CellRapeutics™ Retrovirus Packaging Kit (CART-019CL)

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All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

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Product Name
CellRapeutics™ Retrovirus Packaging Kit

Cat. No.
CART-019CL

General Description
This kit is our premium retroviral packaging system featuring the GP2-293 packaging cell line. GP2-293 is a HEK 293-based retroviral packaging cell line, containing only the MoMuLV gag and pol genes. All four commonly used envelopes are supplied on separate vectors that you cotransfect with your transfer vector. Envelope choices include VSV-G, eco, ampho, and 10A1 to allow you to choose the tropism that is most appropriate for your target cells. A LacZ Control Vector which constitutively expresses β-galactosidase in transduced mammalian cells is included in the set.
This kit provides a rapid and convenient method for producing high titers of replication-incompetent retrovirus. The envelope vector supplied in this kit is cotransfected with your retroviral expression vector to GP2-293 packing cell line, and high titers of amphotropic, ecotropic, dualtropic, or pantropic virions can be obtained in less than 48 hours.

Principles of the generation of recombinant retroviruses
Retroviral gene transfer systems depend on packaging cell lines to supply necessary viral structural proteins. The provided GP2-293 cell line contains the Moloney Murine Leukemia Virus (MMLV) gag and pol genes stably integrated into its genome.
1. Co-transfect GP2-293 cells with the retrovirus vector containing your gene of interest and an envelope plasmid such as pVSV-G.
2. Resulting production of the corresponding recombinant retroviral genome and viral packaging proteins.
3. GP2-293 Cells express gag and pol from genomic locations.
4. Recognition of the packaging sequence (Ψ) on the recombinant viral RNA genome by the packaging proteins. Resulting assembly of viral cores, which are transported to the cell membrane.
5. Cores are then enveloped by cellular membrane containing aggregated VSV-G or other envelope proteins. Mature, infectious virions then bud from the cell.
6. Infectious virions are collected in the medium.

Components
This kit contains a packaging vector set and GP2-293 packaging cell line.
•A Packaging Vector Set
p10A1 Vector (500 ng/µl) 20 μg
pAmpho Vector (500 ng /µl) 20 μg
pEco Vector (500 ng /µl) 20 μg
pVSV-G Vector (500 ng /µl) 20 μg
Positive Control Vector (500 ng /µl)) 20 μg
•1 ml GP2-293 Packaging Cell Line (2 x 10⁶ cells/ml)

Storage Conditions
Store cell lines at -196°C and all other components at -20°C.

Reagent and instrument requirements
Instruments and Materials
•Biological Safety Cabinet
•Humidified CO2 incubator
•-80°C freezer
•Tissue culture plates (100 mm)
•15 ml polystyrene conical centrifuge tube
•Sterile microfuge tubes (1.5 ml)
•Sterilized 0.45 μm filters
Reagents
•Cell growth medium
•Penicillin/streptomycin solution
•Trypsin-EDTA
•Dulbecco's phosphate-buffered saline
•Transfection Reagents

Procedure
1. Approximately 24 h before transfection, seed 4-5 x 10^6 cells/100 mm plate, in 10 ml of growth medium. Make sure that the cells are plated evenly. Incubate at 37°C, 5% CO2 overnight. The cells should be 80-90% confluent at the time of transfection.
2. In a microcentrifuge tube, dilute your retroviral plasmid DNA with transfection reagent reaction buffer to a final volume of 600 µl. Use the following amounts of DNA for GP2-293 cell lines: 15 µg retroviral plasmid + 15 µg envelope plasmid.
3. Add the indicated amounts of transfection reagent to the diluted retroviral plasmid DNA solution and mix well.
4. Incubate DNA-transfection reagent mixture for 10 min at room temperature to allow nanoparticle complexes to form.
5. Add the entire mixture (Step 4) dropwise to the cell culture medium.
6. After 4 h to overnight, replace the transfection medium with 10 ml fresh complete growth medium and incubate at 37°C for an additional 24-48 h. Virus titers will generally be highest 48 h after the start of transfection. Caution: discarded medium contains infectious retrovirus.
7. Harvest the retroviral supernatants and pool similar stocks, if desired. Caution: supernatants contain infectious retrovirus. Centrifuge briefly (500g for 10 min) or filter through a 0.45 μm filter to remove cellular debris.
8. Verify virus production by titrating the virus stock, then use the virus to transduce target cells, or aliquot and store at -80°C. Avoid repeating freeze-thaw cycles.

Product Use Statement
•Please follow the guideline for experiments using recombinant DNA issued by the relevant authorities and the safety committee of your organization or your country in using this product.
•The viral supernatants produced by the retroviral system could contain potentially hazardous recombinant virus
•Retrovirus requires the use of a Biosafety Level 2 facility. Know and use appropriate safety precautions.
•The user is strongly advised not to create retroviruses capable of expressing known oncogenes in amphotropic or polytropic host range viruses.
•Titers can drop as much as 2-4 fold with each freeze-thaw cycle.
•Our products are to be used for research purposes only.

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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

CellRapeutics™ Retrovirus Packaging Kit (CART-019CL)

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