expression of NtFer1 cleady and similady, namely NtFer1 expression was responsive to iron-ovedoad, and the abundance of NtFer1 mRNA was greatly increased after iron loaded for 6 h. To compare the effect and sensitivity of two methods, results revealed that Northern blot need 30 μg of total RNA and at least 3 days for the total protocol performance, whereas real-time RT-PCR only need 2 μg of total RNA and 1.5 h. The real-time RT-PCR is rather sensitive and effective than Northern blot. Real-time RT-PCR analysis can be used to rapidly detect the relative abundance of mRNA expression instead of Northern blot analysis.
基金supported by the National Natural Science Foundation of China,No.81771892(to JHC).
文摘The neuronal differentiation of mesenchymal stem cells offers a new strategy for the treatment of neurological disorders.Thus,there is a need to identify a noninvasive and sensitive in vivo imaging approach for real-time monitoring of transplanted stem cells.Our previous study confirmed that magnetic resonance imaging,with a focus on the ferritin heavy chain 1 reporter gene,could track the proliferation and differentiation of bone marrow mesenchymal stem cells that had been transduced with lentivirus carrying the ferritin heavy chain 1 reporter gene.However,we could not determine whether or when bone marrow mesenchymal stem cells had undergone neuronal differentiation based on changes in the magnetic resonance imaging signal.To solve this problem,we identified a neuron-specific enolase that can be differentially expressed before and after neuronal differentiation in stem cells.In this study,we successfully constructed a lentivirus carrying the neuron-specific enolase promoter and expressing the ferritin heavy chain 1 reporter gene;we used this lentivirus to transduce bone marrow mesenchymal stem cells.Cellular and animal studies showed that the neuron-specific enolase promoter effectively drove the expression of ferritin heavy chain 1 after neuronal differentiation of bone marrow mesenchymal stem cells;this led to intracellular accumulation of iron and corresponding changes in the magnetic resonance imaging signal.In summary,we established an innovative magnetic resonance imaging approach focused on the induction of reporter gene expression by a neuron-specific promoter.This imaging method can be used to noninvasively and sensitively detect neuronal differentiation in stem cells,which may be useful in stem cell-based therapies.
文摘目的利用昆虫细胞/杆状病毒系统表达细粒棘球蚴Eg95-Eg.ferritin融合蛋白,用于开发包虫病新型疫苗以及建立相关血清学诊断方法等研究。方法从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA为模板,通过RT-PCR扩增细粒棘球蚴Eg95和Eg.ferritin基因,采用基因拼接法将Eg95和Eg.ferritin融合,将该融合基因Eg95-Eg.ferritin插入到p Fast Bac DUAL载体中,构建重组转座载体后转化DH10Bac感受态细胞,获得重组Bacmid质粒后转染Sf-9昆虫细胞,传毒3代,对表达蛋白进行Western blot鉴定。结果成功克隆了Eg95和Eg.ferritin基因,通过柔性氨基酸linker成功获得了融合基因Eg95-Eg.ferritin,经PCR和酶切鉴定成功构建了重组质粒p Fast Bac DUAL-Eg95-Eg.ferritin,Western blot结果证实表达蛋白能够被包虫病人标准阳性血清识别。结论在Bac-to-Bac杆状病毒表达系统中成功表达了细粒棘球蚴Eg95-Eg.ferritin融合蛋白,与包虫病人标准阳性血清具有良好的反应性。
文摘Congenital cataract is a crystallin severe blinding disease and genetic factors in disease development are important. Crystallin growth is under a combination of genes and their products in time and space to complete the coordination role of the guidance. Congenital cataract-related genes, included crystallin protein gene (CRYAA, CRYAB, CRYBA1/A3, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD, CRYGS), gap junction channel protein gene (GJA1, GJA3, GJA8), membrane protein gene (GJA3, GJA8, MIP, LIM2), cytoskeletal protein gene (BF-SP2), transcription factor genes (HSF4, MAF, PITX3, PAX6), ferritin light chain gene (FTL), fibroblast growth factor (FGF) and so on. Currently, there are about 39 genetic loci isolated to which primary cataracts have been mapped, although the number is constantly increasing and depends to some extent on definition. We summarized the recent advances on epidemiology and genetic locations of congenital cataract in this review.
