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BACKGROUND:The polymorphisms of dopamine receptor in promoter region wil affect the expression of the receptor, thereby affecting the dopaminergic neurotransmitter, final y lead to related diseases. OBJECTIVE:To construct the dual luciferase reporter vector containing human DRD1 promoter region and determine its activity, which could provide the basic tool for studying the transcriptional regulation of DRD1 gene. METHODS:DRD1 promoter sequence was amplified by PCR using the human blood genomic DNA and cloned into pGM-T vector. After sequencing, the correctly constructed vectors were ligated to the firefly luciferase reporter plasmid pGL3-Basic. The cloned pGL3-Basic vectors were transfected into HEK293 using cationic liposome method. In the meanwhile, PGL3-Basic vector with no promoter was co-transfected with pGL3-TK plasmid as negative control group. The relative fluorescence intensity was measured by chemiluminescence. RESULTS AND CONCLUSION:(1) Recombinant luciferase reporter gene vectors were confirmed by restriction analysis and sequencing. (2) Compared with the negative control group, the HEK293 cel s transfected by recombinant vectors presented transcriptional activity. (3) In conclusion, luciferase reporter gene vectors containing DRD1 promoter region are successful y constructed and can provide the basic tool for further study on the transcriptional regulation of DRD1.
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Objective To develop a specific trans-splicing intron ribozyme type Ⅰ-mediated dual reporter gene system (Rib53-Fluc-tk) for targeting CEA.Methods The novel CEA-targeting trans-splicing ribozyme with the downstream reporter system (Rib53-Fluc-tk) was constructed by genetic engineering technology.The trans-splicing reaction product was evaluated using the 131I-5-iodo-2'-fluro-l-beta-D-arabinofuranosy-luracil (FIAU) cellular uptake rates and the bioluminescence.Two-sample t test,the analysis of variance and the least significant difference (LSD) t test was performed for data analysis.Results The sequence of Rib53-Fluc-tk was proved by gene-sequencing test.Human MCF-7 breast cancer cells showed a high ratio of firefly luciferase/renilla luciferase (0.64±0.10,n =4).A 520 bp band of product existed,which matched with the predicted size using RNA from cells transfected with Rib53-Fluc-tk in MCF-7.Signals were detected by bioluminescence in human embryonic kidney 293T cells co-transfected with Rib53-Fluc-tk and pCDNA3.1-CEA.The labelling rate of 131I-FIAU was (64.02±4.79)％ (n =3).The radiochemical purity was (95.96± 1.07)％ (n=3),and the stability of the radiocompound remained high in human serum at least for 24 h.The uptake of 131I-FIAU in 293T cells transfected with Rib53-Fluc-tk was (0.31±0.01)％ (n=4),while it increased with the incubation time in 293T cells co-transfected with pCDNA3.1-CEA and Rib53-Fluc-tk and reached (1.40±0.06)％ at 4.5 h (F=1 007.29,t=136.34,both P＜0.01).Conelusions A novel and specific reporter gene in the cellular level was established.Taking advantage of trans-splicing reaction of the ribozyme,it could improve the specificity of the reporter gene imaging.
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Objective To explore the specificity and efficiency of YFP labeled natural killer (NK) cells through Vav-Cre induced YFP reporter system in mice. Methods ROSA26R-YFP and Vav-Cre mice were crossed, and their YFP and Cre gene double positive progeny were screened by genotyping. The specificity of YFP in hematopoietic cells from im-mune organs including lymph nodes, spleen, thymus and bone marrow were analyzed by flow cytometry. The percentages of YFP positive cells in NK cells from lymph nodes, spleen and bone marrow were also analyzed by flow cytometry. Results A total of 11 double positive mice (ROSA26R-YFP-(+/-)VavCre) were obtained in 17 mouse offspring by crossing ROSA26R-YFP mice with Vav-Cre mice. The percentages of YFP positive cells in immune organs including lymph nodes, spleen, thy-mus and bone marrow were 73.87%± 1.51%, 56.07%± 1.47%, 86.17%± 1.74%and 53.60%± 3.56%, and there were signifi-cant differences compared with the corresponding negative control cells(0.27%±0.01%, 1.33%±0.91%, 0.11%±0.01%and 0.29%± 0.03%, P0.05). The positive rates of YFP were significantly higher in NK cells in lymph nodes, spleen and bone marrow (76.94%±0.84%、81.66%±1.18%and 88.92%±0.77%) compared with those of control (P<0.01). Conclusion YFP marked NK cells through Vav-Cre induced YFP reporter system in mice have high specificity and efficiency.
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Objective To construct adenovirus vector containing firefly luciferase reporter gene (AdLuc) and infect bone marrow mesenchymal stem cells (BMSC),then to take bioluminescence imaging in vitro and in vivo for identification.Methods The luciferase gene was amplified with PCR from psiCHECK-2 plasmid and cloned into the adenoviral shuttle vector (pShuttle-CMV).It was confirmed by Nhe Ⅰ/Xba Ⅰ digestion and sequencing.PShuttle-CMV-Luc and backbone vector (pAdeno) were homologous recombined.Then the recombinant plasmid was packaged in HEK293 cells and the virus titer was detected.The BMSC were infected by the recombinant adenovirus.The bioluminescence imaging in vitro was performed to determine the best multiplicity of infection (MOI),and the relationship between bioluminescence intensity and MOI was analyzed by curve fitting regression analysis.Viability was evaluated via Trypan blue staining.The transfected BMSC (l× 106) were implanted into the muscles of forelimb of SD rats,and then tracked by bioluminescence imaging in vivo.Cell viability was compared using two-way repeated measures analysis of variance between groups.Results Enzyme digestion and sequence analysis indicated that Ad-Luc was successfully constructed.The virus titer was 1 × 1010 plaque forming unit (PFU)/ml.The bioluminescence detection in vitro showed that Ad-Luc could infect BMSC high efficiently to express luciferase and the best MOI was 50.The bioluminescence intensity enhanced with increase of MOI (R2 =0.98).No statistically significant difference was found in cell viability between transfected and untransfected BMSC at 1,3,5,7 d.The cell survival rates were (92.5±2.3)％ vs (94.1±1.8)％,(91.4±0.9)％ vs (92.7±2.0)％,(92.1±1.6)％ vs (93.3± 2.4) ％,(91.9 ± 1.5) ％ vs (93.0 ± 3.1) ％,respectively (F =4.38,P ＞ 0.05).The bioluminescence imaging in vivo showed that BMSC survived 1,3,7 d after implantation.However,bioluminescence signal decreased gradually over time.Conclusion It is feasible to apply the optical reporter gene imaging for tracing transplanted stem cells in vitro and in vivo due to the effective transformation of luciferase reporter gene into BMSC by adenovirus vector.
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ObjectiveTo develop a cell culture system with consistent expression of whole hepatitis C virus (HCV) gene and Renilla luciferase gene and to facilitate the study on HCV pathogenesis and the screening of new antiviral drugs.MethodsRenilla luciferase (RLuc) reporter gene and a mutation that could yield higher virus gene expression were introduced into the C-terminus of non-structural protein 5A (NS5A) of the JFH1 viral genome by using recombinant PCR.The viral RNA was transfected into Huh7.5 cells.Naǐve Huh7.5 cells were infected by the supernatant from the viral RNA transfected cells.HCV replication and infection were determined by virus titration,Renilla luciferase assay,immunofluorescence assay and western blotting.IFN-α was used to evaluate the feasibility of this system for anti-HCV new drug screening.ResultsThe viral RNA replicated efficiently in transfected cells.These cells could produce high titer of HCV-Rluc reporter virus and the virus titer reached to 1.5 × 104 FFU/ml at day 15 of posttransfection.The activity of Renilla luciferase was inhibited by IFN-α in a dose dependent manner in Huh7.5 cells infected by HCV-Rluc reporter virus.ConclusionThe recombinant HCV-JFH1-Rluc reporter gene system is sensitive and efficient.It can be a useful tool for high throughput screening of anti-HCV drugs.
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Objective To study the biodistribution of 131 I-2'-deoxy-1-β-D-arabinofuranosy1-5-iodouracil (FIAU) in the rat middle cerebral artery occlusion model and the expression of thymidine kinase (TK) gene in brain tissue after gene-modified stem cell transplantation,and thus evaluate the possibility of further noninvasive monitoring of stem cell transplantation therapy in cerebral infarction.Methods Adenovirus recombinant Ad5-TK-intemal ribosome entry site-brain derived heurotrophic factor-enhanced green florecent protein(IRES-BDNF-EGFP) carrying TK-IRES-BDNF gene was prepared.Cerebral infarction model was established in rats by intraluminal middle cerebral artery occlusion with nylon monofilament.Gene modified bone marrow mesenchymal stem cells were transplanted via intraparenchymal route,lateral ventricle,carotid artery and tail vein,respectively.The normal rats were used as controls.131 I- FAU was prepared to be the tracer for biodistribution study and the ％ ID/g was calculated based on measurement of the tissue radioactivity counts.The expression of TK gene was evaluated by quantitative real-time PCR (QR-PCR) and Western blot analysis.Data were analyzed with independent-samples t-test,one-way analysis of variance (ANOVA) test,and Pearson linear correlation test.Results The ％ ID/g of infarcted brain tissue in the intraparenchymal group was 0.124 ± 0.013,which was significantly higher than that in lateral ventricle group (0.052 ±0.004),carotid artery group (0.061 ±0.002),tail vein group (0.059 ±0.005) and control group (0.005 ±0.001) (t =2.913 - 5.652,all P＜0.05),while there were no statistically significant differences among the other route transplanted groups ( t =0.694 - 1.448,all P ＞ 0.05 ).The differences of ％ ID/g between the infarcted and contralateral sides of brain tissue in all transplanted groups were statistically significant (t =9.004 - 15.734,all P ＜ 0.05 ),while there was no statistically significant difference of this parameter between both sides of brain tissue in control group (t =1.511,P =0.182).The expression of TK gene in intraparenchymal group was significantly higher than other groups (t =7.482 -12.371,all P ＜0.05).The expression levels ofTK gene on QR-PCR showed a positive correlation with ％ID/g of the brain tissue ( r =0.971,P ＜ 0.001 ).Similarly,the ratio of TK/β-actin by the Western blot analysis correlated with the ％ ID/g ( r =0.899,P =0.002 ).Conclusion Intraparenchymal route may be the way of choice for cell transplantation therapy of cerebral infarction.If suitable radionuclide tracer is available,PET or SPECT may be potentially used for noninvasive monitoring of stem cell transplantation in cerebral infarction in vivo.
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AIM: To observe the effect of exogenous androgen responsive element decoy on the promoter of prostate specific antigen (PSA) and the growth of LNCaP cells for searching the possibility of gene therapy for prostate cancer. METHODS: Firstly, pGL3 - PSA luciferase expression vector containing 640bp - promoter fragment of PSA gene was constructed. Then, a 23 -mer phosphorothioated ARE decoy based on the deduced ARE sequence at the promoter region of PSA gene was synthesized. pGL3 - PSA and ARE decoy DNA were cotransfected into PC3 - M cell by lipofectamineTM 2000. Through detecting the activity of luciferase, the effect of ARE decoy on the promoter of PSA was studied. Then the ARE decoy DNA was transfected into LNCaP cells. The effect of decoy DNA on the proliferation of LNCaP cells was examined by using MTT assay. The effects of apoptosis were detected by phase contrast microscopy, DNA agrose gel electrophoresis and flow cytometry. Meanwhile, the nuclear extract was prepared from LNCaP cells and DNA - protein interactions were examined by electrophoretic mobility shift assay (EMSA). RESULTS: The reporter assay showed that the aetivity of luciferase was significantly reduced in the ARE decoy - transfected cells, bnt not in the cells transfected with the control decoy. EMSA demonstrated specific binding of the ARE decoy to androgen receptor. The growth of LNCaP was remarkably inhibited and apoptotic morphological changes as well as DNA fragmentation were observed in the ARE decoy- transfected cells. The rate of apoptosis was 22.4% detected by FCM. CONCLUSION: The ARE decoy is capable of inhibiting the promoter of PSA gene and inducing the apoptosis in prostate cancer cells. It may become a potential therapeutic tool for prostate cancers.
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Objective To investigate the effects of SLB elements presented at 5' UTR of dengue virus genome on viral translation and RNA replication.Methods The 5 end of RNA secondary structures of dengue virus genome were predicted using mfold 3.2,and three mutants with different modifications in SLB elements were designed:SLB1,part deletion of the 5' UAR sequence(82-87nt);SLB2,mutation of the sequence on stem(Mut 77G/C);SLB3,mutation of the sequence on stem(M77-78AG/GA),respectively.The mutants described above were constructed by OL-PCR based on DEN-R.luc2A-RP,respectively.Replicon RNAs corresponding to DEN-R.luc2A-RP,DEN-R.luc2AGDD-RP and the 3 mutants mentioned above were in vitro transcribed and equal amounts of RNA were transfected into BHK cells with Lipofectamine 2000.After RNA transfection,the replicons were detected and characterized by RT-PCR,IFA,Renilla Luciferase assay system and real time RT-PCR,respectively.Results It was shown that SLB1 mutant did not significantly affect the translation of the input RNA,but seriously compromised RNA synthesis;SLB2 mutant did not significantly affect the translation of the input RNA either,but its RNA replication was abolished;both the translation and replication of SLB3 mutant were abolished.Conclusion Both the nucleotide sequence and the RNA secondary structure of the second loop and short stem of SLB element are highly conserved.SLB element may play an essential role on viral translation and RNA replication.The findings of present study may set a foundation for elucidating the role of SLB during viral translation and replication.
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Objective To clone the 5′ non-coding region (NCR) of human interferon-?-inducible protein 10(IP-10), and to identify the transcriptional activity of IP-10 promoter induced by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVEC). Methods Genomic DNA of lymphocytes was isolated from the human blood. With above DNA as the template, the 5'NCR of human IP-10 was amplified by nest polymerase chain reaction (PCR) method. Then, the IP-10 promoter was cloned into luciferase reporter vector, pGL3. The recombined vector was transfected into HUVEC, and then the activity of the luciferase was determined after the cells were stimulated by LPS. Results Human IP-10 promoter was obtained and the pGL3/IP-10 was successfully constructed. Moreover, the activity of luciferase driven by human IP-10 promoter was observed to obviously increase in the HUVEC stimulated by LPS. Conclusion We successfully cloned human IP-10 promoter, constructed luciferase reporter vector driven by the human IP-10 promoter, and confirmed that high transcriptional activity of human IP-10 promoter was induced by LPS in HUVEC. The results supplied an experimental base for the further study of the transcriptional regulation of human IP-10.