Pre-made Lentiviral Particles for Fluorescent Proteins

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1 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 LVP023 LVP023-PBS LVP299 LVP299-PBS LVP300 LVP300-PBS LVP306 LVP306-PBS GFP (CMV-Bsd) GFP (CMV-Bsd) GFP-His-2A-RFP (Bsd) GFP-His-2A-RFP (Bsd) CFP (CMV-Bsd) CFP (CMV-Bsd) YFP (CMV-Bsd) YFP (CMV-Bsd) RFP (CMV-Bsd) Lentiviral particles RFP (CMV-Bsd) RFP (CMV-Neo) RFP (CMV-Neo) GFP (CMV-Neo) GFP (CMV-Neo) CFP (CMV-Neo) CFP (CMV-Neo)

2 LVP307 LVP307-PBS LVP309 LVP309-PBS LVP340 LVP340-PBS LVP342 LVP342-PBS LVP471 LVP471-PBS LVP361 LVP361-PBS LVP362 LVP362-PBS LVP363 LVP363-PBS YFP (CMV-Neo) YFP (CMV-Neo) RFP (CMV-Puro) RFP (CMV-Puro) GFP (CMV-puro) GFP (CMV-Puro) CFP (CMV-puro) CFP (CMV-Puro) YFP (CMV-puro) YFP (CMV-Puro) BFP (CMV-Puro) BFP (CMV-Puro) BFP (CMV-Bsd) Lentiviral particles BFP (CMV-Bsd) BFP (CMV-Neo) BFP (CMV-Neo) Catalog# Product Name Amounts Fluorescent proteins expressed under EF1a promoter: LVP310 GFP (EF1a-Bsd)

3 LVP310-PBS LVP425 LVP425-PBS LVP426 LVP426-PBS LVP427 LVP427-PBS LVP428 LVP428-PBS LVP429 LVP429-PBS LVP430 LVP430-PBS LVP431 LVP431-PBS LVP432 LVP432-PBS LVP468 LVP468-PBS GFP (EF1a-Bsd) GFP (EF1a-Neo) GFP (EF1a-Neo) GFP (EF1a-Puro) GFP (EF1a-Puro) RFP (EF1a-Bsd) RFP (EF1a-Bsd) RFP (EF1a-Neo) RFP (EF1a-Neo) RFP (EF1a-Puro) RFP (EF1a-Puro) CFP (EF1a-Bsd) CFP (EF1a-Bsd) CFP (EF1a-Neo) CFP (EF1a-Neo) CFP (EF1a-Puro) CFP (EF1a-Puro) CFP (EF1a-RB) CFP (EF1a-RB)

4 LVP465 LVP465-PBS LVP466 LVP466-PBS LVP467 LVP467-PBS LVP364 LVP364-PBS LVP365 LVP365-PBS LVP366 LVP366-PBS YFP (EF1a-Bsd) YFP (EF1a-Bsd) YFP (EF1a-Neo) YFP (EF1a-Neo) YFP (EF1a-Puro) YFP (EF1a-Puro) BFP (EF1a-Puro) BFP (EF1a-Puro) BFP (EF1a-Bsd) Lentiviral particles BFP (EF1a-Bsd) BFP (EF1a-Neo) BFP (EF1a-Neo) Catalog# Product Name Amounts Fluorescent proteins expressed under CAG promoter: LVP579 LVP579-PBS LVP580 LVP580-PBS GFP (CAG-Puro) GFP (CAG-Puro) GFP (CAG-Bsd) GFP (CAG-Bsd)

5 LVP581 LVP581-PBS LVP582 LVP582-PBS LVP583 LVP583-PBS LVP584 LVP584-PBS LVP585 LVP585-PBS LVP586 LVP586-PBS LVP587 LVP587-PBS GFP (CAG-Neo) GFP (CAG-Neo) RFP (CAG-Puro) RFP (CAG-Puro) RFP (CAG-Bsd) RFP (CAG-Bsd) RFP (CAG-Neo) RFP (CAG-Neo) CFP (CAG-Puro) CFP (CAG-Puro) CFP (CAG-Bsd) CFP (CAG-Bsd) CFP (CAG-Neo) CFP (CAG-Neo) Catalog# Product Name Amounts Fluorescent proteins expressed under the optional inducible TetCMV promoter *: LVP800 LVP800-PBS GFP (TetCMV-Puro) GFP (TetCMV-Puro)

6 LVP801 LVP801-PBS LVP802 LVP802-PBS LVP803 LVP803-PBS RFP (TetCMV-Puro) RFP (TetCMV-Puro) CFP (TetCMV-Puro) CFP (TetCMV-Puro) BFP (TetCMV-Puro) BFP (TetCMV-Puro) Fluorescent proteins expressed under the optional inducible TetCMV promoter containing a constitutive fluorescent marker*: LVP024 LVP024-PBS LVP357 LVP357-PBS LVP531 LVP531-PBS GFP (RFP-Bsd) inducible control lentivirus GFP (RFP-Bsd) inducible control lentivirus in PBS YFP (RFP-Bsd) inducible control lentivirus YFP (RFP-Bsd) inducible control lentivirus in PBS RFP (GFP-Puro) inducible control lentivirus RFP (GFP-Puro) inducible control lentivirus in PBS *: The TetCMV promoter displays a tetracycline inducible expression only when its repressor protein, TetR, is present. Without TetR presence, the TetCMV is a constitutive promoter. The TetR protein can be delivered by the premade TetR lentivirus. The TetCMV driven expression is first stopped by TetR, then induced by addition of tetracycline or doxycycline. Storage: Store at < -70 C, avoid repeat freeze/thaw cycles. Stable for > 6 months. Product Description: Lentiviral particles or lentivirus is a gene delivery tool produced from lentivectors for gene expression or knockdown. AMSBIO s lentivector system are Human Immunodeficiency Virus-1 (HIV) based

7 plasmids for gene expression and knockdown. The lentivectors are used to generate (lentivirus) that can be transduced into almost all kinds of mammalian cells, including stem cells, primary cells, and non-dividing cells both in vivo and in vitro. Lentiviral particles stably integrate into the transduced cell s genome for long term expression, making it a great gene transfer agent. Pre-made expressing fluorescent proteins are generated from AMSBIO s high expression lentiviral system. Each fluorescent protein is codon-optimized to demonstrate the strongest possible fluorescence signal and is available expressed under a selection of promoters. These lentivirus are available with a selection of different antibiotic markers, allowing selection of transduced stable cells. Please see the schematic lentivector structure below. The proprietary sucmv promoter demonstrates high expression level in the majority of cell types. The engineered EF1a promoter is non-tissue specific, highly expressed in all cell types, and less likely to be silenced after long-term culture. The CAG promoter--a hybrid of the CMV enhancer and the chicken beta-actin promoter--has the highest expression level in embryonic stem cells. The optional inducible CMV promoter (TetCMV) can be used for either constitutive high expression or tetracycline-inducible expression.

8 Expression lentivectors were co-transfected with packaging mix (Cat# HT-pack) into 293T cells (cat# TLV-C). The pre-made are VSV-G pseudotyped viruses. Each lot of virus is validated and quality is guaranteed. Particles are provided in two formats: Regular particles in DMEM medium with 10% FBS and 60 μg/ml polybrene (10 x stock) Particles concentrated and buffer exchanged into PBS for in vivo use AMSBIO also provides lentiviral services for cloning your gene of interest and generates ready-to-use viral particles with the best prices and fastest turnaround time. Please see our website for details. Transduction Protocols: 1) Transduction Protocol for Adhesive cells: Note: Pre-made lentivirus is provided ready to use, so it can be simply added into your cell culture; the amount of virus to add depends on the cell type. For quick transduction, add 50 µl of virus into each well of 24-well-plate where cell density is 50% to 75%. After 72 hours (no need to change medium), visualize positive transduction rate by fluorescent microscopy. For stable cell line generation, pass cells into medium containing antibiotic or perform fluorescent cell sorting followed by antibiotic selection. Day 0: Seed cells in complete medium at the appropriate density and incubate overnight. Note: at the time of transduction, cells should be 50%-75% confluent. For example, seed HeLa cells at 0.5 x 10 5 /ml x 0.5ml in a well of a 24-well plate.

9 Day 1: Remove the culture medium and add 0.5ml of fresh, warm, complete medium. Thaw the pre-made lentiviral stock at room temperature and add the appropriate amount of virus stock to obtain the desired MOI. Return cells to 37 C, CO2 incubator. Note: Try to avoid freezing and thawing. If you do not use all of the virus at one time, you may re-freeze the virus at -80 o C for future use; virus titer will decrease by ~10% for each freeze/thaw cycle. Day 3: At ~72hr after transduction, check the transduction rate by fluorescent microscopy or calculate the exact transduction rate by flow cytometer (FACS or Guava). Day 3 + (optional): Sort transduced cells by FACS, and select for antibiotic resistance. 2) Transduction Protocol for Suspension Cells: Grow cells in complete suspension culture medium; use a shaking flask in a CO2 incubator if necessary. Measure cell density. When density has reached ~3 x 10 6 cells/ml, measured viability should be > 90%. Dilute cells into 1 x 10 6 cell/ml in complete medium. Day 1: Thaw at room temperature. Add premade into the diluted cells at a ratio of: 50 to 100 µl virus per 0.5 ml of cells (Note: depending on cell type, you may need to use more or less virus). Grow cells in a shaking flask in a CO2 incubator. Day 2: Day 3: At 24 hours after transduction, add an equal amount of fresh medium containing relevant antibiotics. Note: amount of antibiotic depends on cell type. Continue growing cells in CO2 incubator. At 72 hours after transduction, check fluorescence with a fluorescence microscope or calculate the transduction efficiency using a cell sorter such as FACS or Guava. Sort for fluorescence positive cells and maintain antibiotic selection to generate a stable cell line.

10 Transduction Example A: Figure 1: GFP Expression in HeLa cells. HeLa cells were transduced with 5 µl (Right image) or 50 µl (Left image) of Pre-made GFP lentivirus (LVP001) in 24-well plate (see protocol above). GFP signal was visualized at 72 hours after transduction. Transduction Example B:. Figure 2: RFP Expression in A549 cells. A549 cells were transduced with 50 µl Pre-made RFP lentivirus (LVP023) in 24-well plate. RFP signal was visualized at 72 hours after transduction. Images were taken under microscope, the left image under RFP filter, the right image under bright light. Transduction Example C: Figure 3: CFP Expression in HEK293 cells: Quick transduction protocol: add 50ul CFP lentivirus (LVP430) into one well in 24-well-plate where cell density is at 50% ~ 75%.

11 Image taken at ~72 hours after virus added (no medium changed). Result: The positive transduced cells are >90%. Note: Filter wavelength settings: BFP filter: ~Ex380 ~Em460; CFP filter: ~Ex436 ~Em480; GFP filter: ~Ex ~Em525; YFP filter: ~Ex500 ~Em535; RFP filter: ~Ex545 ~Em620; nirfp filter: ~Ex690 ~Em715 Safety Precaution: Amsbio have adopted the most advanced lentiviral safety features (using the third generation vectors with self-inactivation SIN-3UTR), and the premade lentivirus is replication incompetent. However, please use extra caution when using. Use the in Bio-safety II cabinet. Ware gloves at all times when handling lentiviral particles! Please refer CDC and NIH s guidelines for more details regarding to safety issues. References: 1. BioTechniques 38: (June 2005); 2. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 5, Issue of January 30, pp , 2004; 3. Biosci. Biotechnol. Biochem., 68(3), , 2004; 4. Annu Rev Microbiol. 1994;48: Microbiol Mol Biol Rev Jun;69(2): APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2005, p ; 7. Molecular & Biochemical Parasitology 155 (2007) ; 8. Biosci. Biotechnol. Biochem., 68(3), , 2004; 9. NIH Guidelines for Biosafety Considerations for Research with Lentiviral Vectors link 10. CDC guidelines for Lab Biosafety levels link Warranty: This product is warranted to meet its quality as described when used accordance with its instructions. Amsbio disclaims any implied warranty of this product for particular application. In no event shall Amsbio be liable for any incidental or consequential damages in connection with the products. Amsbio s sole remedy for breach of this warranty should be, at Amsbio s option, to replace the products. Those products are provided for research use only!

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