Optimal Promoter Choices For Lentiviral Vector-mediated Transduction Of Different Prostate Cancer Cell Lines And Design Of The Dual Luciferase Labeled Lentiviral Vector With Its Character Of Expression In Vitro And In Vivo

BACKGROUNDProstate cancer(PCa)is a common disease in aged men. In America, PCa is the most common malignant illness in men, and the second leading cause of cancer death. Most PCa was not detected and diagnosed in the first time due to the deep in of prostate and the lack of obvious symptoms. Many patients were diagnosised too lated to take an redical operation. At present, there is still no effective method to treat prostate cancer, especially the androgen independent prostate cancer. Chemotherapy had led to stabilization of disease and improvement of symptoms but did not increase survival rate. The method of gene therapy is to transduct specific gene into target cells and fixes the problem in gene expression through exogenous gene. In the recent 10 years, as the development of molecular biology, gene therapy has made great pogoress in cancer therapy and succeeded in curing some monogenic diseases. Gene therapy has several advantages:①high selectivity comparing to Chemotherapy and radiotherapy, which can be obtained by using targeting gene.②less side effect③efficient even to the advanced stage cancer and metastasis. How ever, lots of things are still to be solved:①the lack of tumor-specific technology to direct the expression of therapeutic genes specifically to the tumor;①the antigenicity of the viral vector, which can deduce the immune response of body, makes it a temporary theatment;③the management of the exogenous gene expression.In the progress of exogenous gene transduction and expression in target cells, the keypoint is to tranduce the vector into target cells and the get stable expression, so it is critical to choose the suitable vector. For now, there are mainly two kinds of vectors:non-viral vectors and viral vectors. Non-viral vectors includes gene sequence and plasmid vector. This kind of vectors is used in limited areas for the low expression efficiency. In contrast, the viral vectors have been widely used, it is estimated that 85% of the total gene therapy was carried on viral vectors. Amoung them, lentiviral vector is the most perspective one with great potential:①no limitation of the cell types:either dividing cell or not;②transducing the exogenous gene into target cell to obtain stable expression;③free of immunogenicity;④free of pathopoiesia areas.The purpose of regulation of exogenous gene in cancer gene therapy is to target the exogenous gene efficiently and specically expressed in tumor cells without hurting the normal tissues. Transcriptional targeting of gene therapy is a novel and prospect method for cancer's treatment.It is a strategy to enhance the specificity of gene expression to target tumor cells of interest.This targeting approach is based on the use of tissue or tumor-specific promoters(TSP)in a heterologous context to direct the expression of therapeutic genes specifically to the tumor. An efficient gene therapy regimen requires transgene expression in the tumor and absence of expression in relevant normal tissues, especially in the liver.This combination of'tumor on" and "liver off" profile can result in increasing the therapeutic index and limiting the toxicity of vectors and transgenes in vivo.Many promoters have already been evaluated for transcriptional targeting in cancer gene therapy to be more sufficient specific to avoid gene expression in normal tissues,while shows noticeable expression levels in tumor cells. Promotor is the key of the efficiency of exogenous gene. It has been demonstrated in many researches that the same promotor shows obvious difference in efficiency in the different cell lines. Take the cytomegalovirus(CMV) promoter for example, it is highly effective in 293 cell lines, in C127 cell lines,however, its efficiency drops sharply. So it is crucial to choose the effective promoter according to different cancer cells. By doing this,the effective and selective expression of exogenous gene can be crchieved.In the evalution of the gene therapeutic effect in animal models, we still a method of ex-vivo monitoring of in-vivo processes of the cancer cells,without killing the subjects until the end of our investigation. This means less animals will be needed during the total experiment and more exact diction. The luciferase is just this kind of marks we need.Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence. There is no endogenous expression in mammalian cell and its detection is not affected by the influence of other substances in cells.Luciferase has characteristics of high sensitivity and specificity,rapid response,simple operation,wide detection range, so as a reporter gene has been widely used in medicine, biology, environmental science,etc. One famous example is firefly luciferase (Fluc) from firefly Photinus pyralis. As early as in 1956, Green and MeElroy got the Flue crystallization. Flue is a molecular weight of 60～64kD polypeptide chain. In a reaction that requires oxygen, magnesium, and ATP, Fluc oxidizes its substrate, luciferin, to produce light with a broad emission spectrum and a peak at approximately 560 nm. Only in living cells produce light-emitting phenomenon, and the light intensity is proportional to the number of labeled cells. As a most commonly used luciferase, Fluc has been widespread application in ATP rapid microbiology, infectious invasion,drug screening,cell marking and tracing as well as in vivo animal imaging, etc. Flue, optimized for expression in mammalian cells, is the most commonly used luciferase for in vivo imaging.Discovered in recent years as a new type of luciferase, Gaussia luciferase (Glue) derived from the Gaussia princeps, its distinguishing feature is efficiently secreted from cells, also has a higher detection sensitivity.Gluc reacts with substrate, coelenterazine, to produce blue light with an emission peak at approximately 480nm,which is ATP-independent.As Glue has small molecular weight, secretion characteristic, detection sensitivity, a short half-life, etc,it has been successfully applied in the experimental study of in vitro and in vivo.By testing animal blood or other body fluids of Glue activity,Gluc can be quantitatively reflected tumor burden in animals,which not only track the progress of tumor metastasis, but also can serve as an effective marker of tumor response to treatment reflects.Luciferase with a specific substrate light-emitting reactions are chemiluminescent,which is specific and no cross-interference.Based on these characteristics,in order to combine the advantages of secreted luciferase Glue and non-secreted luciferase Fluc,this study was to construct a novel dual luciferase co-expression vector and studied its expression characteristics in vivo and in vitro, for the next step to establish dual-luciferase gene markers of tumor cells and animal models, and lay the foundation for monitoring and tracing study later.The article is component of two parts, the first part will discuss the optimal promoter choices for lentiviral vector-mediated transduction of different prostate cell lines; the second part details the design of the dual luciferase lebeled lentivirus vector with its character of expression in vitro and in vivo.Part 1:optimal promoter choices for lentiviral vector-mediated transduction of different prostate cell linesObjectivesTo compare the expression of GFP driven by three different promoters carried by lentiviral vectors in 293A, MOLT-4, DU145, PC3 and RM1 cell lines. Demonstrate that different promoters showed various driven activities among different cell lines. So that promoter selection and proper host cells could be considered in order to achieve high level transgene expression in the further experiment.Methods1. construction of PTY-EF1α-EGFP, PTY-CMV-GFP and PTY-UBIQUITIN-GFPPTY-EF1α-EGFP (EF1α-GFP) was constructed by excising the IRES sequence from PTY-EF1α-IRES-GFP. PTY-CMV-GFP (CMV-GFP) were constructed by replacing the EF1αpromoter in PTY-EF1α-GFP with human cytomegalovirus promoter.2. package and titer lentivirus with different promotor293T cells were planted in T25 flask and transfected with shuttle plasmid PMD2G, PSPAX2 and packaging plasmid PPAX2 following the instruction of lipofectamineTM2000. Harvest virus 60h after transfection. Purified the lentivirus with ultracentrifuge, stored at -80℃. Some samples was subjected to quantitative PCR analysis using the stragene 3500 system, to quantify genomic titer. Lentivirus was serially diluted and sequentially digested with DNase I. Viral RNA was extracted with Roche viral RNAmini kit. A standard amplification curve was set up at range from 105 to 108 copies and the amplification curve corresponding to each initial template copy number was obtained.3. vitro transduction of different cells293T and 293A cells, DU145, PC3 cells and RM1 cells were cultured according to the instruction of ATCC.293A, DU145, PC3, RM1 and MOLT-4 cells were plated at the appropriate density (70-80% confluence the following day). Medium was removed and cells were transduced with maintaining medium containing 108 particles of each recombinant lenti virus per well for 6-well plates and incubated at 37℃,5% CO2 for 3 days before assay.4. Visualization of GFPGFP fluorescence were directly imaged on an Olympus Model BX41 fluorescent microscope (Olympus, Tokyo, Japan). All cell images were captured at×10 magnification and All fluorescent images were collected using an identical exposure time.5. Flow cytometry Analysis of EGFP fluorescence in transduced cells was performed by flow cytometry. Briefly, cells were harvested with Trypsin-EDTA Solution. Cells were washed and resuspended in PBS for analysis on a FACS Calibur (Becton Dickinson, MA, USA). Samples were analyzed using CellQuest oftware (Becton Dickinson, MA, USA).6. Total RNA extraction and quantitative real-time PCR analysesTotal RNA was isolated from the transduced cultures using RNeasy Mini Kit (Qiagen,Valencia,CA). RNA concentration was quantified by the ultraviolet spectrum at 260 nm, and reverse transcribed using PrimeScript 1st Strand cDNA Synthesis Kits (TaKaRa) according to the manufacturer's instructions. Synthesized cDNA corresponding to 100 ng of total RNA, was used for real-time PCR. Specific primers for the GFP and ribosomal RNA (18sRNA; internalcontrol) genes were used in real-time RT-PCR analysis. All primers were synthesized from Invitrogen company. The primers used were as follows:EGFP-F:5'-GAGCTGAAGGGCATCGACTT-3', EGFP-R:5'-CTTGTGCCCCAGGATGTTG-3'; 18sRNA, forward primer: 5'-CAGCCACCCGAGATTGAGCA-3', reverse primer:5'-TAGTAGCGACGGGCGGTGTG-3'.The SYBR green real-time PCR assays for each target gene were performed on cDNA samples using an Stragene 3005 Detection system (Agilent Biosystems).18s RNA assays were run in parallel for each sample. Amplication was carried out in optical 96-well reaction plates (Agilent Biosystems)with each well containing 2μl of cDNA template,1μl of sense primer (10μM),1μl of antisense primer (10μM),12.5μl of SYBR Premix Ex TaqTM (TaKaRa),0.4μl of ROX and DEPC- treated water (8.1μl). The conditions for real-time PCR were one cycle of 95℃for 30 s, followed by 40 cycles of 95℃for 5 s and 60℃for 30 s.Results1. Identification of lentiviral vectorRecombinant plasmid PTY-EF1α-EGFP and PTY-CMV-EGFP was identified by restriction enzyme digestion with Mlu和SmaI. No fragment can be obtained again, which indicates that the vectors have been built successfully.2. Titer of lentiviraus producedCompare the cTvalue of samples with standard curve to get the exact copy in every ml of viral solution. The titers of the lentivirus are listed below: LV-UBIQUITIN-EGFP 5.71*108copy/ml, LV-EFla-EGFP5.53*108copy/ml, LV-CMV-EGFP 5.87*108.3. Evaluation of transgene expression by lentiviral vectors in vitro using flourscent microscopy 293A, DU145, PC3, RM1 and MOLT-4 cells were transduced with 109 copies/ml of either LV-UBIQUITIN-EGFP, LV-EFla-EGFP or LV-CMV-EGFP and,72h later, we compared activity of the UBIQUITIN, EF1αand CMV promoters in various cell lines. GFP expression was analyzed by visualizing green fluorescence. All three lentiviral vectors were capable of producing gene transfer to cell lines, but in various cell lines, they showed different driven activities. In 293A cell lines, all three CMV, EF1αand ubiquitin promoters showed high activities. EGFP expression driven by CMV promoter peaked earlier than the two other promoters (not shown) and showed the highest activity. Also in PC3 cells, CMV promoter showed the highest activity. In the contrast, the CMV promoter showed much lower activity than the two other promoters in MOLT-4 cells. In the present study the CMV promoter showed a different driven activity in vitro among different cells tested, in fact, we found that it is the weakest promoter tested in vitro among MOLT-4 cells and DU145 cells. However, in DU145 cells, EF1αpromoter showed the highest activity, UBIQUITIN promoter comes the second. The CMV promoter shows the weakest activity among them. In RM1 cells, no promoter showed high activity, but the EF1αpromoter activity is higher than the other two.4. Evaluation of transgene expression by lentiviral vectors in vitro using flow cytometryTo determine whether the level of EGFP expression is only dependent on the strength of promoters,293 T cells were transduced with. LV-UBIQUITIN-EGFP, LV-EF1α-EGFP or LV-CMV-EGFP vector.72h after transfection, the cells are digested and resuspended in PBS. Cell concentration per milliliter was determined using a hemocytometer. All the cells were tested using a Aliquots of 200μL of each sample was analyzed on a FACScan cytometer (Becton Dickinson, San Jose, CA) equipped with an argon ion laser set at 488 nm line and fluorescence detector wavelength of 530/30 bandpass using Summit Software with the Cicero upgrade (Cytomation, Ft. Collins, CO). The results shows that the GFP expression from LV-UBIQUITIN-EGFP, LV-EFla-EGFP and LV-CMV-EGFP vectors are varied. The data showed that 293A cells harboring CMV-EGFP, UBIQUITIN-EGFP and EF1α-EGFP vectors displayed high, medium, and low intensity of green fluorescence, respectively. The results suggests that the CMV-EGFP, UBIQUITIN-EGFP and EF1α-EGFP promoters are listed in the order of promoter activity from the highest to the lowest in the 293 A cells, totally corresponds well with the results in the past chapter. Similar results can be draw in other cells. In MOLT4 cells, the order from high to low is; while CMV EF1α, UBIQUITIN in descending order in PC3 and EF1α, UBIQUITIN, CMV in DU145. In RM1 still no promoter shows high activity, but the EF1αpromoter is still higher, then comes the UBIQUITIN promoter, CMV promotor is the last one.5. Evaluation of transgene expression by lentiviral vectors in vitro by Realtime-PCRThe lentiviral stocks were further analyzed byquantitative PCR to determine the DNA titer and the same DNA titers of LV-UBIQUITIN-EGFP, LV-EF1α-EGFP and LV-CMV-EGFP lentiviral stocks were used to transducer 293A, MOLT4, PC3, DU145 and RM1 cells. Negative controls were set up with DEPC H2O correspondingly. The results showed that the EF1αpromoter exhibited the highest activity in MOLT4, PC3, DU145 and RM1 cell lines. The CMV promoter appeared to be the strongest in 293A cells. All the results corresponded well with the results of flow cytometry except the order in PC3. This may be caused of the different expression in mRNA and protein.DiscussionThe lentiviral transduction is one of the most effective methods to overexpress transgenes. However, the influence of the lentiviral DNA context on overexpression still has to be considered. In this study, we have shown that the GFP expression from the UBIQUITIN, EF1αand CMV promoters encoded in three lentiviral vectors, reveals sigficant differences in 293A, MOLT4, PC3, DU145 and RM1 cells. The differential expression of GFP reporter may be due to the specific characters of these promotors as the other parts of the lentivius are the same. The results indicate that the differential expression of GFP between the three promotors in different cells is due to the lentiviral genomic sequences but not the plasmid topology. In PC3, the results of Realtime-PCR did not correlate well with that of flow cytometry and flourscent microscopy. This may be caused by the differenct expression in mRNA and protein. Some mRNA seemed were not translated into protein. The CMV promoter may not always be the best choice for transgene overexpression in all cell types. By comparing to other promoters, while CMV promoter gave much more than 6-fold of GFP expression than human house keeping gene EFla promoter in 293A and PC3 cells, the GFP production by EF1αpromoter revealed the highest intensity in MOLT4, DU145 and RM1 cells.Part 2:design of the dual luciferase lebeled lentivirus vector with its character of expression in vitro and in vivoObjectivesTo constructe a novel dual luciferase co-expression lentiviral vector, which expressed simultaneously secreted luciferase Gluc and non-secreted luciferase Fluc, transducer 293T and PC3 cells with it, establish Subcutaneous tumor model of prostate cancer and study its expression characteristics in vitro and in vivo.Methods1. Construction of co-expressed plasmid PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc with its identificationThe sequence CAG-Gluc-2A-Fluc was deprived from plasmid pAAV2neoCAG-Gluc-2A-Fluc and incerted ito PTY-UBIQUITIN-EGFP vector deprived off GFP coding gene. Sequence GFP-HSV-Tk-neo was then deprived from plasmid pTR-UF11 and cloned into it. The production was identified and then transformed into E. coli DH5a, picked a single colony which is ampicillin resistance,extracted plasmid, and then identified by restriction enzyme digestion and sequencing.2. PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc transient transfection and character of expression293T cells in 10% FBS DMEM medium, placed in 37℃,5% CO2 of incubator for adherent culture. When degree of 293T cells confluence reached 90%, the plasmid was transfected into 293 T cells and the operation steps were carried out according to the instructions of lipofectamineTM2000, control plasmid PTY- UBIQUITIN-EGFP was co-transfected with the equal copies. Expression and distribution characters of Gluc and Fluc in PTY- UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc in vitro were carried out in 24-well plates. Transfection after a 24h, cell culture supernatant and cell lysate was collected. Steps of cell lysis were as follows:washed the cells three times with PBS, added 200μl cell lysis buffer to each hole, placed on ice 30min, then moved cell suspension to 1.5 ml centrifuge tubes, centrifuged for 15 sec at 12 000 rpm,after that collected supernatant and measured its volume. Detection of Gluc and Fluc activity in cell culture supernatant or cell lysate was done in accordance with instructions of Gaussia luciferase Assay kit and Luciferase Assay System. Coelenterazine (concentration of 5ug/ml,200μl) or of Fluc substrate luciferin (150μg/ml,200ul) were added, images were taken instantly with XenogenIVIS vivo imaging system. Time for collecting photon counts was 10 sec. 3. Bioluminescence imaging of mice injected with PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc through tail vein6 six to eight-week-old, weighing 16～20 grams of male BALB/c mice were randomly divided into two groups,3 mice each group. Within 5-7 sec, through tail vein, each mouse in the experimental group was hydrodynamically injected 2.0ml normal saline containing 10μg UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc through tail vein expression plasmid, while only injecting 2.0ml normal saline per mouse in the control group.4. Detect activity of Gluc and Flue in vitroUsing three-plasmid packaging system, and lipfectimine transfection method, the PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc plasmid was packaged into lentiviral vector LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc, then purified and Realtime-PCR method was used for the determination of its titer.293T cells was panted with a density of 1×105/hole in 24-well plates, transduced with 1×107copy per well of virus solution (approximately 50μl), incubated in a 37℃,5% CO2 incubator for 72 hours. Dual Glue and Flue activity dynamic monitoring was carried out in 24 well plates. After lentiviral transduction,293T cells were plated in 24-well plates,1×104/hole,48 hours later the cells were collected every 12 hours for both culture supernatants and cell lysates. According to kit instructions, add Coelenterazine (concentration of 5ug/ml,200μl) or of Flue substrate luciferin (150μg/ml,200ul), images were taken instantly with XenogenIVIS vivo imaging system. The photon collection time is 10 sec. Continuous observation was taken on for 72h.5. Establishment of dual luciferase labeled subcutaneous transplantation tumor model in nude mice and the dynamic monitoring of their expressionsTransduce PC3 cells in the same way which 293T cells were transduced as described above. Negative control was set up with transduction of lentivral vector LV-UBIQUITIN-EGFP, without neo gene, into PC3 cells. Replace the medium with DMEM containing 10% fetal bovine serum medium 72 hours after transduction, while adding G418, with the concentration of 300ug/ml. Adjust the G418 concentration gradually to 700ug/ml. Negative control group was treated the same with experiment group. When 95% of negative control cells died, replace the medium with DMEM containing 10% fetal bovine serum medium free of G418. GFP was observed under fluorescent microscope. The cells left was PC3-Gluc-2A-Fluc with stable expression of dual luciferase. Coelenterazine (concentration of 5ug/ml,200μl) or of Fluc substrate luciferin (150μg/ml,200ul), images were taken instantly with XenogenIVIS vivo imaging system. Exposure time is set to 3min, repeat this triplex.6 nude mice (male,4-6 week old) were fed in the SPF environment. PC3-Gluc-2A-Fluc resuspended in PBS (5×106/ml,200μl/mice) was injected subcutaneously into the flank of nude mice. The control group mices were inject with 200μl NS at the same location. Photos were taken every 3 days with XenogenIVIS system to observe the tumor growth, the observation lasted continuously for 2 weeks. Extract 20μl blood of the mice via the tail vein every 3 days since day 7, add Gaussia luciferase Assay Kit substrate 50μl, Coelenterazine (concentration of 5ug/ml,200μl) images were taken instantly with XenogenIVIS vivo imaging system, with a collection time of 1min.Results1. Identification of PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A--Fluc plasmidRecombinant plasmid PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc was identified by restriction enzyme digestion with KpnⅠand SacⅠ. We obtained 3 fragments, a size of 2274bp fragment was our target fragment. sequencing results showed that the direction and sequence of GFP-HSV-Tk-neo-CAG-GIuc-2A-Fluc fragment was entirely correct.2. Expression and distribution characters of Glue and Flue in vitroExpression and distribution characteristics of Glue and Flue of transient transfection with PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc in 293T cells were namely:24 hours post transfection, the expression of Glue and Flue can both be detected in cell supernatants and cell lysates, most of Glue was mainly detected in the culture media most Flue was mostly within cells;the activity of Glue in the supernatant increased gradually with time while the Flue activity in cells almost kept stable.3. Bioluminescence imaging of miceIn vivo imaging results showed that, both Glue and Flue can take imaged in vivo imaging system, but they have distinctly different imaging characteristics:after the injection of Glue substrate coelenterazine, images show that luminescence can be seen throughout the body, the signal from liver shows no difference in contrast with other organs, while in superficial exposed parts (such as the nose and mouth, and limbs) signals were strong, and the imaging signals decreased sharply within 10 minutes; Liver imaging was showed when Flue specific substrate named D-Luciferin was injected, and the imaging remained stable at least for half an hour.4.Stable expression of Glue and Flue in vitroLV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc transducd 293T cells in 96-well plates,, in 24h,36h,48h,60h,72h detect Glue activity in cell culture supernatant and Flue activity in cell lysate respectively. The results showed that after transduction of 293T cells with dual luciferase, Glue activity and Flue activity can either be detected in cell culture or cell lysates. intracellular expression of and Glue activities in the supernatant gradually increased with time, while Flue activity in cells remained stable. Cells and cell supernatant were added respectively with Glue substrate Coelenterazine and Flue substrate Luciferin, then instantly taken images in XenogenIVIS imaging system. The results showed:in the cell supernatant and the cells the expression of GLuc and Fluc activity can both be detected respectively. GLuc signal in supernatant is stronger than signal of Flue intracellular. With Continuous observation of 30min, Glue images disappeared within 15min, Fluc image remained stable within 30min.5. Dynamic observation of tumor growth in vivo with dual luciferasePC3-Gluc-2A-Fluc cells were into the flank of nude mice subcutaneously, the growth of tumors was observed every 3 days with the Xenogen IVISTM Lumina Imaging system imaging system. The results show that Glue and Flue can both catalyze the light-emitting substrate respectively. As the tumor growth, Flue and Glue light-emitting gradually increased. Because Glue substrate Coelenterazine was directly jnjected into blood, the signal of Glue lasted for just a short time, while the signal of Flue lasted much longer. In the luminous intensity, Glue signal is slightly stronger than Fluc signal; both are in the same order of magnitude.6. Dynamic monitoring of Gluc activity in blood20μl of blood taken through tail-vein was assayed for luciferase activity with Gaussia luciferase Assay kit using a luminometer at various time points. The results showed that at least within 21 days 20μl whole blood by tail vein was able to detect Glue activity, Glue signal growed as the tumor growed.Conclusions1.We have successfully constructed a dual-luciferase expression vector PTY-UBIQUITIN-GFP-HSV-Tk-neo-CAG-GIuc-2A-Fluc.2.Cells in vitro experimental verificationco-expression LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc lentival vector can mediate simultaneously high-level expression of secreted report gene Glue and the non-secreted report gene Flue, and Glue mainly distributed in the cell culture supernatant, Flue mainly exists in the cell lysate, indicating expression and distribution characteristics of Glue and Flue were not changed. After transduced with LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc, cells expressed dual luciferase stably and consistantly.3.In vivo imaging of dual luciferaseTail vein injection of plasmid transfection method confirmed, Flue as a reporter gene is more suitable for in vivo localization of gene expression, while Glue has the character of secretion into the blood with high efficiency. Because of this, if Glue was used in ways like hydrodynamic method, errors may occur when using it for localization. Glue may also not be suitable for establishment and imaging localization of the tumor metastasis animal model of blood through tail vein or atrial injection. After injection of Glue substrate, the luminescence signal appears to be instability and temporary, which is not quite convenient to imaging analysis. Subcutaneous PC3-Gluc-2a-Fluc tumor surveillance luciferase expression confirmed that Flue and Glue subcutaneous tumor model can both be used to monitor the positioning of the tumor in vivo. Both of them can correctly reflect the position and scale of tumor growth. Their luminous intensity are in the same order of magnitude. Therefore, Glue and Flue contributes equally to the monitoring of subcutaneous tumor model.4. Test of blood through tail vein of tumor model in nude mice to monitor tumor growthDynamic monitoring of Glue in blood through tail vein in reflacting tumor growth shows that:expression of Glue in peripheral blood gradually increased with tumor growth, with a significant correlation between the two. Peripheral blood can be used to monitor Gluc activity in reflacting tumor growth indirectly.In conclusion, we successfully construceted the dual luciferase expression lentiviral vector LV-UBIQUITIN-GFP-HSV-Tk-neo-CAG-Gluc-2A-Fluc, established the 293T-Gluc-2A-Fluc cell lines and PC3-Gluc-2A-Fluc cell lines and established animal models with these cell lines. The new lentiviral vector combines the advantages of the secreted report gene Gluc and the non-secreted report gene Fluc, and will provide a new tool for cell labeling and tracing. On one hand, we can use Gluc, which has secretion characteristics and high sensitivity, to dynamically monitor the expression levels of Gluc by detecting Gluc activit in cell culture supernatant or blood without clearaging cells or sacrificing animals; On the other hand, we can use Fluc, which distributes in the intracellular and emits spectra long, penetrates well, and imaged stablily, to localize biological processes in vivo animal imaging.Part 3:A Comparison and Analysis of Methods for Titering Adenoviruses1. Object:To compare different methods commonly used for titering adenovirus and discuss the advantages and drawbacks of each method.2. Methods:Four types of recombined adenovirus were amplified and purified. Each is then simultaneously titered by optical absorbance, Real-time PCR, green fluorescent protein (GFP) labeled method, immunoassay, and cytopathic effect (CPE). Results are then compared.3. Results:No significant difference was found between the titer amounts derived from the GFP labeled method, immunoassay, and cytopathic effect method (P>0.1) There is a position correlation between the titer amounts (r=0.965), even though Real-Time PCR resulted in ten times the vg/ml amount compared to the ifu/ml amount obtained from the immunoassay.4. Conclusion:These results indicate that the GFP labeled method and the immunoassay can quickly determine the titer amount. Real-time PCR cannot titer the real infectious titer of the adenovirus, but the results indicate that its approach has good correlation with that of immunoassay, so it can also reflect the infectious titer of adenovirus, though not that accurate. As the Realtime PCR method takes the least time, it still worth using.