Description

Retrovirus Packaging Kits are 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.
This kit contains a gag-pol expression vector and an ecotropic or amphotropic env expression vector. The recombinant virus obtained using Eco kit (Cat.# 6160) is able to infect most mouse and rat cells. On the other hand, the recombinant virus obtained using Ampho kit (Cat.# 6161) can infect most mammalian cells.
The packaging vectors have retrovirus structural genes (gag-pol and env gene) which are necessary for construction/ replication of virus particles, but do not 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 (calcium phosphate method), transiently high-titer recombinant retrovirus particles are obtained after 48 hours.
These packaging vectors are highly purified and ready to be used for transfection. There is little possibility of yielding the replication competent retrovirus, because these packaging vectors do not have any other DNA sequence originating from retrovirus except for gag-pol and env genes.

Seed the cells on plates on the day before experiment. Prepare recombinant retrovirus vector plasmid with target gene. Prepare mixture of recombinant retrovirus vector plasmid, pGP vector and pE-eco or pE-ampho vector. Add Transfection Buffer (supplied in this kit) to the vector mixture and pour onto the cultured cells. After 48 hours, collect the supernatant of cell culture and filtrate it. Recombinant retrovirus particles are constructed.

Principles of the generation of recombinant retrovirus particles

Components

Retrovirus Packaging Kit Eco (Cat.# 6160)

1. pGP Vector (1 µg/µl)	50 µl
2. pE-eco Vector (1 µg/µl)	50 µl
3. Transfection Buffer	500 µl × 10 tubes
4. 2 M CaCl2	620 µl
5. 25 mM Chloroquine	40 µl

Retrovirus Packaging Kit Ampho (Cat.# 6161)

1. pGP Vector (1 µg/µl)	50 µl
2. pE-ampho Vector (1 µg/µl)	50 µl
3. Transfection Buffer	500 µl × 10 tubes
4. 2 M CaCl2	620 µl
5. 25 mM Chloroquine	40 µl

Storage

-20°C
The components 3, 4, 5 should be stored at 4°C once they are thawed.
Avoid long term exposure of Transfection Buffer to dry ice.

Feature

1. High titer	The titer is transiently 104-107 infectious units/ml.
2. Ready to use	Highly purified packaging vectors and transfection reagents are included in this kit.
3. Safety	There is little possibility of yielding the replication competent retrovirus.
4. Selectivity	The appropriate envelope type can be chosen between ecotropic env expression vector and amphotropic env expression vector.