CellRapeutics™ Retrovirus Packaging Kit(CART-019CL)


Product Name
CellRapeutics™ Retrovirus Packaging Kit
Cat. No.
CART-019CL
General Description
This kit is designed to obtain transient high-titer recombinant retrovirus particles by co-transfection of retrovirus vector plasmid with target gene and two unique vectors for packaging, using calcium phosphate method. This kit contains agag-pol expression vector and an amphotropic env expression vector.
The recombinant virus obtained using this kit are able to infect most mammalian cells. The packaging vectors have retrovirus structural genes (gag-pol and env gene) which are necessary to construction/replication of virus particles, but don't have ψ (packaging signal) and LTR sequence.
By co-transfecting the recombinant retrovirus vector plasmid which has ψ and LTR with these packaging vectors to 293 cells or 293T cells using transfection reagents supplied in this kit,transiently high-titer recombinant retrovirus particles are obtained after 48 hours.
These packaging vectors are highly purified and ready to use for transfection. There is little possibility of getting replication competent retrovirus, because these packaging vectors don't have any DNA sequence from retrovirus except for gag-pol andenv gene.
Principles of the generation of recombinant retroviruses
1. Seed the cells on plates in the previous day.And prepare recombinant retrovirus vector plasmid with target gene.
2. Prepare mixture of recombinant retrovirus vector plasmid, pGP vector and pEampho vector.
3. Add Transfection Buffer (supplied in this kit) to the vector mixture and pour onto the cultured cells.
4. After 48 hours, collect the supernatant of cell culture and filtrate it.
5. Recombinant retrovirus particles are completed.
Components
Contents of the kit (for 10 reactions) This kit contains two packaging vectors and other necessary reagents for transfection.
Since vectors may be damaged by repeating freeze-thaw cycles, store at 4°C once thawing.
1. pGP Vector (1 μg/μl) 50 μl
2. pE-ampho Vector (1 μg/μl) 50 μl
3. Transfection Buffer 10 × 500 μl
4. 2M CaCl2 620 μl
5. 25mM Chloroquine 40 μl
Storage Conditions
4°C
(Store at -20°C for long term.)
Avoid long term exposure of Transfection Buffer to dry ice.
Reagent and instrument requirements
Instruments and equipments
・ Safety cabinet
・ Microscope for cell observation
・ Humidified CO2 incubator
・ - 80°C freezer
・ Sterilized pipette tips with filters
・ Sterilized 5.0 ml round-bottomed tubes (polystylene)
・ Sterilized 2.0 ml tubes (for storage of viruses)
・ Sterilized 0.45 μm filters (low adsorption)
・ Gelatin or collagen coated dishes (6 cmφ)
・ Sterilized pipettes
・ Electoric pipetter Reagents
・ Recombinant retrovirus vector plasmid (highly purified)
・ Fetal Bovine Serum (FBS)
・ Trypsin-EDTA solution
・ 293 cells, 293T cells2) etc.
・ Dulbecco's Modified Eagle's Medium (DMEM) with Glucose (4.5 g/L) and L-Glutamine (584 mg/L)
・ Penicillin/Streptomycin
・ Sterilized distilled water
Procedure
A. Preparation (previous day)
Seed 2 - 3 × 106 of 293 cells or 293T cells into one 6 cm φ gelatin or collagen coated dish, and prepare recombinant retrovirus vector plasmid.
Purify the vector plasmid to a degree sufficient for transfection. Use CsCl gradients equilibrium ultracentrifugation method or an appropriate DNA preparation kit. B. Transfection (1st day)
Bring the transfection reagents and sterilized distilled water to room temperature. Operate the following steps avoiding any contamination.
1. Prepare the following vector mixture in a sterilized 5.0 ml round-bottomed tube (polystylene). 2. Change the culture medium to DMEM with 10% FBS and 25μM chloroquine (3 ml).
3. Form calcium phosphate precipitation and transfect to cells.
1) Suck up 500 μl of Transfection Buffer using electoric pipetter.
2) Add the buffer gently to the vector mixture with shaking the tube.
3) Bubble the solution immediately by using exhaust of pipetter for 10 - 20 sec. to accelerate the formation of calcium phosphate precipitation.
4) Within 1 - 2 min., drop the solution onto the cell dish evenly and rock the dish gently several times.
5) Incubate for 7 - 11 hours in 5% CO2 incubator at 37°C.
When calcium phosphate precipitation is formed successfully,it looks like powder-snow on the surface of cells under a microscope.
6) Remove 3 ml of the medium from the dish, and then add 4 ml of fresh DMEM with 10% FBS. (Complete removal of culture medium may reduce the transfection efficiency, so it is recommended to leave 1 ml of medium in the dish. C. Exchange of medium (2nd day)
After 24 hours from transfection, change the culture medium again to DMEM with 10% FBS (4 ml).
D. Virus solution (3rd day)
After 48 hours from transfection, collect the supernatant of culture medium and filtrate with 0.45 μm sterilized filter. For storage, the virus solution must be dispensed each in a small amount and stored at - 80°C. Avoid repeating freeze-thaw cycles.
Note : The titer of the recombinant virus prepared using this kit is varied by size of inserted target gene in retrovirus vector, efficiency of transfection,etc. Generally, the titer is transiently 105 - 107 infectious units/ml. 293T cells, which were made by introducing SV40 T antigen gene into 293 cells, are useful for transient transfection and can produce higher titer virus than 293 cells.
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 use of this product is limited only for research purposes. It must not be used for clinical purposes or for in vitro diagnosis.
・ Individual license agreement must be concluded when this product is used for industrial purposes.
・ Recombinant retrovirus solution may include virus with unknown hazardous gene. Please use a safety cabinet and gloves to prevent inhalation or adhesion of the virus.
・ Basic techniques of genetic engineering and cell cultivation are needed for the use of this product.
・ The user is strongly advised not to generate recombinant retrovirus capable of expressing known oncogenes and any genes known to be hazardous to the mammals.
・ Biolabs is not liable for any accidents or damages caused by the use of this product.

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.

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