Platinum-GP (Plat-GP) Retroviral Packaging Cell Line逆转录病毒包装细胞株 BioVector NTCC质粒载体菌种细胞基因保藏中心

Platinum-GP (Plat-GP) Retroviral Packaging Cell Line逆转录病毒包装细胞株 BioVector NTCC质粒载体菌种细胞基因保藏中心

Higher retroviral yields: average titer 106 to 107 infectious units/mL with transient transfection
Longer stability: up to 4 months in the presence of drug selection
Produce VSVG-pseudotyped pantropic retrovirus, which can easily infect cells of nearly any species
Conventional cells used for retrovirus packaging, such as those based on NIH3T3 cells, have limited stability and produce relatively low yields of retrovirus, mainly due to the poor expression of retroviral structure proteins (gag, pol and env) in the cells.

The Platinum Retroviral Packaging Cell Lines are based on the 293T cell line. They exhibit longer stability and produce higher yields of retroviral structure proteins. Plat-GP cells contain the gag and pol genes required for retroviral packaging; an expression vector is co-transfected with a VSVG envelope vector.
Background
Retroviruses are efficient tools for delivering heritable genes into the genome of dividing cells. However,
conventional NIH-3T3 based retroviral packaging cell lines have limited stability and produce low viral
yields, mainly due to poor expression level of the retroviral structure proteins (gag, pol, env) in the
packaging cells.
The Platinum-GP (Plat-GP) Cell Line, a potent retrovirus packaging cell line based on the 293T cell line,
was generated using novel packaging constructs with an EF1α promoter to ensure longer stability and
high-yield retroviral structure protein expression (gag and pol). Plat-GP cells can be kept in good
condition in for at least 4 months in the presence of drug selection, and can produce retroviruses with an
average titer of 1 x 107
infectious units/mL by transient transfection. In addition, replication competent
retroviruses (RCR) are virtually nonexistent because only coding sequences of viral structural genes are
used, avoiding any unnecessary retroviral sequences.
The Plat-GP cell line allows you to select the envelop according to the tropism needed. The viral env
gene, such as VSV-G, must be cotransfected with the retroviral expression vector. The Plat-GP cell line is
designed for rapid, transient production of high-titer retrovirus.

Figure 1. Jurkat cells were infected with GFP retrovirus supernatant produced in Plat-GP cells after
transfection.

Medium
1. Culture Medium: DMEM, 10% fetal calf serum (FCS), 10 µg/mL blasticidin, penicillin and
streptomycin
2. Freeze Medium: 70% DMEM, 20% FBS, 10% DMSO
Methods
I. Establishing Plat-GP Cultures from Frozen Cells
1. After quickly thawing the cells in a 37ºC water bath, immediately transfer the thawed cell
suspension into a 15 mL tube containing 10 mL of culture medium.
2. Centrifuge the tube for 5 min at 1300 to 1500 rpm.
3. Discard the supernatant and break the cell pellet by finger tapping.
4. Add a few drops of culture medium with gentle shaking and finger tap the tube a few times.
5. Add 2 mL of culture medium to the tube and gently pipet the cell suspension up and down twice.
6. Transfer the cell suspension to a 10 cm culture dish (Falcon® #3003 works well) containing 8 mL
of culture medium.
7. Swirl the culture plate well to mix the cells, then incubate the cells for three days before
expansion.
Important Notes:
 Don’t change the culture medium during the first three days. It is normal to see some
cells floating after the first 24 hours.
 Don’t culture cells to complete confluency. Split cells 4X to 6X every two to three days
when the culture reaches 70-90% confluency.
II. Splitting the Cells
Note: Avoid forming bubbles as much as possible during this procedure.
1. Wash cells once with PBS.
2. Add 4 mL of 0.05% Trypsin/0.5 mM EDTA solution to a 10 cm dish and incubate at 37ºC for 3-5
min.
3. Remove the cells from the dish surface by tapping the rim of the culture dish.
4. Transfer 10 mL of the culture medium to a 50 mL tube.
5. Using the same pipette with some residual culture medium, wash the dish surface gently three
times in 4 mL of theTrypsin/EDTA solution.
6. Gently pipette the cell suspension up and down 7 times and transfer the cell suspension into the 50
mL tube containing 10 mL medium from step 4.
7. Centrifuge the cells for 5 min at 1300-1500 rpm.
8. Discard the supernatant and break the cell pellet by finger tapping.
9. Add a few drops of culture medium with gentle shaking and finger tap the tube a few times.
10. Add 5 mL of culture medium and gently pipet the cell suspension up and down twice.
11. Add 15 mL of culture medium, then count and seed the cells. Typically 107
cells can be harvested
from one 10 cm culture dish.
Transfection
1. Seed 2 x 106
cells in a 60 mm culture dish without antibiotics including blasticidin one day before
transfection.

2. After 16 to 24 hours, start transfection when the culture becomes 70-80% confluent.
Note: We suggest transfecting cells with FuGENE® Transfection Reagent (Roche Applied
Science) or Lipofectamine™ Plus (Invitrogen). For example, 3 μg DNA is mixed with 9 μL
FuGENE® Transfection Reagent according to the manufacturer’s recommendation. The mixed
DNA- FuGENE® complex is added by dropwise into the culture media.
3. Harvest retroviral supernatant 48 hours after transfection.

References
1. Morita, S., Kojim, T., and Kitamura, T. (2000) Gene Therapy 7: 1063-1066.

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