A method to mathematically determine transduction efficiency of lentivirus in HeLa cells Research Article
|
|
- Meredith Watkins
- 6 years ago
- Views:
Transcription
1 Gene Therapy and Molecular Biology Vol 15, page 138 Gene Ther Mol Biol Vol 15, , 2013 A method to mathematically determine transduction efficiency of lentivirus in HeLa cells Research Article Zhipin Liang 1, Bin Liu 1, Xuechao Zhao 1, Chang Liu 1,*, Xiaohong Kong 1,* 1 Laboratory of Medical Molecular Virology, School of Medicine, Nankai University, Weijin Road No.94, Nankai District, Tianjin, P.R. China *Correspondence: Chang Liu and Xiaohong Kong, Laboratory of Medical Molecular Virology, School of Medicine, Nankai University, Weijin Road No.94, Nankai District, Tianjin, P.R. China. Tel: ; Fax: changliu@nankai.edu.cn; kongxh@nankai.edu.cn. Keywords: 50% Transduction efficiency (TE50), Lentiviral vector, Gene therapy, Flow cytometry Received: 18 December 2013; Revised: 29 December 2013 Accepted: 30 December 2013; electronically published: 31 December 2013 Summary Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors are widely applied in gene transfer and gene therapy because of their high transduction efficiency and stable expression. There are various quantification methods for the transduction efficiency (TE) calculation of lentiviral vectors, while most of them usually need serial dilutions and experimental materials costing. So it is required to develop a feasible quantification method for lentiviral vectors' TE calculation. Here, we deduced a math equation between the number of infectious viral particles (v) and the transduction efficiency (TE): v = a ln (1-TE) + b. An HIV-1 based lentiviral vector FG12 encoding the GFP reporter gene was used to evaluate practicability of this method. According to the math equation, TE50 of FG12 was verified in different number of HeLa cells. Our results documented that the math equation was adopted into the TE calculation. Comparing with routine TE50 determination method, this method needed fewer serial dilutions and was more feasible. I. Introduction: Viral vectors are widely applied in gene transfer and gene therapy due to their inherent ability of viruses to introduce genetic material into target cells (Machida 2003; Papayannakos and Daniel 2012; Apolloni et al., 2013). Especially, human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors are important tools of gene transduction because of their stable integration in dividing and nondividing cells and allowing transgene longterm expression (Ikeda et al., 2003; Leyva et al., 2011; Gay et al., 2012). Lentiviral vectors biological and safety properties have been improved by multiple genetic modifications (Pauwels K Fau - Gijsbers et al., 2009; Matrai et al., 2010; Persons 2010; Dropulic 2011), 138
2 Liu et al: A method to mathematically determine transduction efficiency of lentivirus in HeLa cells and now they have been developed into the third generation (Salmon and Trono 2006). FG12 used in this study is one of the third generation HIV-based lentiviral vectors. Security and transduction efficiency of FG12 have been improved through the following optimizations: 1) deletion of the U3 region to generate self-inactivation (SIN) vectors (Miyoshi et al., 1998), 2) addition of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to increase expression levels, addition a reporter gene of GFP to be observed and quantified easily (Qin et al., 2003), 3) pseudotyping with vesicular stomatitis virus (VSV) glycoproteins extending cell tropism (Lois et al., 2002). Because of FG12 s features described above, it has been successfully applied in the field of immunization and anti-hiv-1 infection (Qin et al., 2003; Yang et al., 2008). All of lentiviral vectors applications require an accurate and reliable method of determination of the transduction efficiency. For this purpose, several methods have been developed (Sanburn and Cornetta 1999; Geraerts et al., 2006; Yamamoto et al., 2006; Leyva et al., 2011). Most of them are focusing on quantification of reporter gene expression or integration and involving a cell transduction step to provide the proportion of infectious particles contained in the vector stocks. The transduction efficiency (TE) is usually evaluated based on the doses of viral vector required to obtain 50% (TE50) of positive cells being transduced (Gay et al., 2012). Quantification methods of TE50 routinely need the dose-response curve of transduced positive cells versus vector input reached the 100% value. They usually need tedious serial dilutions and adequate amount of vector stocks (Salmon and Trono 2006). In our study, HeLa cells infected with the lentiviral vector FG12 were taken as a research model. Based on the simple hypothesis that infections by individual viral particles are individual events (Sigal et al., 2011), a math equation between the infectious viral particle number and the transduction efficiency was deduced. According to this equation, TE50 of FG12 to HeLa cells was easily calculated and also verified in different numbers of HeLa cells. II. Materials and Methods: A. Cell culture The HEK293T cell line, derived from a transformation of HEK293 cell with the SV40 large T gene, is used for virus packaging (Salmon and Trono 2006). HeLa cell line was often used in virus titer assay (Naldini et al., 1996; Salmon and Trono 2006). Both kinds of cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10 % fetal bovine serum (FBS), 100 U/ ml penicillin, and 100 μg/ ml streptomycin at 37 o C with 5 % CO 2. B. Virus packaging and ultracentrifugation Viral vector plasmid pfg12 (25 μg) and helper plasmids (prsvrev 6.25 μg, phcmv-g 12.5 μg and pmdlg/prre 7.5 μg) were cotransfected into the HEK293T cells ( cells in 15 cm dish). Total plasmids and polyethylenimine (PEI) were mixed for 10 min in the serum-free DMEM with a ratio of μg: 205 μg. Medium was refreshed at 8 hours post transfection. Then 48 hours after transfection, the cultured supernatant was harvested, filtered through a 0.45 μm filter, and concentrated with ultracentrifugation at 4 o C 82,000 g for 1.5 hours. The supernatant was discarded gently after ultracentrifugation, and added 200 µl serum-free DMEM medium into the tube. The pellet was resuspended at 4 o C for overnight (Salmon and Trono 2006; Liang et al., 2012). Next day, medium contained virus was dispensed into 25µl per tube and stored at - 80 o C for further study. 139
3 Gene Therapy and Molecular Biology Vol 15, page 140 C. Lentiviral transduction and fluorescence determination HeLa cells were seeded in the 24-well plates ( cells in each well) the day before infection. A total of 15 wells were needed for the infection at a ratio of 1μl/ 2μl/ 4μl/ 8μl concentrated virus medium and a negative control with three repeats, respectively. At the time of infection, the old medium was discarded and add 500 μl fresh DMEM which contains 40 μg/ ml DEAE-dextran. Different volumes of FG12 viruses were added into the wells with three repeat, respectively. Medium was refreshed at 24 hours post infection. About 48 hours after infection, cells were treated in accordance with the requirements by flow cytometry. Mock infected cells were used as negative controls. Discarded the DMEM and digested for 3 min with 300 μl 0.25 % trypsin each well; terminated the reaction with 300 μl DMEM (10 % serum) and mixed by pipette; pooled the cell suspensions into a 1.5 ml tube; pelleted the cells at 850 g for 5 min; discarded the supernatant and resuspended with 500 μl phosphate buffer solution (PBS); re-pelleted the cell at 850 g for 5 min, discarded the supernatant and resuspended with 500 μl 1 % paraformaldehyde fixed for 30 min. At last, green fluorescent was checked by the flow cytometer (FACSCalibur, BD). D. Statistical analysis Flow cytometry assay data were analyzed by the WinMDI Version 2.9 software provided by Joseph Trotter of the Scripps Research Institute, La Jolla, CA. M1 was determined based on the cells without virus infection, and M1 stands for the GFP negative ratio as shown in Figure 1A. M2 was the GFP positive ratio, and got by M1 minused by 100 % as shown in Figure 1A. Checking time of fluorescence was optimized. Statistical analyses and linear figures (Figure 2, 3) were performed using Prism 5 for Windows version 5.02 (GraphPad Software, Inc., La Jolla, CA). Figure 1: Definition of GFP positive cells and HeLa cellular permissivity to FG12 virus. (A) The above three panels are HeLa cells mock- infected, and M1 was determined based on these cells. Following three panels are HeLa cells infected with 2 μl FG12 virus, M2 was the ratio of GFP positive cells. Green fluorescence was observed at 48 hours post FG12 infection. Each bar scale = 50 μm in the figure. Peak value of diagram moved to the higher position of FL1- H. (B) Transduction efficiency of HeLa cells by different volumes of FG12 viruses. In the permissivity assay, HeLa cells ( ) were infected with increasing doses of FG12 stocks ( μl). The percentage of GFP- expressing cells was determined by flow cytometry at 48 hours post infection. All experiments were done in triplicate. 140
4 Liu et al: A method to mathematically determine transduction efficiency of lentivirus in HeLa cells Figure 2: TE equation acquiring and testing with flow cytometry results. (A) In the equation acquiring assay, HeLa cells ( ) were infected with 1 μl, 2 μl, 4 μl, 8 μl FG12 viruses, mocked infection as a control. Different volumes of FG12 viruses were added into the 24- well plate with 500 μl DMEM which contains 40 μg/ ml DEAE- dextran. Flow cytometry assay was performed at 48 hours post infection. Twenty thousand cells were collected as total events as shown in panel A of the above figure. (B) A linear chart was acquired according to the transduction efficiency of different volumes FG12 infection by linear regression method. Meanwhile, the equation and R- squared value were shown on the chart. All experiments were done in triplicate. III. Results A. Transduction efficiency and HeLa cellular permissivity to FG12 vectors To measure the transduction efficiency of FG12 to HeLa cells, HeLa cells in one well of 24-well plate were infected or mockinfected with 2 μl concentrated FG12 viral stocks. After HeLa cells were infected by FG12 vectors, the GFP gene was transcribed and expressed in host cells. After 48 hours, infected or mock-infected groups were analysed using flow cytometry for GFP expression (Hawley and Hawley 2004). 141
5 Gene Therapy and Molecular Biology Vol 15, page 142 Readout of flow cytometry standard (FCS) format files were analyzed with the software WinMDI, and the GFP positive threshold was determined by the marker value of the mockinfected group. As shown in Figure 1A, marker M1 is set around the negative peak of the subclass control on the histogram of mock infection. Marker M2 is set to the right of M1 to designate GFP positive events. M1 in the mock-infected group should be 100%. The percentage of GFP positive cells represented the transduction efficiency of FG12, so the transduction efficiency is equal to M2, the percentage of GFP positive cells, numerically. To explore the permissivity of HeLa cells to the FG12 vector, a dose-response assay of GFP expression processed routinely. HeLa cells were seeded at cells per well of a 24-wells plate. Then the cells were infected with increasing doses of FG12, and the percentage of GFP-expressing cells was determined by flow cytometry at 48 hours post infection according the method described above. As shown in Figure 1B, the doseresponse curve of GFP positive cells versus vector was drawn. The curve showed that to reach the 100% GFP positive the lowest vector dose was μl, and to obtain 50% (TE50) GFP positive cells 2-3 μl FG12 was need. These results suggested that HeLa cells were highly permissive to FG12 vector, with TE50=2-3 μl. B. Transduction efficiency equation deduction The transduction efficiency (TE) equation was deduced based on the hypothesis that HeLa cells infections by individual FG12 vector are individual events (Sigal et al., 2011). When the fraction of infected target cells (I) is low relative to the total target cell population (T), I is a possible product of the total target cell population and the probability (Prob) is that each target cell is infected: I = T Prob (infection per cell) (Sigal et al., 2011) The probability of a single viral infection in a cell is r, and the probability that non-infected cell is 1-r. We suppose that infection probability per viral vector is individual event that is independent of other infections. Given λ, a frequency that viral vector contacting a single cell, the probability that no infection is Prob (no infection by λ viruses) = (1-r) λ. Therefore, the probability of successful infection with λ viruses per cell is 1-(1-r) λ and the total number of infected cell is I = T (1-(1-r) λ ). The transduction efficiency (TE) is the percentage of successful infected cell in total cells, so TE = I/T = 1-(1-r) λ. The following equation could be deduced as ln (1-TE) = λ ln (1-r). We suppose that the probability of a single viral vector infecting a cell (r) is a constant, so ln (1-r) is a constant. There is linear correlation between ln (1-TE) and λ. In a certain viral vector infection, λ is proportional to the volume of FG12 infecting the HeLa cells (v). Finally, v and ln (1-TE) are linear correlation and the TE equation is v = a ln (1-TE) + b. In the equation: v indicated the viral vector volume; TE indicated the transduction efficiency that could be obtained by flow cytometry as above descriptions; a and b are two constants got in the calculation by linear regression method based on value of the v and TE. C. TE equation calculation and verification Theoretically, there are two paired parameters of v and TE needed to determine the value of TE. To test whether the equation could reflect the FG12 infecting HeLa cells in practical application, we took 142
6 Liu et al: A method to mathematically determine transduction efficiency of lentivirus in HeLa cells FG12 in a series of volumes (1 μl, 2 μl, 4 μl, 8 μl) respectively, to infect HeLa cells. GFP positives cells were observed using fluorescence microscopic and further quantified by flow cytometry assay at 48 hours post infection of FG12. As shown in Figure 2A, GFP positive cells increased along with the volumes of FG12. Transduction efficiency of FG12 to HeLa cells were calculated according to the definition presented previously. As shown in Table 1, the volume of FG12 (v) and the transduction efficiency (TE) were listed, and ln (1-TE) was also calculated. Table 1: Equation testing in a gradient amount of viruses infection. HeLa cells ( ) were infected by a gradient amount of FG12 (1 μl, 2 μl, 4 μl, 8 μl) viruses. At 48 hours post infection, transduction efficiency was determined by flow cytometry assay. v: virus volume; TE: transduction efficiency (GFP positive percentage); ln (1-TE): natural logarithm of negative transduction efficiency. All experiments were done in triplicate. According to the TE equation, v and ln (1-TE) have a linear relationship. As shown in Figure 2B, according to our experimental data and based on the method of least squares which linear curve determination from different points, the TE equation in this study was got v = ln (1-TE) The regression coefficient R 2 = 0.97 showed that the strong linear correlation between v and ln (1-TE). D. TE50 calculation and application From the TE equation v = ln (1-TE) , TE50 could be easily calculated. As the definition of TE50 described, TE50 should be the vector doses required to achieve 50% positive infected cells (Chen et al., ). In TE equation, supposing TE is equal to 0.5 (50%), and then v was calculated to 2.78μl. Moreover, supposing a serial values of TE, for example, TE= 40%, 50%, 60% transduction efficiency was evaluated. According to the equation, HeLa cells ( ) should be infected by 1.69 μl (40%), 2.78 μl (50%), 4.09 μl (60%) FG12, respectively. At 48 hours post infection, GFP expression of HeLa cells were analyzed by flow cytometry and transduction efficiency was calculated. As shown in Figure 3A, the 50% infection efficiency was accurate for a zero error with the equation derived value, experimentally derived values of 40% and 60% infection efficiency were in the vicinity of corresponding equation derived values with 0.01 to 0.03 standard deviations. Consequently the equation could be applied to determine virus transduction efficiency in HeLa cells. During virus infection and fluorescence determination HeLa cells were seeded in one well of the 24-well plates to validate whether this method is practicable. We next checked the application of this equation in different cells number. A series of cell numbers in , , HeLa cells were respectively passaged in one well of 24- well plates for determination of 50% infection efficiency. We presumed that 50% infection efficiency in HeLa cells was composed of three-fold of HeLa cells infection, and infection of HeLa cells was two-fold of HeLa cells infection. Volumes of FG12 viruses used to infect corresponding amount of cells were 1.39 μl ( cells), 2.78 μl ( cells), 4.17 μl ( cells) respectively. An expectable result was got from the results of flow cytometry assay. As shown in Figure 3B, transduction efficiencies of three groups were close to the 50% infection efficiency, and only with a 0 to 0.10 standard deviations with the equation derived values. According to these results, equation can be adapted to different cell numbers within certain errors. 143
7 Gene Therapy and Molecular Biology Vol 15, page 144 Figure 3: Equation application and a series of tests of transduction efficiency. (A) Different transduction efficiency can be got in a certain amount of cells by virus quantities. HeLa cells ( cells) were infected by 1.69 μl, 2.78 μl, 4.09 μl FG12 viruses to get a series of 40 %, 50 %, 60 % transduction efficiency. (B) In order to check the application in different cell numbers, a series of , and HeLa cells were checked for a 50 % transduction efficiency with 1.39 μl ( cells), 2.78 μl ( cells) and 4.17 μl ( cells) FG12 viruses infection. Experimentally derived values got from the flow cytometry results were compared with the equation derived values. Blue bars stand for the equation derived value, red bars stand for the experimentally derived value got from the flow cytometry assay. All experiments were done in triplicate. IV. Discussion and conclusion Lentivirus can permanently integrate into a chromosome of an infected cell and subsequently express viral genes in that cell and its progeny, thereby lentiviral vector was widely used in gene delivery and gene therapy (Buchschacher and Wong-Staal 2000; Federico 2003; Campbell and Hope 2005). To facilitate their applications in gene therapy, it is required to develop a feasible quantification method for lentiviral vectors' transduction efficiency determination. Many methods were based on the characters induced by viruses or expression of viral DNA or RNA (Sanburn and Cornetta 1999; Pourianfar et al., 2012). Transduction efficiency of fluorescence virus is determined according to the expression level of fluorescence gene in target cells. The fluorescence intensity can be checked by flow cytometry, which is easy and convenient for fluorescence virus quantification. Additionally, the dose of lentiviral vectors or the cells transduced by lentiviral vector is very important in clinical application. So it s necessary to quantify a virtual transduction efficiency of lentiviral vector before used in the clinical gene therapy. This study provided a feasible method of quantification virtual TE50 of HIV-based lentiviral vector by flow cytometry. In this work, an HIV-1 based lentiviral vector FG12 was used to evaluate the TE50 calculating method. According to our method, an accurate TE50 of variable amount of cells could be obtained without any tedious serial dilutions. Such as TE50 of HeLa cells from to in 24-well plate was acquired easily and exactly in our study. In conclusion, our method was based on the hypothesis that HeLa cells infections by individual FG12 are individual events. A formula v = a ln (1-TE) + b could be obtained in the range of FG12 permissivity to HeLa cells. This equation was calculated by linear regression method based on the measured value of the v and TE. According to the calculated equation, different transduction efficiency and TE50 of different amount of cells were verified. Experimentally derived values were consistent with equation derived values. What s more, virus infection didn t need serial dilutions in TE calculation. This makes TE50 calculation of 144
8 Liu et al: A method to mathematically determine transduction efficiency of lentivirus in HeLa cells predetermined viral transduction efficiencies became feasible. Theoretically, it can be straight forwardly modified to imply that, this method can be used in TE calculation of any replication defective viral vectors with a fluorescent reporter gene. Acknowledgements This work was supported by the National Natural Science Foundation of China ( and ), the Fundamental Research Funds for the Central University ( ), Natural Science Foundation of Tianjin Municipal Science and Technology Commission (13JCQNJC09800), the Ministry of Education of Talents in the New Century (NCET ) and Doctoral Fund of Ministry of Education ( ). Authors' contributions CL and XK made great contributions to equation derivation and design in this study. ZL, BL and XZ carried out all the experiments and statistical analysis. ZL and CL drafted the manuscript. CL and XK revised the manuscript. All the authors have read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. References: Apolloni A., Sivakumaran H., Lin M.H., Li D., Kershaw M.H., Harrich D. (2013). A mutant Tat protein provides strong protection from HIV-1 infection in human CD4+ T cells. Hum Gene Ther 24, Buchschacher G.L., Jr., and Wong-Staal F. (2000). Development of lentiviral vectors for gene therapy for human diseases. Blood 95, Campbell E.M., and Hope T.J. (2005). Gene therapy progress and prospects: viral trafficking during infection. Gene Ther 12, Chen C., Akerstrom V., Baus J., Lan M.S., Breslin M.B. Comparative analysis of the transduction efficiency of five adeno associated virus serotypes and VSV-G pseudotype lentiviral vector in lung cancer cells. Virol J 10, 86. Dropulic B. (2011). Lentiviral vectors: their molecular design, safety, and use in laboratory and preclinical research. Human gene therapy 22, Federico M. (2003). Lentivirus Gene Engineering Protocols. In Methods in Molecular Biology Gay V., Moreau K., Hong S.-S., Ronfort C. (2012). Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity. Arch Virol 157, Geraerts M., Willems S., Baekelandt V., Debyser Z., Gijsbers R. (2006). Comparison of lentiviral vector titration methods. BMC Biotechnol 6, 34. Hawley T.S., and Hawley R.G. (2004). Flow Cytometry Protocols. Humana Press, Totowa, N.J. Ikeda Y., Takeuchi Y., Martin F., Cosset F.L., Mitrophanous K., Collins M. (2003). Continuous high-titer HIV-1 vector production. Nat Biotechnol 21, Leyva F.J., Anzinger J.J., McCoy J.P., Jr., Kruth H.S. (2011). Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors. BMC Biotechnol 11, 13. Liang Z., Guo Z., Wang X., Kong X., Liu C. (2012). Two retroviruses packaged in one cell line can combined inhibit the replication of HIV-1 in TZM-bl cells. 145
9 Gene Therapy and Molecular Biology Vol 15, page 146 Virol Sin 27, Lois C., Hong E.J., Pease S., Brown E.J., Baltimore D. (2002). Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, Machida C.A. (2003). Viral vectors for gene therapy : methods and protocols. Humana Press, Totowa, N.J. Matrai J., Chuah M.K., and VandenDriessche T. (2010). Recent advances in lentiviral vector development and applications. Mol Ther 18, Miyoshi H., Blomer U., Takahashi M., Gage F.H., Verma I.M. (1998). Development of a self-inactivating lentivirus vector. J Virol 72, Naldini L., Blomer U., Gallay P., Ory D., Mulligan R., Gage F.H., Verma I.M., Trono D. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, Papayannakos C., and Daniel R. (2012). Understanding lentiviral vector chromatin targeting: working to reduce insertional mutagenic potential for gene therapy. Gene Ther 20, Pauwels K Fau - Gijsbers R., Gijsbers R Fau - Toelen J., Toelen J Fau - Schambach A., Schambach A Fau - Willard-Gallo K., Willard-Gallo K Fau - Verheust C., Verheust C Fau - Debyser Z., Debyser Z Fau - Herman P., Herman P. (2009). State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations. Current Gene Therapy 9, Persons D.A. (2010). Lentiviral Vector Gene Therapy: Effective and Safe? Mol Ther 18, Pourianfar H.R., Javadi A., and Grollo L. (2012). A Colorimetric-Based Accurate Method for the Determination of Enterovirus 71 Titer. Indian Journal of Virology 23, Qin X.F., An D.S., Chen I.S., Baltimore D. (2003). Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A 100, Salmon P., and Trono D. (2006). Design and Production of Human Immunodeficiency Virus-Derived Vectors. In Cell Biology: A Laboratory Handbook. Celis J.E., ed. Elsevier Inc. pp Sanburn N., and Cornetta K. (1999). Rapid titer determination using quantitative real-time PCR. Gene Ther 6, Sigal A., Kim J.T., Balazs A.B., Dekel E., Mayo A., Milo R., Baltimore D. (2011). Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy. Nature 477, Yamamoto N., Tanaka C., Wu Y., Chang M., Inagaki Y., Saito Y., Naito T., Ogasawara H., Sekigawa I., Hayashida Y. (2006). Analysis of human immunodeficiency virus type 1 integration by using a specific, sensitive and quantitative assay based on realtime polymerase chain reaction. Virus Genes 32, Yang L., Yang H., Rideout K., Cho T., Joo K.I., Ziegler L., Elliot A., Walls A., Yu D., Baltimore D., Wang P. (2008). Engineered lentivector targeting of dendritic cells for in vivo immunization. Nat Biotechnol 26,
DNA context and promoter activity affect gene expression in lentiviral vectors
ACTA BIOMED 2008; 79: 192-196 Mattioli 1885 O R I G I N A L A R T I C L E DNA context and promoter activity affect gene expression in lentiviral vectors Gensheng Mao 1, Francesco Marotta 2, Jia Yu 3, Liang
More informationPre-made Lentiviral Particles for Fluorescent Proteins
Pre-made Lentiviral Particles for Fluorescent Proteins Catalog# Product Name Amounts Fluorescent proteins expressed under sucmv promoter: LVP001 LVP001-PBS LVP002 LVP002-PBS LVP011 LVP011-PBS LVP012 LVP012-PBS
More informationPre-made Reporter Lentivirus for MAPK/ERK Signal Pathway
Pre-made Reporter for MAPK/ERK Signal Pathway Cat# Product Name Amounts LVP957-P or: LVP957-P-PBS SRE-GFP (Puro) LVP958-P or: LVP958-P-PBS SRE-RFP (Puro) LVP959-P or: LVP959-P-PBS SRE-Luc (Puro) LVP960-P
More informationPre-made Reporter Lentivirus for JAK-STAT Signaling Pathway
Pre-made Reporter for JAK-STAT Signaling Pathway Cat# Product Name Amounts LVP937-P or: LVP937-P-PBS ISRE-GFP (Puro) LVP938-P or: LVP938-P-PBS ISRE-RFP (Puro) LVP939-P or: LVP939-P-PBS ISRE-Luc (Puro)
More informationQuickTiter Lentivirus Titer Kit (Lentivirus-Associated HIV p24)
Product Manual QuickTiter Lentivirus Titer Kit (Lentivirus-Associated HIV p24) Catalog Number VPK-107 VPK-107-5 96 assays 5 x 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction
More information~Lentivirus production~
~Lentivirus production~ May 30, 2008 RNAi core R&D group member Lentivirus Production Session Lentivirus!!! Is it health threatening to lab technician? What s so good about this RNAi library? How to produce
More informationVIRAL TITER COUNTS. The best methods of measuring infectious lentiviral titer
VIRAL TITER COUNTS The best methods of measuring infectious lentiviral titer FLUORESCENCE CYCLES qpcr of Viral RNA SUMMARY Viral vectors are now routinely used for gene transduction in a wide variety of
More informationQuickTiter Lentivirus Quantitation Kit (HIV p24 ELISA)
New and Improved Product Manual QuickTiter Lentivirus Quantitation Kit (HIV p24 ELISA) Catalog Numbers VPK-108-HIV-p24 96 tests VPK-108-HIV-p24-5 5 x 96 tests FOR RESEARCH USE ONLY Not for use in diagnostic
More informationVIROLOGY. Engineering Viral Genomes: Retrovirus Vectors
VIROLOGY Engineering Viral Genomes: Retrovirus Vectors Viral vectors Retrovirus replicative cycle Most mammalian retroviruses use trna PRO, trna Lys3, trna Lys1,2 The partially unfolded trna is annealed
More informationPre-made Reporter Lentivirus for NF-κB Signal Pathway
Pre-made Reporter for NF-κB Signal Pathway Cat# Product Name Amounts LVP965-P or: LVP965-P-PBS NFKB-GFP (Puro) LVP966-P or: LVP966-P-PBS NFKB-RFP (Puro) LVP967-P or: LVP967-P-PBS NFKB-Luc (Puro) LVP968-P
More informationConstitutive Reporter Lentiviral Vectors Expressing Fluorescent Proteins
Constitutive Reporter Lentiviral Vectors Expressing Fluorescent Proteins www.vectalys.com/products/ Constitutive Reporter Lentiviral Vectors Catalog Number referring to this User Manual: 0008VCT; 0009VCT;
More informationCRISPRaTest Functional dcas9-activator Assay Kit v1 Last update: 2018/07/04 Cellecta, Inc.
CRISPRaTest Functional dcas9-activator Assay Kit v1 Last update: 2018/07/04 Cellecta, Inc. Copyright (c) 2018 Cellecta, Inc. All Rights Reserved. Table of Contents 1. CRISPRaTest Functional dcas9-activator
More informationPROTOCOL: OPTIMIZATION OF LENTIVIRAL TRANSDUCTION USING SPINFECTION
Last Modified: April 2018 Last Review: October 2018 PROTOCOL: OPTIMIZATION OF LENTIVIRAL TRANSDUCTION USING SPINFECTION Table of Contents 1. Brief Description 1 2. Materials and Reagents.1 3. Optimization
More informationPre-made Lentiviral Particles for intracelular labeling: (LocLight TM Living cell imaging lentivirus for sub-cellular localization)
Pre-made Lentiviral Particles for intracelular labeling: (LocLight TM Living cell imaging lentivirus for sub-cellular localization) LocLight TM cell organelle labeling lentivirus is provided as 200ul/per
More informationCertificate of Analysis
Certificate of Analysis Catalog No. Amount Lot Number 631987 10 μg Specified on product label. Product Information plvx-ef1α-ires-mcherry is a bicistronic lentiviral expression vector that can be used
More informationCertificate of Analysis
Certificate of Analysis plvx-ef1α-ires-puro Vector Table of Contents Product Information... 1 Description... 2 Location of Features... 3 Additional Information... 3 Quality Control Data... 4 Catalog No.
More informationTable of Contents. Product and Service Item of Genemedi Lentivirus...3 Product Character of Genemedi Lentivirus...3
User Manual Lentivirus USER GUIDE Table of Contents Table of Contents... 1 Safe Use of Lentivirus (Lv)...2 Storage and Dilution of Lentivirus... 2 Introduction of Lentivirus... 3 Lentivirus Product, Service
More informationLarge Scale Infection for Pooled Screens of shrna libraries
Last modified 01/11/09 Large Scale Infection for Pooled Screens of shrna libraries Biao Luo, Glenn Cowley, Michael Okamoto, Tanaz Sharifnia This protocol can be further optimized if cells being used are
More informationChoosing Between Lentivirus and Adeno-associated Virus For DNA Delivery
Choosing Between Lentivirus and Adeno-associated Virus For DNA Delivery Presenter: April 12, 2017 Ed Davis, Ph.D. Senior Application Scientist GeneCopoeia, Inc. Outline Introduction to GeneCopoeia Lentiviral
More informationProduct Information: CellPlayer NeuroLight Red Lentivirus - Synapsin Promoter Catalog Number: 4584
Product Information: CellPlayer NeuroLight Red Lentivirus - Synapsin Promoter Catalog Number: 4584 Contents 1x vial of CellPlayer NeuroLight Red Lentivirus - Synapsin promoter (0.45 ml/vial) Lot #: Titer:
More informationReady-to-use Lentiviral Particles for intracelular labeling
Ready-to-use Lentiviral Particles for intracelular labeling (LocLight TM Living cell imaging lentivirus for sub-cellular localization) LocLight TM cell organelle labeling lentivirus are provided as 200ul/per
More informationAdenovirus Manual 1. Table of Contents. Large Scale Prep 2. Quick MOI Test 4. Infection of MNT-1 Cells 8. Adenovirus Stocks 9
Adenovirus Manual 1 Table of Contents Large Scale Prep 2 Quick MOI Test 4 TCID 50 Titration 5 Infection of MNT-1 Cells 8 Adenovirus Stocks 9 CAUTION: Always use filter tips and bleach everything!!! Adenovirus
More informationA Stable System for the High-Titer Production of Multiply Attenuated Lentiviral Vectors
doi:10.1006/mthe.2000.0103, available online at http://www.idealibrary.com on IDEAL A Stable System for the High-Titer Production of Multiply Attenuated Lentiviral Vectors Natacha Klages, Romain Zufferey,
More informationHIV-1 Virus-like Particle Budding Assay Nathan H Vande Burgt, Luis J Cocka * and Paul Bates
HIV-1 Virus-like Particle Budding Assay Nathan H Vande Burgt, Luis J Cocka * and Paul Bates Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
More informationQuickTiter Lentivirus Titer Kit (Lentivirus-Associated HIV p24)
Product Manual QuickTiter Lentivirus Titer Kit (Lentivirus-Associated HIV p24) Catalog Number VPK-107 VPK-107-5 96 assays 5 x 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction
More informationSOP History Number Date Reason for Change v1 10/10/2014 Original V2 10/10/2016 Update V3 10/10/2018 Update
Document Number: SASoM/METHOD/073.v3 Title: Version: Author: Lentivirus production from HEK293T cells v3 Paul Reynolds Effective from: 10/10/2018 Valid to: 09/10/2020 SOP History Number Date Reason for
More informationMulti-plasmid approach
MISSION Lentiviral Packaging Mix Catalog Number SHP001 Storage Temperature 20 C TECHNICAL BULLETIN Product Description The MISSION Lentiviral Packaging Mix is an optimized formulation of two plasmids expressing
More informationSupplementary information. MARCH8 inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins
Supplementary information inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins Takuya Tada, Yanzhao Zhang, Takayoshi Koyama, Minoru Tobiume, Yasuko Tsunetsugu-Yokota, Shoji
More informationSupplemental Materials and Methods Plasmids and viruses Quantitative Reverse Transcription PCR Generation of molecular standard for quantitative PCR
Supplemental Materials and Methods Plasmids and viruses To generate pseudotyped viruses, the previously described recombinant plasmids pnl4-3-δnef-gfp or pnl4-3-δ6-drgfp and a vector expressing HIV-1 X4
More informationViral Vectors In The Research Laboratory: Just How Safe Are They? Dawn P. Wooley, Ph.D., SM(NRM), RBP, CBSP
Viral Vectors In The Research Laboratory: Just How Safe Are They? Dawn P. Wooley, Ph.D., SM(NRM), RBP, CBSP 1 Learning Objectives Recognize hazards associated with viral vectors in research and animal
More informationJumpstart your research with ViraPower Lentiviral Expression Systems
ViraPower Lentiviral Expression Systems Jumpstart your research with ViraPower Lentiviral Expression Systems With ViraPower Lentiviral Systems you can: Efficiently transduce both dividing and non-dividing
More informationRabies virus-like particles expressed in HEK293 cells
Engineering Conferences International ECI Digital Archives Vaccine Technology IV Proceedings Spring 5-21-2012 Rabies virus-like particles expressed in HEK293 cells Diego Fontana Cell Culture Laboratory
More informationMyBioSource.com. QuickTiter Lentivirus Titer Kit (Lentivirus-Associated HIV p24) Product Manual. Catalog Number. MBS x 96 assays
Product Manual QuickTiter Lentivirus Titer Kit (Lentivirus-Associated HIV p24) Catalog Number MBS168055 96 assays MBS168055-5 5 x 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction
More informationTrans-Lentiviral TM Packaging System
Trans-Lentiviral TM Packaging System The safest lentiviral system for expression TLP4614 - Trans-Lentiviral Packaging System, shrna TLP4615 - Trans-Lentiviral Packaging System, shrna (contains cell line)
More informationPhosphate buffered saline (PBS) for washing the cells TE buffer (nuclease-free) ph 7.5 for use with the PrimePCR Reverse Transcription Control Assay
Catalog # Description 172-5080 SingleShot Cell Lysis Kit, 100 x 50 µl reactions 172-5081 SingleShot Cell Lysis Kit, 500 x 50 µl reactions For research purposes only. Introduction The SingleShot Cell Lysis
More informationRecombinant Protein Expression Retroviral system
Recombinant Protein Expression Retroviral system Viruses Contains genome DNA or RNA Genome encased in a protein coat or capsid. Some viruses have membrane covering protein coat enveloped virus Ø Essential
More informationOCCUPATIONAL HEALTH CONSIDERATIONS FOR WORK WITH VIRAL VECTORS
OCCUPATIONAL HEALTH CONSIDERATIONS FOR WORK WITH VIRAL VECTORS GARY R. FUJIMOTO, M.D. PALO ALTO MEDICAL FOUNDATION ADJUNCT ASSOCIATE CLINICAL PROFESSOR OF MEDICINE DIVISION OF INFECTIOUS DISEASES AND GEOGRAPHIC
More informationSupplemental Information
Cell Host & Microbe, Volume 14 Supplemental Information HIV-1 Induces the Formation of Stable Microtubules to Enhance Early Infection Yosef Sabo, Derek Walsh, Denis S. Barry, Sedef Tinaztepe, Kenia de
More informationLentiBoost Lentiviral Transduction Enhancer
Product Manual Lot number: IP1108_LM_R_5 LentiBoost Lentiviral Transduction Enhancer SB-P-LV-101-01 SB-P-LV-101-02 Catalogue number: 100 standard transductions 300 standard transductions Shipped at room
More informationA phase I pilot study of safety and feasibility of stem cell therapy for AIDS lymphoma using stem cells treated with a lentivirus vector encoding
A phase I pilot study of safety and feasibility of stem cell therapy for AIDS lymphoma using stem cells treated with a lentivirus vector encoding multiple anti-hiv RNAs John A. Zaia, M.D. John J. Rossi,
More informationxcelligence Real-Time Cell Analyzers
xcelligence Real-Time Cell Analyzers Application Note No. 9 A New Way to Monitor Virus-Mediated Cytopathogenicity Introduction One of the most important procedures in virology is the measurement of viral
More informationDevelopment of a Sensitive Assay for Detection of Replication-Competent Recombinant Lentivirus in Large-Scale HIV-Based Vector Preparations
METHOD doi:10.1016/s1525-0016(03)00167-9 Development of a Sensitive Assay for Detection of Replication-Competent Recombinant Lentivirus in Large-Scale HIV-Based Vector Preparations Paul Escarpe, Nathalie
More informationA protocol for enhancement of the AAV-mediated expression of transgenes
A protocol for enhancement of the AAV-mediated expression of transgenes Hiroaki Mizukami, Takeharu Kanazawa, Takashi Okada, and Keiya Ozawa Division of Genetic Therapeutics, Center for Molecular Medicine,
More informationReplication-competent Lentivirus Analysis of Clinical Grade Vector Products
original article Replication-competent Lentivirus Analysis of Clinical Grade Vector Products Kenneth Cornetta 1 3, Jing Yao 1, Aparna Jasti 1, Sue Koop 1, Makhaila Douglas 1, David Hsu 4, Larry A Couture
More informationSupplementary Material
Supplementary Material Nuclear import of purified HIV-1 Integrase. Integrase remains associated to the RTC throughout the infection process until provirus integration occurs and is therefore one likely
More informationLentiviral Delivery of Combinatorial mirna Expression Constructs Provides Efficient Target Gene Repression.
Supplementary Figure 1 Lentiviral Delivery of Combinatorial mirna Expression Constructs Provides Efficient Target Gene Repression. a, Design for lentiviral combinatorial mirna expression and sensor constructs.
More informationLenti-Bicistronic & Tricistronic Expression Systems
Lenti-Bicistronic & Tricistronic Expression Systems Lenti-Bicistronic Vector Lenti-Tricistronic Vector Complete Lenti-Bicistronic Expression Kit Complete Lenti-Tricistronic Expression Kit Partial Lenti-Bicistronic
More informationFeb 11, Gene Therapy. Sam K.P. Kung Immunology Rm 417 Apotex Center
Gene Therapy Sam K.P. Kung Immunology Rm 417 Apotex Center Objectives: The concept of gene therapy, and an introduction of some of the currently used gene therapy vector Undesirable immune responses to
More informationYour Gene ATG GGT. pd1118 EF1a-ORF, Lenti-ElecD 7803 bp
Mammalian Expression Vectors has mammalian expression vectors suitable for transient or stable expression. These vectors are available with features including various promoters, markers, and fusions. Lentiviral
More informationSupplementary data Supplementary Figure 1 Supplementary Figure 2
Supplementary data Supplementary Figure 1 SPHK1 sirna increases RANKL-induced osteoclastogenesis in RAW264.7 cell culture. (A) RAW264.7 cells were transfected with oligocassettes containing SPHK1 sirna
More informationNature Medicine: doi: /nm.2109
HIV 1 Infects Multipotent Progenitor Cells Causing Cell Death and Establishing Latent Cellular Reservoirs Christoph C. Carter, Adewunmi Onafuwa Nuga, Lucy A. M c Namara, James Riddell IV, Dale Bixby, Michael
More informationGene Transfer Vector Derived from Jembrana Disease Virus: A Review
American Journal of Biochemistry and Biotechnology Review Articles Gene Transfer Vector Derived from Jembrana Disease Virus: A Review 1,2 Asmarani Kusumawati, 3 Tenri A. Wanahari, 4 Pudji Astuti, 3 Basofi
More informationActivation of Gene Expression by Human Herpes Virus 6
Activation of Gene Expression by Human Herpes Virus 6 M. E. M. Campbell and S. McCorkindale 1 Introduction Human herpes virus type 6 (HHV-6) was first detected by Salahuddin et al. [6] and has been isolated
More informationChiachen Chen 1,2, Victoria Akerstrom 1,2, James Baus 1,2, Michael S Lan 1,2,3 and Mary B Breslin 1,2,3*
Chen et al. Virology Journal 213, 1:86 RESEARCH Open Access Comparative analysis of the transduction efficiency of five adeno associated virus serotypes and VSV-G pseudotype lentiviral vector in lung cancer
More informationLenti-miRNA Expression Systems
Lenti-miRNA Expression Systems Lenti-GFP-miRNA Vector Lenti-GFP-miRNA Premade Virus Lenti-miRNA Vector Lenti-miRNA Premade Virus Lenti-miRNA-Off Vector Lenti-miRNA-Off Premade Virus mh10001-mh14999 mh15001-mh19999
More informationACTG Laboratory Technologist Committee Revised Version 2.0 ACTG Lab Man HIV Syncytium-Inducing (MT-2) assay 29 April 2004
HIV SYNCYTIUM-INDUCING (MT-2) ASSAY 1. BACKGROUND and CLINICAL SIGNIFICANCE Host and viral factors may play a role in determining the way in which an individual responds to anti-retroviral therapy. Presence
More informationIdentification of Mutation(s) in. Associated with Neutralization Resistance. Miah Blomquist
Identification of Mutation(s) in the HIV 1 gp41 Subunit Associated with Neutralization Resistance Miah Blomquist What is HIV 1? HIV-1 is an epidemic that affects over 34 million people worldwide. HIV-1
More informationProtocol for A-549 VIM RFP (ATCC CCL-185EMT) TGFβ1 EMT Induction and Drug Screening
Protocol for A-549 VIM RFP (ATCC CCL-185EMT) TGFβ1 EMT Induction and Drug Screening Introduction: Vimentin (VIM) intermediate filament (IF) proteins are associated with EMT in lung cancer and its metastatic
More informationNBP Protocol. Orders: Support: Web: NBP
NBP2-29541 NBP2-29541 Protocol Orders: orders@novusbio.com Support: technical@novusbio.com Web: www.novusbio.com Protocols, Publications, Related Products, Reviews and more: www.novusbio.com/nbp2-29541
More informationLENTI-Smart. For the generation of lentiviral particles USER MANUAL. FOR RESEARCH PURPOSES ONLY
LENTI-Smart For the generation of lentiviral particles FOR RESEARCH PURPOSES ONLY USER MANUAL Catalog # ltsint-5, ltsint-10, ltsnil-5, ltsnil-10 Version # 16K25-MM INVIVOGEN USA INVIVOGEN EUROPE INVIVOGEN
More informationIncuCyte NeuroPrime Cell Kit
IncuCyte NeuroPrime Cell Kit Catalog number: 4585 Contents 1x vial IncuCyte NeuroPrime rcortical Neurons (2 x 10 6 cells/vial) 1x vial IncuCyte NeuroPrime rastrocytes (2 x 10 6 cells/vial) 1x vial IncuCyte
More informationA VLP vaccine for epidemic Chikungunya virus protects non-human primates against infection
A VLP vaccine for epidemic Chikungunya virus protects non-human primates against infection Wataru Akahata, Zhi-yong Yang, Hanne Andersen, Siyang Sun, Heather A. Holdaway, Wing-Pui Kong, Mark G. Lewis,
More informationPersistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization
J. gen. Virol. (199), 70, 341-345. Printed in Great Britain 341 Key words: influenza C virus/interferon/persistent infection Persistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization
More informationLENTI-Smart OSKM USER MANUAL FOR RESEARCH PURPOSES ONLY.
LENTI-Smart OSKM FOR RESEARCH PURPOSES ONLY /lenti USER MANUAL Catalog # ltsint-hoskm-5, ltsint-hoskm-10, ltsint-moskm-5, ltsint-moskm-10, ltsnil-hoskm-5, ltsnil-hoskm-10, ltsnil-moskm-5, ltsnil-moskm-10
More informationSupplementary Information. Supplementary Figure 1
Supplementary Information Supplementary Figure 1 1 Supplementary Figure 1. Functional assay of the hcas9-2a-mcherry construct (a) Gene correction of a mutant EGFP reporter cell line mediated by hcas9 or
More informationSupplementary Information. Novel lentiviral vectors with mutated reverse transcriptase for mrna delivery of TALE nucleases
Supplementary Information Novel lentiviral vectors with mutated reverse transcriptase for mrna delivery of TALE nucleases Ulrike Mock 1, Kristoffer Riecken 1, Belinda Berdien 1, Waseem Qasim 2, Emma Chan
More informationSUPPLEMENTARY MATERIAL
SUPPLEMENTARY MATERIAL Acetone and methanol fruit extracts of Terminalia paniculata inhibit HIV-1 infection in vitro Ankita Durge a, Pratiksha Jadaun a, Ashish Wadhwani a, Ashish A. Chinchansure b, Madhukar
More informationRetro-X qrt-pcr Titration Kit User Manual
Takara Bio USA Retro-X qrt-pcr Titration Kit User Manual Cat. No. 631453 PT3952-1 (030218) 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: techus@takarabio.com United States/Canada
More informationB16-F10 (Mus musculus skin melanoma), NCI-H460 (human non-small cell lung cancer
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2017 Experimental Methods Cell culture B16-F10 (Mus musculus skin melanoma), NCI-H460 (human non-small
More informationIncuCyte NeuroPrime Cell Kit Catalog Number: 4585
IncuCyte NeuroPrime Cell Kit Catalog Number: 4585 IncuCyte Cell Kits Contents 1x vial IncuCyte NeuroPrime rforebrain Neurons* (2 x 106 cells/vial) 1x vial IncuCyte NeuroPrime rastrocytes* (2 x 106 cells/vial)
More informationReview and Public RAC Discussion of Protocol #
Review and Public RAC Discussion of Protocol #0508 725 A phase I pilot study of safety and feasibility of stem cell therapy for AIDS lymphoma using stem cells treated with a lentivirus vector encoding
More informationTel: ; Fax: ;
Tel.: +98 216 696 9291; Fax: +98 216 696 9291; E-mail: mrasadeghi@pasteur.ac.ir Tel: +98 916 113 7679; Fax: +98 613 333 6380; E-mail: abakhshi_e@ajums.ac.ir A Soluble Chromatin-bound MOI 0 1 5 0 1 5 HDAC2
More informationLentivirus Titer Determination. Speaker: Chi-long Lin May.,2008
Lentivirus Titer Determination Speaker: Chi-long Lin May.,2008 Why do we need to get virus titer? RNAi knockdown vs. Viral dose Incomplete infection Ideal RNAi KD effect Cytotoxicity / Off target 0 X Y
More informationLentiviral vectors titration using real-time PCR
Lentiviral vectors titration using real-time PCR Amir Allahverdi 1, Fatemeh Eskandari 1, Mohammad Hossein Moghadasi 1, Mehdi Azad 2, Mehdi Goudarzi 3, Saied Abroun*,1, Masoud Soleimani 1 1 Department of
More informationGladstone Institutes, University of California (UCSF), San Francisco, USA
Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24 Gag Marianne Gesner, Mekhala Maiti, Robert Grant and Marielle Cavrois * Gladstone Institutes, University of
More informationComparison of Young and Old Cardiac Telocytes Using Atomic Force Microscopy
Comparison of Young and Old Cardiac Telocytes Using Atomic Force Microscopy Jiali Luo 1, 2, 3, 4, a, Shanshan Feng 1, 2, 3, 4, b 1Key Laboratory of Regenerative Medicine, Ministry of Education, Jinan University,
More informationProtocol for Thawing Cryopreserved Hepatocytes
cell and tissue-based products Protocol for Thawing Cryopreserved Hepatocytes Product Instruction The following procedure may be carried out in a biosafety containment hood to reduce the risk of contamination
More informationTransfection of Sf9 cells with recombinant Bacmid DNA
Transposition Bacmid DNA Mini Culturing baculo cells Transfection of Sf9 cells with recombinant Bacmid DNA Amplification of the virus Titration of baculo stocks Testing the expression Transposition 1.
More informationBMC Biotechnology. Open Access. Abstract
BMC Biotechnology BioMed Central Research article Comparison of lentiviral vector titration methods Martine Geraerts 1, Sofie Willems 1, Veerle Baekelandt 2, Zeger Debyser* 1 and Rik Gijsbers 1 Open Access
More informationGraveley Lab shrna knockdown followed by RNA-seq Biosample Preparation and Characterization Document
Graveley Lab shrna knockdown followed by RNA-seq Biosample Preparation and Characterization Document Wet Lab: Sara Olson and Lijun Zhan Computational Lab: Xintao Wei and Michael Duff PI: Brenton Graveley
More informationThis training module is required for all personnel listed on an IBC protocol that describes work utilizing viral vectors (both replication competent
This training module is required for all personnel listed on an IBC protocol that describes work utilizing viral vectors (both replication competent and incompetent) regardless of the biosafety level used
More informationChromatin Immunoprecipitation (ChIPs) Protocol (Mirmira Lab)
Chromatin Immunoprecipitation (ChIPs) Protocol (Mirmira Lab) Updated 12/3/02 Reagents: ChIP sonication Buffer (1% Triton X-100, 0.1% Deoxycholate, 50 mm Tris 8.1, 150 mm NaCl, 5 mm EDTA): 10 ml 10 % Triton
More informationGene expression scaled by distance to the genome replication site. Ying et al. Supporting Information. Contents. Supplementary note I p.
Gene expression scaled by distance to the genome replication site Ying et al Supporting Information Contents Supplementary note I p. 2 Supplementary note II p. 3-5 Supplementary note III p. 5-7 Supplementary
More informationHepatitis B Antiviral Drug Development Multi-Marker Screening Assay
Hepatitis B Antiviral Drug Development Multi-Marker Screening Assay Background ImQuest BioSciences has developed and qualified a single-plate method to expedite the screening of antiviral agents against
More informationCaspase-3 Assay Cat. No. 8228, 100 tests. Introduction
Introduction Caspase-3 Assay Cat. No. 8228, 100 tests Caspase-3 is a member of caspases that plays a key role in mediating apoptosis, or programmed cell death. Upon activation, it cleaves a variety of
More informationAnnexin V-Cy3 Apoptosis Detection Reagent
ab14143 Annexin V-Cy3 Apoptosis Detection Reagent Instructions for Use For the rapid, sensitive and accurate measurement of apoptosis in various samples This product is for research use only and is not
More informationGraveley Lab shrna knockdown followed by RNA-seq Biosample Preparation and Characterization Document
Graveley Lab shrna knockdown followed by RNA-seq Biosample Preparation and Characterization Document Wet Lab: Sara Olson and Lijun Zhan Computational Lab: Xintao Wei and Michael Duff PI: Brenton Graveley
More informationCertification Assays for HIV-1-Based Vectors: Frequent Passage of Gag Sequences without Evidence of Replication-Competent Viruses
ARTICLE doi:10.1016/j.ymthe.2003.08.003 Certification Assays for HIV-1-Based Vectors: Frequent Passage of Gag Sequences without Evidence of Replication-Competent Viruses Lakshmi Sastry, 1 Yi Xu, 2 Terry
More informationHuman ipsc-derived Ventricular Cardiomyocytes. Protocol version 3.1
Human ipsc-derived Ventricular Cardiomyocytes Protocol version 3.1 Protocol version 3.1 Table of Contents Product Information 2 Recommendations 2 Preparing Cardiomyocyte Maintenance Medium 3 Cardiomyocyte
More informationL6 GLUT4myc Cell Growth Protocol
L6 GLUT4myc Cell Growth Protocol Background: Parental L6 cells selected for high fusion (2, 3) were stably transfected with a rat GLUT4 cdna carrying a myc epitope (recognized by the commercially available
More informationOncolytic Immunotherapy: A Local and Systemic Antitumor Approach
Oncolytic Immunotherapy: A Local and Systemic Antitumor Approach Oncolytic immunotherapy Oncolytic immunotherapy the use of a genetically modified virus to attack tumors and induce a systemic immune response
More informationBiodegradable Zwitterionic Nanogels with Long. Circulation for Antitumor Drug Delivery
Supporting Information Biodegradable Zwitterionic Nanogels with Long Circulation for Antitumor Drug Delivery Yongzhi Men, Shaojun Peng, Peng Yang, Qin Jiang, Yanhui Zhang, Bin Shen, Pin Dong, *, Zhiqing
More informationSupporting Information
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2015 Supporting Information Enzyme-activatable Probe with a Self-immolative Linker for Rapid and Sensitive
More informationab Adipogenesis Assay Kit (Cell-Based)
ab133102 Adipogenesis Assay Kit (Cell-Based) Instructions for Use For the study of induction and inhibition of adipogenesis in adherent cells. This product is for research use only and is not intended
More information1ME A17R11 WI238 DMS2114 JC L2929 P815 PT67
20 1 2004 1 Chinese Journal of Biotechnology Vol. 20 No. 1 January 2004 3 (, 361005), BacV2CMV2EGFPA,Sf9, 20, 12,7,1 CMV,,,LipofectAMINE CMV EGFP pcdna3112egfp, CMV EGFP, CMV CMV,,,,,, Bac2to2Bac 2, CMV,,,
More informationFor the rapid, sensitive and accurate measurement of apoptosis in various samples.
ab14082 500X Annexin V-FITC Apoptosis Detection Reagent Instructions for Use For the rapid, sensitive and accurate measurement of apoptosis in various samples. This product is for research use only and
More informationTest Report. Efficacy of A New JM Nanocomposite Material in Inhibiting Respiratory Syncytial Virus Cellular Infection
Test Report Efficacy of A New JM Nanocomposite Material in Inhibiting Respiratory Syncytial Virus Cellular Infection Test Reagent New JM Nanocomposite Material Project Commissioner JM Material Technology,
More informationFigure S1. Schematic presentation of genomic replication of idsiv after transfection and infection. After transfection of idsiv plasmid DNA into 293T
Figure S1. Schematic presentation of genomic replication of idsiv after transfection and infection. After transfection of idsiv plasmid DNA into 293T cells, the RNA genomes with all modifications are generated
More informationSimplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells
Cribbs et al. BMC Biotechnology 2013, 13:98 METHODOLOGY ARTICLE Open Access Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells Adam P Cribbs
More information