Vankyrin-Enhanced insect cell line BioVector NTCC质粒载体菌种细胞基因保藏中心

Vankyrin-Enhanced insect cell line BioVector NTCC质粒载体菌种细胞基因保藏中心 Vankyrin-Enhanced Insect Cell Line
Product No.: 10010; 10020; 10030
Content
Product No. 10010 (previously VE-CL-01), 10020 (previously
VE-CL-02) and 10030 (previously VE-CL-03) contain >1x107
cells in 50% fresh Sf-900 II serum free medium (InvitrogenTM),
50% conditioned Sf-900 II serum free medium and Dimethyl
Sulfoxide (DMSO) to a final concentration of 7.5%.
• 10010 has a cell doubling time of 37.6 hrs in Sf-900 II
SFM and an average cell size of 21.6
μm.
• 10020 has a cell doubling time of 29.7 hrs in Sf-900 II
SFM and an average cell size of 21.3
μm.
• 10030 has a cell doubling time of 43.6 hrs in Sf-900 II
SFM and an average cell size of 21.7
μm.
Shipping and Storage
Cells are shipped on dry ice and are supplied in a cryogenic vial
containing >1x107cells/mL. Cells were frozen in a freezing
medium composed of 50% fresh Sf-900 II serum free medium,
50% conditioned Sf-900 II serum free medium and Dimethyl
Sulfoxide (DMSO) to a final concentration of 7.5%. Store cells
in liquid nitrogen (vapor phase).
Live cells can be shipped upon request.
Product Qualification
To qualify for sales cells must be in logarithmic growth with
98% viability and less than 20 passages before they are frozen.
Cells have been shown to recover as healthy logarithmically
growing cells within 3 days after thawing.
Caution
DMSO is a hazardous material and caution has to be taken when
handling this substance.
Cell Maintenance and Handling of Cells
Medium Requirement
Use of Sf-900 II SFM (Invitrogen) medium is recommended but
cells also grow well in TNM-FH Insect Cell Culture Medium
supplemented with 10% heat-inactivated FBS. NOTE: The
addition of 400µg/mL neomycin should not be done until the
first passage after thawing. Neomycin is optional for cell
maintenance. We recommend starting the cells in adherent
culture and then adapting to shaker culture after 2 passages.
Thawing Cells
Thaw frozen cells rapidly in a 370C water bath. Decontaminate
the outside of the vial with 70% ethanol before transferring the 1
mL cell suspension into two T-25 cm2 flasks.
Adherent Culture: Put 0.5 mL of thawed cells into 5 mL of
medium and transfer flask to a 270C incubator and allow the cells
to attach for 30-45 minutes before replacing the medium with 5
mL fresh Sf-900 II SFM. Subculture cells when they have
reached a density of >80% confluency. Release cells from the
flask’s surface by tapping the flask sharply against your palm 3-4
times (> 75% of the cells should be detached) and transfer 2 mL
cells into a new T25 flask containing 3 mL of medium.
Suspension Culture: To start a suspension culture, release the
cells from two T25 monolayer cultures and transfer the entire
volume from one flask and 3 mL from the second flask (a total
volume of 8 ml) into a 125 mL shaker flask containing12 mL of
fresh Sf-900 II SFM. Use the remaining 2 mL of cells to
continue the cell line as adherent culture in a T-25 flask.
Incubate Erlenmeyer flask in a 270C incubator on an orbital
shaker platform rotating at 100-110 rpm. Loosen caps of flasks
to allow proper oxygenation/aeration. When a cell density of
2x106 viable cells/mL has been reached, cells should be seeded
to a density of 1x106 cells in 50 mL of Sf-900 II SFM in a 125
mL shaker flask. Once a suspension culture has been established
VE cells are routinely diluted to a cell density of 7-8x105 viable
cells/mL with Sf-900 II SFM.
VE cells have an average diameter of approximately 21 µm
which is bigger than Sf9 cells. In addition, VE cells grow slower
than Sf9 cells.
Freezing Cells
Freeze cells at a density of
>2x107 viable cells/mL in a freezing
medium composed of fresh Sf-900 II serum free medium
(Invitrogen), 10% heat-inactivated FBS and DMSO to a final
concentration of 7.5%. (Optional freezing media: 50%
conditioned Sf-900 II media: 50% fresh Sf-900 II and DMSO to
a final concentration of 7.5%). Centrifuge cells at 100g at 4oC
for 5-10 minutes, remove the supernatant and resuspend the
pellet in an appropriate volume of chilled freezing medium to
reach a density of
>2x107 viable cells/mL. Transfer suspension
into a cryovial. Place cells in a styrofoam container and place at
-20oC for one hour, then transfer the styrofoam container with
cells to –80oC overnight before transferring the cells to liquid
nitrogen (vapor phase). Frozen cells remain viable if properly
stored in liquid nitrogen.
Overview of Vankyrin-Enhanced (VE) Insect Cell Line
Vankyrin-Enhanced Insect Cells (VE cells) are transgenic insect
Sf9 cells that have been engineered to stably express the
Campoletis sonorensis ichnovirus P-vank-1 protein (FathGoodin
et al., 2006; Kroemer and Webb, 2006). Sf9 cells
originated from the IPLBSF-21 cell line, derived from the pupal
ovarian tissue of the fall army worm, Spodoptera frugiperda
(Vaughn et al., 1977).
The stably transformed VE insect cell line was obtained by
transfecting Sf9 cells with ParaTechs’ proprietary transformation
vector harboring the P-vank-1 gene and the neomycin resistance
gene. Neomycin was then used to select for stable cell lines. The
expression of the P-vank-1 transcript was confirmed by RTPCR.

The presence of the P-vank-1 protein leads to prolonged
longevity and increased recombinant protein production of
baculovirus infected VE cells compared to regular Sf9 cells.
This cell line has been developed for enhanced recombinant
protein production using the baculovirus expression vector
system (BEVS).
• Modified insect Sf9 cells stably expressing a Campoletis
sonorensis ichnovirus vankyrin gene
• Use of neomycin for selection of stable lines
• Prolonged longevity of cells after infection with a BEVS
• Up to 14-fold increase in protein yield as compared to
regular Sf9 cells. Further enhancement (up to more than
20-fold) can be obtained by using modified cells in
combination with the VE-BEVS transfer vector
• Compatible with all conventional BEVS
• Essentially no additional work or adaptation required
• Expression of recombinant protein may need to be
optimized
This product is intended for research purposes only
CAUTION: Not intended for human or animal diagnostic or
therapeutic uses.
BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心
电话：+86-010-53513060
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