Transfected Stable Cell Lines
Reliable | High-Performance | Wide Rage
Precision reporter, kinase, immune receptor, biosimilar, Cas9, and knockout stable cell lines for diverse applications.
Premade Virus Particles
Ready-to-Use | High Titer | Versatile Applications
Premade AAV, adenovirus, lentivirus particles, safe, stable, in stock.
Virus-Like Particles (VLPs)
Stable | Scalable | Customizable
Advanced VLPs for vaccine development (Chikungunya, Dengue, SARS-CoV-2), gene therapy (AAV1 & AAV9), and drug screening (SSTR2, CCR5).
Oligonucleotide Products
Precise | High Yield | Tailored Solutions
Accelerate your research with cost-effective LncRNA qPCR Array Technology.
RNA Interference Products
Targeted | Potent | High Specificity
Human Druggable Genome siRNA Library enables efficient drug target screening.
Recombinant Drug Target Proteins
Authentic | Versatile | Accelerated
Providing functional, high-purity recombinant proteins—including membrane proteins and nanodiscs—to overcome bottlenecks in drug screening and target validation.
Clones
Validated | Reliable | Comprehensive Collection
Ready-to-use clones for streamlined research and development.
Kits
Complete | Convenient | High Sensitivity
Chromogenic LAL Endotoxin Assay Kit ensures precise, FDA-compliant endotoxin quantification for biosafety testing.
Enzymes
Purified | Stable | Efficient
Powerful Tn5 Transposase for DNA insertion and random library construction.
Aptamers
Highly Specific | Robust | Versatile
Aptamers for key proteins like ACVR1A, Akt, EGFR, and VEGFR.
Adjuvants
Enhancing | Synergistic | Effective
Enhance immune responses with high-purity, potent CpG ODNs.
Laboratory Equipment
Innovative | Reliable | High-Precision
Effortlessly streamline DNA extraction with CB™ Magnetic-Nanoparticle Systems.
Stable Cell Line Generation
Reliable | Scalable | Customizable
Fast proposals, regular updates, and detailed reports; strict quality control, and contamination-free cells; knockout results in 4-6 weeks.
Target-based Drug Discovery Service
Innovative | Comprehensive | Efficient
Target identification, validation, and screening for drug discovery and therapeutic development.
Custom Viral Service
Versatile | High-Yield | Safe
Unbeatable pricing, fully customizable viral packaging services (covering 30,000+ human genes, 200+ mammals, 50+ protein tags).
Custom Antibody Service
Precise | Flexible | Efficient
End-to-end antibody development support, from target to validation, enabling clients to rapidly obtain application-ready antibodies.
Antibody-Drug Conjugation Service
Integrated | Controlled | Translational
Comprehensive solutions covering design, development, and validation to ensure conjugated drugs with consistent quality and clinical potential.
Protein Degrader Service
Efficient | High-Precision | Advanced Therapeutics
Harness the power of protein degraders for precise protein degradation, expanding druggable targets and enhancing therapeutic effectiveness for cutting-edge drug discovery.
Nucleotides Service
Accurate | Flexible | High-Quality
Custom synthesis of oligonucleotides, primers, and probes for gene editing, PCR, and RNA studies.
Custom RNA Service
Custom RNA ServicePrecise | Flexible | GMP-ReadyCustom
RNA design, synthesis, and manufacturing—covering mRNA, saRNA, circRNA, and RNAi. Fast turnaround, rigorous QC, and seamless transition from research to GMP production.
Custom Libraries Construction Service
Comprehensive | High-throughput | Accurate
Custom cDNA, genomic, and mutagenesis libraries for drug discovery, screening, and functional genomics.
Gene Editing Services
Precise | Efficient | Targeted
Gene editing solutions for gene editing, knockouts, knock-ins, and customized genetic modifications. Integrated multi-platform solutions for one-stop CRISPR sgRNA library synthesis and gene screening services
Microbe Genome Editing Service
Precise | Scalable | Customizable
Enhance microbial productivity with advanced genome editing using Rec-mediated recombination and CRISPR/Cas9 technologies.
Biosafety Testing Service
Reliable | Comprehensive | Regulated
Complete biosafety testing solutions for gene therapy, viral vectors, and biologics development.
Plant Genetic Modification Service
Advanced | Sustainable | Tailored
Genetic modification for crop improvement, biotechnology, and plant-based research solutions.
Plant-based Protein Production Service
Efficient | Scalable | Customizable
Plant-based protein expression systems for biopharmaceuticals, enzyme production, and research.
Aptamers Service
Innovative | Fast | Cost-Effective
Revolutionizing drug delivery and diagnostic development with next-generation high-throughput aptamer selection and synthesis technologies.
CGT Biosafety Testing
Comprehensive | Accurate | Regulatory-compliant
Internationally certified evaluation system for biologics, gene therapies, nucleic acid drugs, and vaccines.
Pandemic Detection Solutions
Rapid | Precise | Scalable
Balancing accuracy, accessibility, affordability, and rapid detection to safeguard public health and strengthen global response to infectious diseases.
cGMP Cell Line Development
Reliable | Scalable | Industry-leading
Stable expression over 15 generations with rapid cell line development in just 3 months.
Supports adherent and suspension cell lines, offering MCB, WCB, and PCB establishment.
GMP mRNA Production
Efficient | Scalable | Precise
Scalable mRNA production from milligrams to grams, with personalized process design for sequence optimization, cap selection, and nucleotide modifications, all in one service.
GMP Plasmid Production
High Quality | Scalable | Regulatory-compliant
Our plasmid production services span Non-GMP, GMP-Like, and GMP-Grade levels, with specialized options for linearized plasmids.
GMP Viral Vector Manufacturing
Scalable | High Yield | Quality-driven
Advanced platforms for AAV, adenovirus, lentivirus, and retrovirus production, with strict adherence to GMP guidelines and robust quality control.
AI-Driven Gene Editing and Therapy
Innovative | Precision | Transformative
AI-powered one-click design for customized CRISPR gene editing strategy development.
AI-Antibody Engineering Fusion
Next-Generation | Targeted | Efficient
AI and ML algorithms accelerate antibody screening and predict new structures, unlocking unprecedented possibilities in antibody engineering.
AI-Driven Enzyme Engineering
Smart | Efficient | Tailored
High-throughput enzyme activity testing with proprietary datasets and deep learning models for standardized and precise enzyme engineering design.
AI-Enhanced Small Molecule Screening
Predictive | Efficient | Insightful
Leverage AI to uncover hidden high-potential small molecules, prioritize leads intelligently, and reduce costly trial-and-error in early drug discovery.
AI-Driven Protein Degrader Drug Development
Innovative | Targeted | Accelerated
Use AI-guided design to optimize protein degraders, addressing design complexity and enhancing efficacy while shortening development timelines.
Our promise to you:
Guaranteed product quality, expert customer support.
24x7 CUSTOMER SERVICE
CONTACT US TO ORDER
Stable cell lines generated using lentivirus exhibit long term protein expression and the system is highly reproducible. In this protocol, we describe the use of the 3rd generation lentiviral system which uses three different plasmids for generating stable cell lines. First plasmid contains your gene of interest usually flanked by Long Terminal Repeat (LTR) sequences, that are integrated into the host genome. Second is the pCMV Delta R8.2 plasmid that encodes all components necessary for packaging the lentivirus viz. HIV-1 Gag, Pol, Tat and Rev. Third and the final plasmid is the pCMV VSVG that encodes the viral envelope protein.
Fig. 1 Schematic representation of generation of stable cell lines using lentivirus.
1. 10 cm2 tissue culture treated plates
2. Low protein binding 0.45 μm PDVF filters
3. 10 ml plastic syringes sterile
4. Sterile Glass jar
5. Disposable plastic pipettes (5 ml and 10 ml)
6. Sterile 1.5 ml tubes
7. Pasteur pipettes
8. HEK293T (293T)
9. Target cell line (any human cancer cell line)
10. Xtreme gene 9 transfection reagents
11. Packaging plasmids: pCMV delta R8.2 and pCMV-VSV-G and your plasmid of interest
12. 10% bleach
13. Polybrene solution: 8 mg/ml in water filter sterilized, made fresh every time
14. Opti-MEM reduced serum medium
15. DMEM complete medium with 10% FBS and pen-strep
16. Selection antibiotic
17. Dry ice or liquid nitrogen
18. DMSO
19. Liquid nitrogen
1. Cell culture incubators (37 °C, 5% CO2 and 32 °C, 5% CO2)
2. Hemocytometer (Standard)
3. Laminar hood BSL-2 type
4. 37 °C water bath
5. Autoclave
6. Micropipettes (1,000 μl, 200 μl)
7. -80 °C freezer
A. Preparation of lentivirus in HEK293T cells
1. Day 0: Seed about 2-3 million HEK293T cells in a 10 cm2 plate.
2. Day 1: HEK293T cells should be about 70-80% confluent. If for some reason they seem less you may wait for another 8-10 h.
3. Prepare transfection mix using the following proportions in a sterile 1.5 ml Eppendorf tube as per manufacturer's instructions:
| Opti-MEM medium | 1 ml |
| pCMV delta R8.2 | 2 μg |
| pCMV-VSV-G | 0.5 μg |
| Target plasmid | 1.5 μg |
| Xtreme gene 9 | 12 μl (1:3, DNA:Xtreme Gene, Ratio) |
Once you add all the five components in a 1.5 ml Eppendorf tube, mix the solution by pipetting or inverting. Do not vortex. Incubate the mix for about 20 min at room temperature.
4. Aspirate the medium from HEK293T cells using Pasteur pipettes and add about 9 ml fresh complete DMEM medium.
5. After 20 min incubation of the transfection mix, add the mix to the HEK293T using a 200 μl micropipette. Make sure to add the mix drop-by-drop to cover the complete area of the plate.
6. Incubate the plate in a 37 °C cell culture incubator for about 48 h.
7. Day 3: Using a 10 ml disposable sterile pipette, collect the supernatant/media off the HEK293T plates in a sterile 15 ml tube. Add another fresh 10 ml DMEM media to the plate and move it back to the incubator. Transfer the collected supernatant into a 10 ml syringe and then filter it through a 0.45 μm PVDF filter inside the laminar hood. This step will result in collection of filtered viral supernatant without any residual HEK293T cells in it.
8. If you do not have your target cells ready or if you plan to use the lentivirus later, you can snap freeze the viral supernatant using dry ice or liquid nitrogen and then store it at -80 °C. The virus is usually stable for several months at this temperature.
9. Day 4: If desired, another round of 10 ml lentiviral supernatant can be collected from still growing HEK293T cell plates, similar to Day 3. Before discarding the HEK293T cell plates in the biohazard bin, they should be treated with 10% bleach for about 5-10 min.
B. Preparation of target cells
1. Day 2 (if you want to do this in parallel with the virus preparation, Recommended): Seed about 0.5-1 x 106 target cells each in two 10 cm2 tissue culture plates. One plate is where you will add the viral supernatant, i.e., the lentivirus and the other will be your killing control where you will not add any lentivirus.
2. Day 3: The target cells should be around 40%-50% confluent prior to transduction, i.e., addition of virus. If your lentivirus is in the freezer, make sure you thaw it in a 37 °C water bath before adding it to the cells. Once the cells are ready, prepare a polybrene medium (target cell medium) mix. The final concentration of polybrene should be 8 μg/ml. Prepare 10x polybrene medium mix by adding 2 ml medium with 20 μl of 8 mg/ml Polybrene. Aspirate the medium in the target cells using Pasteur pipette and add 1 ml polybrene medium mix and let it incubate for about 1-2 min.
3. Using a disposable 10 ml pipette, add 9 ml lentivirus to the target cells. Add plain medium to the killing control plate.
4. Day 4: Repeat the same steps as Day 3, to do a second round of transduction to ensure all the cells get the lentivirus. See the note in Step A7.
5. Day 5: Add the selection antibiotic at the killing concentration (e.g., Puromycin 1-2 μg/ml) to both plates. See Notes to determine the killing concentration of the selection antibiotic. Typically, fresh media with antibiotic is added every two days until all the cells in the killing control plate are dead.
6. Days 8-10: Before you do any further experiments, make sure to cryo-freeze about 1/4th portion of cells in 10% DMSO with DMEM in liquid nitrogen. Passage the rest of the cells for RNA/protein isolation, in order to validate for stable expression of protein of interest.
Copyright © Creative Biogene. All rights reserved.