Lentivirus and Retrovirus Services
Delivery of genes or RNAi by lentiviral vectors has been well established and is currently widely used in various the most desirable cell types such as immune systems or primary cells in vitro or in vivo due to several advantages: Lentiviral vectors can accommodate long sequences; the vectors seem to be non-immunogenic due to the lack of viral coding sequences transfer; the vectors are capable of infecting a wide variety of dividing and non-dividing cells; and the products can be stably expressed due to integration into the host genome. But, stringent requirement for lentivirus biosafety, complexity and time-consuming of the experiment process make it uneconomic for most beginners to establish lentiviral experiment system for their researches. Biomega custom service brings to you the most advanced lentivirus packaging service from RNAi design, shRNA cloning, large-scale plasmid preparation, high titer virus packaging, titer test, stable cell lines generating and qRT-PCR or western blot assay.
Custom Lentivector Cloning Service
l ShRNA Design: 5 RNAi targets per gene will be selected using the professional software.
l shRNA Constructs: DNA templates encoding the designed shRNA will be synthesized and cloned into the lentiviral vectors according to the customer's request.
l cDNA Constructs: cDNA will be cloned into the lentiviral vectors or retroviral vectors according to the customer's request.
l Validation of New Constructs: transient expression of cDNA in 293 cells and western blot or qRT-PCR will be performed to verify the construct.
l Large-Scale Plasmid Preparation Suitable for Transfection Purposes: more than 1mg high concentration (>1ug/ml) endotoxin-free advanced transfection grade plasmid will be provided. Plasmid Maxi Kit or CsCl Density Gradient Centrifugation will be used according to the customer's request.
Lentivirus or Retrovirus Production
l Mini-scale Lentivirus or Retrovirus Production: 10 ml - 100 ml lentivirus supernatant (～1 x 106 TU/ml) will be provided using 10-100 ug lentivecvtor or retrovector.
l Midi-scale Lentivirus or Retrovirus Production: 100 ul - 200 ul of concentrated viral stocks (> 1 x 108 TU/ml) will be provided using 200 - 300 ug lentivector or retrovector.
l Large-scale Lentivirus or Retrovirus Production: 1 ml of concentrated viral stocks (>5 x 108 - 5 x 109 TU/ml) will be provided using 3 - 5 mg lentivector.
l Lentivirus or Retrovirus Titering Assay: The functional titer of lentivirus or retrovirus (Transducing Unit / ml) is determined using 293 cells or Hela cells. The assay is usually takes 4-10 business days. Notably, antibody-based (p24 test) and QPCR methods are also used to assess the titer of viral particles in a solution. But titers quantified by these two methods detect transduction-competent as well as empty and defective particles and therefore may overestimate the fraction of particles capable of entering cells and integrating into the host genome. Usually, 1 TU is about 100 to 1000 Lentiviral Particle (LP). In our service, the functional titer we strongly recommended can consistently offer the confidence to the researchers for their experiments.
l In this service, we provide high titer reporter lentivirus (Turbo-GFP, Luciferase and GFP-Luciferase)
Stable Cell Lines Generation
l MOI Determination: The multiplicity of infection (MOI) is the ratio defined by the number of infectious virus particles divided by the number of target cells. In this service, MOI will be assayed using cells provided either by customer or by Promiga. It usually takes two weeks.
l Cell Culture: Customer or our company provides the cells (cell lines or primary culture) and customer provides culture protocol.
l Virus transduction: The target cells will be transduced with different MOI of viral stocks.
l Antibiotics Selection: The selection usually takes 1-2 weeks to get stable cell lines.
l In this service, we also provide reporter cell lines generated with lentivirus or retrovirus as positive control. For example, Hela cells or 293 cells expressing Turbo-GFP, RFP, Luciferase and GFP-Luciferase.
Validation of lentivirus mediated RNAi or gene expression (lentivirus or retrovirus mediated)
l QRT-PCR Examination: We will provide professional services containing gene isoforms analysis, primer design, RNA purification, cDNA reverse transcription, QPCR and data analysis.
l Western Blot Examination: Protein extraction, PAGE gel electrophoresis, transfer and film developing.
l Generation of knockdown or overexpression cell lines from single colony: Pick up the single colonies and propagate the cells. Perform the QRT-PCR and Westernblot to select the best knockdown or overexpression cell lines.
l Gene expression rescue using knockdown cell lines: Construct retrovector expressing target gene cDNA. Perform site directed mutagenesis to change the bases (not change the amino acid codons) in the shRNA targeting site. Make retrovirus and transduced the knockdown cell lines. Select the cells by antibiotics to get stable cell lines. Perform QRT-PCR and Westernblot to verify the target gene rescue.
Lentivirus Titer Assay
Stable Cell Lines or Primary Culture Cells Infected By Lentivirus
P73 Knockdown in Chronic Myelogenous Leukemia Cells Using Lentivirus Mediated RNAi
Gene X knockdown in U87 cells and in single cell lines
Single colonies from sh1 knockdown cells
Human Primary Smooth Muscle Cells (HPSMCs) Xenograft Model
Abl gene knockdown and rescue in HT29 cells