Construction and transcriptional activity of human CREG promoter reporter gene plasmid / 解放军医学杂志

Objective To construct the promoter enhanced green fluorescent protein (pEGFP1) reporter gene vector of different truncated fragments of human cellular repressor of E1A-stimulated genes (hCREG), and compare the transcriptional activity of each promoter to determine the hCREG core promoter region. Methods The promoter fragment with length of 2003 (–1925/+78) bp was obtained by querying the hCREG sequence from US National Center of Biotechnology Information (NCBI) database and combining with the characteristics of the promoter. Five promoter fragments were truncated by PCR and double enzyme digestion and cloned into pEGFP1 to construct pEGFP1_hCREG_2003, pEGFP1_hCREG_945, pEGF P1_hCREG_586, pEGFP1_ hCREG_478 and pEG FP1_hCREG_358 reporter gene vector plasmid. The 293T cells were transiently co-transfected with the internal reference plasmid pGL4.73 [hRluc/SV40] for 48 hours. The green fluorescence expression of pEGFP1_hCREG promoter reporter gene was observed under fluorescence microscope, and the mRNA expression of each promoter was detected by real-time quantitative PCR, and the core promoter region was determined. Bioinformatics was used to predict the transcription factors that might bind to the core promoter region. Results Five hCREG promoter reporter gene vectors were successfully constructed by double enzyme digestion and gene sequencing. The results showed that the transcription activity of pEGFP1_ hCREG_586 was the highest (P0.05), implying that –867/ –509 bp is a negative regulatory region, and there existed enhancer sequences in –400/–281 bp and –508/–401 bp, so the core promoter region of hCREG gene is located in the upstream sequence of –508/–281 bp. Bioinformatics predicted that the possibly bound transcription factors in key promoter region –508/–281 bp were Pax5/P53, C/EBPβ, GR-β, GATA-1, GR-α, c-Jun, PRB/ PRA, YY1, RXR-α, AP-2, FOXP3, GR, TFIID, STAT4 and c-Ets-1. Conclusion The recombinant plasmid of hCREG gene promoter has been successfully constructed, the core promoter of which is located in –508/–281 bp, where several transcription factors might be bound.

Cloning of the Coffea canephora SERK1 promoter and its molecular analysis during the cell-to-embryo transition

Background: Somatic embryogenesis receptor-like kinase 1 (SERK1) is a cell membrane receptor active in different plant tissues and involved in cell differentiation activities including somatic embryogenesis. The identification of promoter elements responsible for SERK1 expression during the onset of somatic embryogenesis can be useful to understand the molecular regulation of the cell-to embryo transition, and these promoter elements represent biotechnological tools in plant organ tissue culture. Results: A −1,620 bp DNA sequence located upstream of the Coffea canephora SERK1 gene homologue (CcSERK1) was isolated, and then, different segments containing key response elements (REs) for somatic embryogenesis onset and development were fused to the uidA (encoding a ß-glucuronidase, GUS) reporter gene to evaluate its expression in transgenic leaf explants. DNA segments of −1,620 and −1048 bp in length directed uidA expression with patterns in leaf explants similar to those occurring during somatic embryogenesis. When a −792-bp fragment was used, uidA expression disappeared only in leaf explants and pro-embryogenic mass but persisted in developing embryos. No uidA expression was detected in any embryogenic stage when a −618-bp fragment was used. Conclusion: DNA deletions showed that a −1048-bp sequence located upstream of the CcSERK1 gene is sufficient to direct gene expression during the onset and the development of C. canephora somatic embryogenesis. The DNA segment located between −1048 and −792 bp (containing BBM and WUS REs) is needed for gene expression before embryogenesis onset but not during embryo development. The promoter segment between −792 and −618 bp (including GATA, ARR1AT, and ANT REs) regulates gene expression in developing embryos.

The Complement Inhibition of Ursolic Acid and Tetrandrine on Tumor Related Signaling Pathways and Their Synergistic Antitumor Proliferation Effects / 浙江中医药大学学报

[Objective]This study was aimed to research the synergistic antitumor proliferation effects and their best proportion of ursolic acid(UA) and tetrandrine(Tet), a pair of compounds isolated from Chinese herbs which showed complement inhibition on the multiple signal pathways. [Methods] The reporter assays on tumor-related signal pathways for MAPK/ERK, MAPK/JNK, NF-κB, Wnt, Notch, Cell Cycle, Myc/Max and Hypoxia were used to study the effect of five different Chinese herbal compounds on tumor proliferation,it was concluded cepharanthine(Cep), Tet, 18α-glycyrrhetinic acid(18α-Gly), UA and luteolin(Lut). MTT assay and crystal violet staining were used to study the antiproliferative effect of 15 different compounds for the tumor cells of MDA-MB-231,SW480,MG63,PC3,DU145,HCT116,143B and MDA-MB-468, which is consisted with Cep, Tet, 18α-Gly, UA and Lut for the 15 different compounds. Coefficient of drug interaction(CDI) method was used to detect the synergistic effect of the two compounds. Combination of index(CI) and isobologram method was used to screen the best ratio of compounds in their antiproliferative effects. [Results] The signal pathway reporter assay showed that UA and Tet could complementarily inhibit tumor-related signaling pathways. And the results also showed that UA and Tet could induce synergetic anti-tumor cell proliferation in vitro. Furthermore, the optimal ratio of UA and Tet was 9:1 by using isobologram and CI method. [Conclusion] UA and Tet can be inhibited and complemented by 8 tumor-related signaling pathways, and we used MTT assay and crystal violet staining or other methods to confirm the synergistic antitumor proliferation effects, furthermore, the optimal proportion for UA and Tet were screened, and it provided a new insight to develop new anticancer formula in research.

Construction of Luciferase Reporter Vector Containing High-mobility Group Box 1 Promoter in Mice and Characterization of Its Transcriptional Activity / 广州中医药大学学报

Objective To construct luciferase reporter vector containing full-length high-mobility group box 1 ( HMGB1, GenBank NM-010439) promoter for the screening of medicine. Methods The full-length HMGB1 promoter was amplified by polymerase chain reaction ( PCR) , and then was inserted into GV238 vector to construct plasmid GV238-HMGB1-P-Luc. GV238-HMGB1-P-Luc combined with internal reference plasmid pRL was co-transfected into Hela cells ( GV238-HMGB1-P-Luc group, which served as positive control group) . Plasmid pGL3-basic combined with pRL was co-transfected into Hela cells (pGL3-basic group, which served as negative control group) . Additionally, lipopolysaccharides ( LPS, 0.2 μg/mL) was used as the activator for the positive control group (LPS group), and then sodium butyrate (SB, 10 mmol/L) was used as the inhibitor for LPS group ( SB group) . At the end of experiment hour 24, luciferase activity was detected. Results The results of digestion, amplification, sequencing and identification showed that the full length of HMGB1 promoter was 2 140 bp, and the DNA sequence was correct, without mutation. Luciferase activity in GV238-HMGB1-P-Luc group was increased as compared with that of the pGL3-basic group ( P<0.05) . Luciferase activity in the LPS group was increased ( P<0.01, compared with that of GV238-HMGB1-P-Luc group) , and then was decreased after the administration of SB ( P<0.01, compared with that of the LPS group) . Conclusion A model of luciferase reporter vector containing HMGB1 promoter has been successfully constructed. Its activity can be increased by LPS, and then is in hibited by SB. The model can be used for further screening of medicine with the activities of regulating HMGB1 promoter.

Bioluminescent Monitoring the Influence of Cepharanthine on Colon Cancer Growth in Mouse Xenograft Tumor Model / 浙江中医药大学学报

[Objective]To investigate the anti-proliferative effects of CEP on HCT116 cells and in mouse xenograft model. [Methods]The in vivo anti-cancer activity of CEP was determined with Xenogen bioluminescence imaging in a xenograft tumor model. The cel-based multiple signaling pathway reporter assays were carried out to determine the effects of CEP on these pathways. [Results] CEP inhibited growth of human cancer cells, the IC 50 was 0.8~11.5 μM. CEP induced cellcycle arrest in S and G2/M phase. CEP also inhibited xenograft tumor growth in athymic nude mice bearing HCT116 cells. The xenograft tumor size was significantly reduced upon the treatment with CEP(10 or 20 mg·kg-1 body weight) for up to 3 weeks. Pathway-spe-cific reporter assays indicated that CEP effectively suppressed the NF-κB and MAPK/ERK signaling pathways. [Conclusions] Our results suggest that the anticancer activity of CEP in colon cancer cells may be mediated through targeting NF-κB and MAPK/ERK signaling pathways.

Cre-loxP recombination vectors for promoter studies

For promoter studies the cloning, subcloning and transfer to different plasmid vectors usually requires use of restriction enzymes and ligation reactions. One obstacle is the nucleotide polymorphisms of eukaryotic genomic DNA, which has the consequence that a sequence often differs from published sequences. Therefore sequencing, rigorous restriction enzyme analysis or introduction of suitable sites has to be performed prior to cloning and subcloning. In addition, conventional methods using restriction enzymes, insert purifications and ligations is expensive and labour demanding. We have developed a fast, efficient and inexpensive Cre recombinase-loxP based method, which allows cloning of promoter regions and subcloning of these into a variety of vectors in a restriction enzyme independent manner. We here demonstrate that expression of a number of reporter genes and a therapeutic gene from both a viral and 2 mammalian promoters cloned by this recombinase method have activities comparable to conventionally cloned plasmids.