ProPak-A.52 Clone #52 [PP-A.52] (ATCC® CRL-12479)

Organism: Homo sapiens, human  /  Cell Type: transformed with adenovirus 5 DNA  /  Disease: Leukemia

Organism Homo sapiens, human
Cell Type transformed with adenovirus 5 DNA
Product Format frozen
Culture Properties adherent
Biosafety Level 2 Cells contain adenovirus and CMV viral sequences
Disease Leukemia
Age fetus
Applications
The ProPak packaging cell lines produce either murine leukemia virus (MLV) xenotropic particles (ProPak-X cells; ATCC CRL-12007) or amphotropic particles (ProPak-A cells; ATCC CRL-12006 and ATCC CRL-12479).
They were derived from the human embryonic kidney line, 293 (see ATCC CRL-1573).
ProPak-based producer cells were demonstrated to be free of replication-competent retrovirus (RCR) by stringent testing.
Consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector.
Storage Conditions liquid nitrogen vapor phase
Derivation
They were derived from the human embryonic kidney line, 293 (see ATCC CRL-1573).
The CMV promoter was excised (EcoRI/XhoI, blunt-ended), and replaced with the MoMLV LTR (Asp 718/HindIII, blunt-ended) from plasmid pVH2.
The amphotropic envelope-encoding plasmid, pCMVEa, was transfected into ProGag cells and clones yielding high transduction efficiencies were isolated. One of these, clone number 52 (ProPak-A.52), was deposited as CRL-12479.
Consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector.
Clinical Data
To construct the ProPak-X and the ProPak-A-52 cell lines, the ATG in the splice donor/splice acceptor of pCMV plasmid was mutated to ACG.
Comments
The ProPak packaging cell lines produce either murine leukemia virus (MLV) xenotropic particles (ProPak-X cells; ATCC CRL-12007) or amphotropic particles (ProPak-A cells; ATCC CRL-12006 and ATCC CRL-12479).
They were derived from the human embryonic kidney line, 293 (see ATCC CRL-1573).
To construct the ProPak-X and the ProPak-A-52 cell lines, the ATG in the splice donor/splice acceptor of pCMV plasmid was mutated to ACG.
The CMV promoter was excised (EcoRI/XhoI, blunt-ended), and replaced with the MoMLV LTR (Asp 718/HindIII, blunt-ended) from plasmid pVH2.
The beta-galactosidase gene was replaced by the gag-pol ORF (NotI fragment) to generate pMoMLVgp. pMoMLVgp was co-transfected with pHA58 into 293 cells by calcium phosphate co-precipitation and hygromycin B-resistant cells were selected.
Clones were screened for the level of Gag secretion and one clone secreting high levels of Gag was selected (designated ProGag); this clone yielded high viral titers in transient transfection.
The amphotropic envelope-encoding plasmid, pCMVEa, was transfected into ProGag cells and clones yielding high transduction efficiencies were isolated. One of these, clone number 52 (ProPak-A.52), was deposited as CRL-12479.
ProPak-based producer cells were demonstrated to be free of replication-competent retrovirus (RCR) by stringent testing.
The highest transduction of human hematopoietic progenitor cells was achieved with vector supernatant generated from a coculture of the ProPak-X and ProPak-A cell lines.
Consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector.
Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Subculturing
Protocol: Remove medium. Do not rinse. Cells detach easily. Add 2.0 to 3.0 ml of 0.25% trypsin, 0.53 mM EDTA solution. Allow the flask to sit at room temperature (or at 37C) until the cells detach. Centrifuge the cell suspension at 1000 rpm for 10 minutes, resuspend the pellet in fresh medium, aspirate and dispense into new flasks.
Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:10 is recommended
Medium Renewal: Every 2 to 3 days
Cryopreservation
Freeze medium: Complete growth medium 95%; DMSO, 5%
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Temperature: 37.0°C
STR Profile
Amelogenin: X
CSF1PO: 11,12
D13S317: 12,14
D16S539: 9
D5S818: 8,9
D7S820: 11,12
THO1: 7,9.3
TPOX: 11
vWA: 16,19
Name of Depositor SyStemix, Inc.
References

Rigg RJ, et al. Method for obtaining retroviral packaging cell lines producing high transducing efficiency retroviral supernatant. US Patent 6,017,761 dated Jan 25 2000

Forestell SP, et al. Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer. Gene Ther. 4: 600-610, 1997. PubMed: 9231077

Basic Documentation
References

Rigg RJ, et al. Method for obtaining retroviral packaging cell lines producing high transducing efficiency retroviral supernatant. US Patent 6,017,761 dated Jan 25 2000

Forestell SP, et al. Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer. Gene Ther. 4: 600-610, 1997. PubMed: 9231077