Diaphanes is the fourth largest genus in Lampyridae, but no luciferase gene from this genus has been reported. In this paper, by PCR amplification of the genomic DNA, the luciferase gene of Diaphanes pectinealis, whic...Diaphanes is the fourth largest genus in Lampyridae, but no luciferase gene from this genus has been reported. In this paper, by PCR amplification of the genomic DNA, the luciferase gene of Diaphanes pectinealis, which is the first case from Diaphanes, was identified and sequenced. The luciferase gene from D. pectinealis spans 1958 base pairs （bp） from the start to the stop codon, including seven exons separated by six introns, and encoding a 547-residuelong polypeptide. Its deduced amino acid sequence showed high protein similarity to those of the Lampyrini tribe （93 - 94% ） and the Cratomorphini tribe （92%）, while low similarity was found with the North American firefly Photinus pyralis （83%） of the Photinini tribe within the same subfamily Lampyrinae. The phylogenetic analysis performed with the deduced amino acid sequences of the luciferase gene further confirms that D. pectinealis, Pyrocoelia, Lampyris, Cratomorphus, and Photinus belong to the same subfamily Lampyrinae, and Diaphanes is closely related to Pyrocoelia, Lampyris, and Cratomorphus. Furthemore, the phylogenetic analysis based on the nucleotide sequences of the luciferase gene indicates Diaphanes is a sister to Lampyris. The phylogenetic analyses are partly consistent with morphological （Branham ＆ Wenzel, 2003） and mitochondrial DNA analyses （Li et al, 2006）.展开更多
The cDNA encoding the luciferase from lantern mRNA of one diurnal firefly Pyrocoelia pygidialis Pic, 1926 has been cloned, sequenced and functionally expressed. The cDNA sequence of P pygidialis luciferase is 1647 bas...The cDNA encoding the luciferase from lantern mRNA of one diurnal firefly Pyrocoelia pygidialis Pic, 1926 has been cloned, sequenced and functionally expressed. The cDNA sequence of P pygidialis luciferase is 1647 base pairs in length, coding a protein of 548 amino acid residues. Sequence analysis of the deduced amino acid sequence showed that this luciferase had 97.8% resemblance to luciferases from the fireflies Lampyris noctiluca, Lampyris turkestanicus and Nyctophila cf. caucasica. Phylogenetic analysis using deduced amino acid sequence showed that P pygidialis located at the base of Lampyris＋Nyctophila clade with robust support （BP=97%）; but did not show a monophyletic relationship with its congeneric species P pectoralis, P tufa and P miyako, all three are strong luminous and nocturnal species. The expression worked in recombinant Escherichia coli. Expression product had a 70kDa band and emitted yellow-green luminescence in the presence of luciferin. Five loops in the P pygidialis luciferase, L1 （NI98-G208）, L2 （T240-G247）, L3 （G317-K322）, L4 （L343-I350） and L5 （G522-D532）, were found from the structure modeling analysis in the cleft, where it was considered the active site for the substrate compound entering and binding. Different amino acid residues between the luciferases of P. pygidialis and the three other known strong luminous species can not explain the situation of weak or strong luminescence. Future study of these loops, residues or crystal structure analysis may be helpful in understanding the real differences between the luciferases between diurnal and nocturnal species.展开更多
Two new types of lentiviral vectors expressing a reporter transgene encoding either firefly luciferase （fLuc） for bioluminescence imaging or the HSV1 thymidine kinase （HSV1-TK） for radiopharmaceutical-based imagin...Two new types of lentiviral vectors expressing a reporter transgene encoding either firefly luciferase （fLuc） for bioluminescence imaging or the HSV1 thymidine kinase （HSV1-TK） for radiopharmaceutical-based imaging were constructed to monitor human embryonic stem cell （hESC） engraftment and proliferation in live mice after trans- plantation. The constitutive expression of either transgene did not alter the properties of hESCs in the culture. We next monitored the formation of teratomas in SCID mice to test （1） whether the gene-modified hESCs maintain their developmental pluripotency, and （2） whether sustained reporter gene expression allows noninvasive, whole-body imaging of hESC derivatives in a live mouse model. We observed teratoma formation from both types of gene-modified cells as well as wild-type hESCs 2-4 months after inoculation. Using an optical imaging system, bioluminescence from the fLuc-transduced hESCs was easily detected in mice bearing teratomas long before palpable tumors could be detected. To develop a noninvasive imaging method more readily translatable to the clinic, we also utilized HSV1-TK and its specific substrate, 1-（2＇-deoxy-2＇-fluoro-β-D-arabinofuranosyl）-5-[^125I]iodouracil（[^125I]FIAU）, as a reporter/ probe pair. After systemic administration, [^125I]FIAU is phosphorylated only by the transgene-encoded HSV1-TK enzyme and retained within transduced （and transplanted） cells, allowing sensitive and quantitative imaging by single-photon emission computed tomography. Noninvasive imaging methods such as these may enable us to monitor the presence and distribution of transplanted human stem cells repetitively within live recipients over a long term through the expression of a reporter gene.展开更多
In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro,we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly lucife...In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro,we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR,from-7 to 1012).The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR.One clone,designated BFVL,was selected from ten neomycin-resistant clones.BFVL showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV infection.Although the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection.A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL.Moreover,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection.These findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.展开更多
In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the f...In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.展开更多
Background p50-associated cyclooxygenase-2 extragenic RNA(PACER)is a recently identified antisense long non-coding RNA(lncRNA)located on the upstream of the promoter region of cyclooxygenase-2(COX-2).Preliminary studi...Background p50-associated cyclooxygenase-2 extragenic RNA(PACER)is a recently identified antisense long non-coding RNA(lncRNA)located on the upstream of the promoter region of cyclooxygenase-2(COX-2).Preliminary studies have suggested that PACER is involved in the regulation of COX-2 expression in macrophagocyte and osteosarcoma cells.However,the role of this lncRNA in colorectal cancer(CRC)remains elusive.Here,we investigated the expression of PACER and its effect on cell proliferation and invasion to explore the role of PACER in CRC.Methods Real-time quantitative PCR(RT-qPCR)analysis was used to evaluate the expression of PACER in CRC tissues and cells.Methyl thiazolyl tetrazolium(MTT)analysis was then used to investigate the inhibition effect of PACER knock-down in cell proliferation.The promoting role of this lncRNA on invasion by CRC cells was analysed by wound-healing assays,colony-formation assay,and transwell assays.We then used fluorescence in situ hybridization(FISH)to establish the subcellular localization of PACER.COX-2 protein levels were quantified by Western blot analysis and grayscale scanning analysis following the knock-down of PACER.Luciferase assay was carried out to monitor the modulation of the COX-2 promoter region by PACER.Tumor xenografts models were used to investigate the impact of PACER on the tumorigenesis of CRC cells in vivo.Enzyme-linked immunosorbent assay(ELISA)was then used to quantify prostaglandin E2(PGE2)production upon knock-down of PACER.Results RT-qPCR analysis revealed that PACER was highly expressed in CRC tissues and cells,and a high PACER-expression level was associated with poor prognosis.MTT assay,wound-healing assay,colony-formation assay,and transwell assay revealed that PACER enhanced CRC-cell proliferation,invasion,and metastasis in vitro.Analysis of lncRNA localization by FISH showed that it mainly resided in the nucleus.RT-qPCR showed that PACER increased mRNA levels of COX-2.Western blot analysis demonstrated,under normal circumstances,that knock-down of PACER decreased the COX-2 protein level.In the case of p50 absence,COX-2 protein increased rapidly and remained highly expressed after knocking down PACER.Luciferase assay revealed that PACER modulated the COX-2 promoter region.Mouse xenograft models of CRC revealed that PACER promoted colorectal tumorigenesis in vivo.ELISA revealed that PACER knock-down inhibited PGE2 production.Conclusions PACER modulates COX-2 expression through the nuclear factor kappa B(NF-jB)pathway in CRC.An increased level of PACER enhances proliferation,migration,and invasion of tumor cells by increasing COX-2 and PGE2 synthesis.展开更多
文摘Diaphanes is the fourth largest genus in Lampyridae, but no luciferase gene from this genus has been reported. In this paper, by PCR amplification of the genomic DNA, the luciferase gene of Diaphanes pectinealis, which is the first case from Diaphanes, was identified and sequenced. The luciferase gene from D. pectinealis spans 1958 base pairs （bp） from the start to the stop codon, including seven exons separated by six introns, and encoding a 547-residuelong polypeptide. Its deduced amino acid sequence showed high protein similarity to those of the Lampyrini tribe （93 - 94% ） and the Cratomorphini tribe （92%）, while low similarity was found with the North American firefly Photinus pyralis （83%） of the Photinini tribe within the same subfamily Lampyrinae. The phylogenetic analysis performed with the deduced amino acid sequences of the luciferase gene further confirms that D. pectinealis, Pyrocoelia, Lampyris, Cratomorphus, and Photinus belong to the same subfamily Lampyrinae, and Diaphanes is closely related to Pyrocoelia, Lampyris, and Cratomorphus. Furthemore, the phylogenetic analysis based on the nucleotide sequences of the luciferase gene indicates Diaphanes is a sister to Lampyris. The phylogenetic analyses are partly consistent with morphological （Branham ＆ Wenzel, 2003） and mitochondrial DNA analyses （Li et al, 2006）.
基金the Natural Foundation of Sciences of Yunnan Province (2006C0046Q)Partly by the Chinese Academy of Sciences (O706551141)
文摘The cDNA encoding the luciferase from lantern mRNA of one diurnal firefly Pyrocoelia pygidialis Pic, 1926 has been cloned, sequenced and functionally expressed. The cDNA sequence of P pygidialis luciferase is 1647 base pairs in length, coding a protein of 548 amino acid residues. Sequence analysis of the deduced amino acid sequence showed that this luciferase had 97.8% resemblance to luciferases from the fireflies Lampyris noctiluca, Lampyris turkestanicus and Nyctophila cf. caucasica. Phylogenetic analysis using deduced amino acid sequence showed that P pygidialis located at the base of Lampyris＋Nyctophila clade with robust support （BP=97%）; but did not show a monophyletic relationship with its congeneric species P pectoralis, P tufa and P miyako, all three are strong luminous and nocturnal species. The expression worked in recombinant Escherichia coli. Expression product had a 70kDa band and emitted yellow-green luminescence in the presence of luciferin. Five loops in the P pygidialis luciferase, L1 （NI98-G208）, L2 （T240-G247）, L3 （G317-K322）, L4 （L343-I350） and L5 （G522-D532）, were found from the structure modeling analysis in the cleft, where it was considered the active site for the substrate compound entering and binding. Different amino acid residues between the luciferases of P. pygidialis and the three other known strong luminous species can not explain the situation of weak or strong luminescence. Future study of these loops, residues or crystal structure analysis may be helpful in understanding the real differences between the luciferases between diurnal and nocturnal species.
文摘Two new types of lentiviral vectors expressing a reporter transgene encoding either firefly luciferase （fLuc） for bioluminescence imaging or the HSV1 thymidine kinase （HSV1-TK） for radiopharmaceutical-based imaging were constructed to monitor human embryonic stem cell （hESC） engraftment and proliferation in live mice after trans- plantation. The constitutive expression of either transgene did not alter the properties of hESCs in the culture. We next monitored the formation of teratomas in SCID mice to test （1） whether the gene-modified hESCs maintain their developmental pluripotency, and （2） whether sustained reporter gene expression allows noninvasive, whole-body imaging of hESC derivatives in a live mouse model. We observed teratoma formation from both types of gene-modified cells as well as wild-type hESCs 2-4 months after inoculation. Using an optical imaging system, bioluminescence from the fLuc-transduced hESCs was easily detected in mice bearing teratomas long before palpable tumors could be detected. To develop a noninvasive imaging method more readily translatable to the clinic, we also utilized HSV1-TK and its specific substrate, 1-（2＇-deoxy-2＇-fluoro-β-D-arabinofuranosyl）-5-[^125I]iodouracil（[^125I]FIAU）, as a reporter/ probe pair. After systemic administration, [^125I]FIAU is phosphorylated only by the transgene-encoded HSV1-TK enzyme and retained within transduced （and transplanted） cells, allowing sensitive and quantitative imaging by single-photon emission computed tomography. Noninvasive imaging methods such as these may enable us to monitor the presence and distribution of transplanted human stem cells repetitively within live recipients over a long term through the expression of a reporter gene.
基金National Natural Science Foundation of China(31070135,81071343)
文摘In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro,we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR,from-7 to 1012).The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR.One clone,designated BFVL,was selected from ten neomycin-resistant clones.BFVL showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV infection.Although the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection.A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL.Moreover,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection.These findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.
基金The General Foundation of Tianjin Science Committee for Applied Basic Research (08JCZDJC21000)Chinese Ministry of Education (30770081)
文摘In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.
基金supported by National Natural Science Foundation of China [No.81572930]Beijing Science and Technology Plan [No.D171100002617004]the Scientific Research Foundation of Graduate School of Harbin Medical University [YJSCX2017-63HYD].
文摘Background p50-associated cyclooxygenase-2 extragenic RNA(PACER)is a recently identified antisense long non-coding RNA(lncRNA)located on the upstream of the promoter region of cyclooxygenase-2(COX-2).Preliminary studies have suggested that PACER is involved in the regulation of COX-2 expression in macrophagocyte and osteosarcoma cells.However,the role of this lncRNA in colorectal cancer(CRC)remains elusive.Here,we investigated the expression of PACER and its effect on cell proliferation and invasion to explore the role of PACER in CRC.Methods Real-time quantitative PCR(RT-qPCR)analysis was used to evaluate the expression of PACER in CRC tissues and cells.Methyl thiazolyl tetrazolium(MTT)analysis was then used to investigate the inhibition effect of PACER knock-down in cell proliferation.The promoting role of this lncRNA on invasion by CRC cells was analysed by wound-healing assays,colony-formation assay,and transwell assays.We then used fluorescence in situ hybridization(FISH)to establish the subcellular localization of PACER.COX-2 protein levels were quantified by Western blot analysis and grayscale scanning analysis following the knock-down of PACER.Luciferase assay was carried out to monitor the modulation of the COX-2 promoter region by PACER.Tumor xenografts models were used to investigate the impact of PACER on the tumorigenesis of CRC cells in vivo.Enzyme-linked immunosorbent assay(ELISA)was then used to quantify prostaglandin E2(PGE2)production upon knock-down of PACER.Results RT-qPCR analysis revealed that PACER was highly expressed in CRC tissues and cells,and a high PACER-expression level was associated with poor prognosis.MTT assay,wound-healing assay,colony-formation assay,and transwell assay revealed that PACER enhanced CRC-cell proliferation,invasion,and metastasis in vitro.Analysis of lncRNA localization by FISH showed that it mainly resided in the nucleus.RT-qPCR showed that PACER increased mRNA levels of COX-2.Western blot analysis demonstrated,under normal circumstances,that knock-down of PACER decreased the COX-2 protein level.In the case of p50 absence,COX-2 protein increased rapidly and remained highly expressed after knocking down PACER.Luciferase assay revealed that PACER modulated the COX-2 promoter region.Mouse xenograft models of CRC revealed that PACER promoted colorectal tumorigenesis in vivo.ELISA revealed that PACER knock-down inhibited PGE2 production.Conclusions PACER modulates COX-2 expression through the nuclear factor kappa B(NF-jB)pathway in CRC.An increased level of PACER enhances proliferation,migration,and invasion of tumor cells by increasing COX-2 and PGE2 synthesis.
