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Flp-InTM System
TABLE OF CONTENTS
PRODUCT DESCRIPTION
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
Vectors
Primers
One Shot TOP10 Competent E. coli
Tetracycline
Flp-In Cell Lines
Flp-In T-REx- 293 Cell Line
PROTOCOL & APPLICATION NOTES
Flp-In vectors
pFRT/lacZeo
pFRT/lacZeo2
pOG44
pcDNA5/FRT
pcDNA5/FRT/V5-His TOPO
pSec/Tag/FRT/V5-His TOPO
pEF5/FRT/V5-D-TOPO and pEF5/FRT/V5-DEST
pcDNA6/FRT/V5-DEST
pcDNA5/FRT/TO, pcDNA5/FRT/TO-TOPO, pcDNA5/FRT/TO-E Echo-Adapted
pcDNA6/TR
pcDNA5/FRT/CAT, pcDNA5/FRT/TO/CAT, pcDNA5/FRT/TO-E/Uni-CAT, pcDNA5/FRT/V5-
His/CAT
Generation of Flp-In Host Cell lines
Suggestions to increase the likelihood of obtaining single integrants
Strategy for optimizing chances of obtaining single integration events of pFRT/lacZeo in CHO cells
Flp-In system not highly recommended for B lymphocyte applications
Flp-In Cell lines
General Guidelines when using Flp-In cell lines
Flp-In-CV-1 cells
Flp-In-BHK cells
Flp-In-3T3 cells
Flp-In-Jurkat cells
Flp-In-CHO cells
Flp-In-293 cells
Flp-In T-REx- 293 Cells
Generation of Flp-In Expression Cell Lines
Maximum size of insert that can be used
Selection with Hygromycin
Transfection protocol for Flp-In Jurkat cells
Transfection protocol for Flp-In -CHO cells
Efficiency of Flp-In integration versus random integration
Stability of Flp-In cell lines containing the gene of interest
Expression in Flp-in Cell Lines
Use of Tetracycline-Tested GIBCO FBS in Flp-in T-Rex expression system
Qualitative and quantitative data to demonstrate that isolated foci from transfection into Flp-in cell
lines will express a reporter at near equivalent levels
Comparison of reporter activities between different Flp-In cell lines
Background bands on Western Blots with the anti-V5 Antibody
Custom Flp-In Host, and Expression Cell Lines Service
ALTERNATE PRODUCTS & COMPATIBILITY
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBER
COMPONENTS
ASSOCIATED PRODUCTS
RELATED TECHNICAL SUPPORT NOTES
PRODUCT DESCRIPTION
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The Flp-In System allows integration and expression of your gene of interest in mammalian cells at a specific genomic
location. The introduction of an Flp Recombination Target (FRT) site into the genome allows the generation of isogenic
stable cell lines.
The major components of the Flp-In System include:
1. A Flp-In target site vector, pFRT/lacZeo or pFRT/lacZeo2, for generation of a host cell line containing an
integrated FRT site
2. An expression plasmid containing a FRT site linked to the hygromycin resistance gene for Flp recombinase-
mediated integration and selection of a stable cell line expressing the gene of interest under control of CMV
immediate-early enhancer/promoter
3. A Flp-recombinase expression plasmid, pOG44, for expression of the Flp-recombinase under the control of the
CMV promoter
4. A control expression plasmid containing the chloramphenicol acetyl transferase (CAT) gene
The Flp-In T-REx System allows the generation of stable mammalian cell lines exhibiting tetracycline-inducible
expression of a gene of interest from a specific genomic location. This system uses regulatory elements from the E. coli
Tn10-encoded tetracycline (Tet) resistance operon to allow tetracycline-regulated expression of your gene of interest
from pcDNA5/FRT/TO.
The mechanism of tetracycline regulation is based on the binding of tetracycline to the Tet repressor and de-repression
of the promoter controlling expression of the gene of interest. Expression of your gene of interest is repressed in the
absence of tetracycline and induced in the presence of tetracycline.
The Flp-In cells lines stably express the lacZ-Zeocin fusion gene and are designed for use with the Flp-In System. Each
cell line contains a single integrated Flp Recombination Target (FRT) site from pFRT/lacZeo or pFRT/lacZeo2. Co-
transfection of Flp-In Cell lines with a Flp-In Expression Vector and the Flp recombinase vector, pOG44, results in
targeted integration of the expression vector to the same locus in every cell.
NOTE: The advantage of the Flp-In system is that equal gene expression is obtained in all clones for that particular cell
line. It does NOT guarantee the same expression in different cell types.
SHIPPING CONDITIONS
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The Flp-In and Flp-In T-Rex Core Kits ship at room temperature. Non-TOPO vectors, primers, and hygromycin box
ship at room temperature. Zeocin ships on blue ice.
TOPO vector Kits and competent E. coli ship on dry ice.
All cell lines ship on dry ice.
STORAGE CONDITIONS
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Store vectors and primers at -20掳C. Store the TOPO vectors at -20掳C and competent E. coli cells at -80掳C.
Store the Core Kits at 鈥?20oC.
Store the tetracycline at +4掳C protected from exposure to light. For long-term storage (> 6 months), store the
tetracycline at -20掳C protected from exposure to light.
Store zeocin at +4掳C or -20掳C. Store Hygromycin at +4掳C.
All mammalian Flp-In cell lines should be stored in liquid nitrogen upon receipt.
STABILITY
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All components guaranteed for 6 months from date of purchase
QC SPECIFICATIONS
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Vectors
Primers
One Shot TOP10 Competent E. coli
Tetracycline
Flp-In Cell Lines
Flp-In T-REx- 293 Cell Line
Vectors
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Each vector is qualified by restriction enzyme digestion with specific restriction enzymes as listed below. Restriction
digests must demonstrate the correct banding pattern when electrophoresed on an agarose gel (see below).
The pcDNA5/FRT/TO supercoiled vector (parental vector of pcDNA5/FRT/TO-TOPO) and pcDNA5/FRT/TO/CAT
are qualified by restriction digest with specific restriction enzymes as listed below. Please note that the
pcDNA5/FRT/TO plasmid is qualified by restriction digest prior to adaptation with topoisomerase I, therefore,
restriction sites used to qualify the parental vector may no longer be present in the topoisomerase I-adapted vector.
Restriction digests must demonstrate the correct banding pattern when electrophoresed on an agarose gel. The table
below lists the restriction enzymes and the expected fragments. The size of the parent pcDNA5/FRT/TO vector is 5137
bp.
Vector Restriction Expected Results (bp)
Enzymes
pFRT/lacZeo Kpn I No site
Xba I 8106
pFRT/lacZeo2 Hind III 2663, 3819
Pst I 6482
pcDNA6/TR Pme I 1701, 4961
Sal I 2185, 4477
pOG44 Kpn I 5438, 347
Xba I 5785
pcDNA5/FRT Kpn I 5069
Xba I 4498, 571
pcDNA5/FRT/CAT Kpn I 5858
Xba I 5286, 572
pcDNA5/FRT/V5-His BamH I 5076
EcoR I 5076
Hind III 5076
Mlu I 749, 4327
pcDNA5/FRT/V5- His/CAT BamH I 5751
EcoR I 1376, 4375
Hind III No site
Mlu I 1424, 4327
pSecTag/FRT/ V5-His BamH I 5167
EcoR I 5167
Mlu I 839, 4328
Pvu II 1804, 3363
pSecTag/FRT/ V5-His/PSA BamH I 741, 5161
EcoR I 5902
Mlu I 1574, 4328
Pvu II 973, 1802, 3125
pcDNA5/FRT/TO EcoR V 5137
Hind III 5137
Hind III/SnaB I 388, 4749
Kpn I 5137
Xba I 572, 4565
pcDNA5/FRT/TO/CAT Hind III/SnaB I 388, 5538
Xba I 572, 5354
TOPO Cloning Efficiency: Once the pcDNA5/FRT/TO vector has been adapted with topoisomerase I, it is lot-qualified
using the control reagents included in the kit. Under conditions described on pages 21-22, a 500 bp control PCR product
is TOPO Cloned into pcDNA5/FRT/TO-TOPO and subsequently transformed into the One Shot TOP10 chemically
competent E. coli included with the kit.
Each lot of vector must yield greater than 85% cloning efficiency.
Primers
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Sequencing primers are lot-qualified by DNA sequencing experiments using the dideoxy-chain termination technique.
One Shot TOP10 Competent E. coli
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50 碌l of competent cells are transformed with 10 pg of supercoiled pUC18 plasmid. Transformed cultures are
plated on LB plates containing 50 碌g/ml ampicillin and the transformation efficiency is calculated. Test
transformations are performed in triplicate. Transformation efficiency should be greater than 1 x 109 cfu/碌g
plasmid DNA.
Untransformed cells are plated on LB plates containing 50 碌g/ml ampicillin to verify the absence of ampicillin
resistant contamination.
SOB plates as a lawn to verify the absence of phage contamination.
Tetracycline
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The pBR322 plasmid is transformed into TOP10 E. coli cells. Greater than 95% of transformants must be viable when
plated on Low Salt LB Agar with and without 15 ug/ml tetracycline.
Flp-In Cell Lines
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The manual states that the number of cells provided is 3 x 10E6 cells, however tubes are labeled as 1 x 10E7.
The following criteria are used to qualify the Flp-In cell lines
Each cell line is tested independently and certified to be free of mycoplasma.
Prior to freezing, cells are greater than 95% viable. Forty-eight hours after thawing, cells are greater than 90%
viable.
Each cell line is tested for 尾-galactosidase activity by plating 1 x 105 cells into 6-well plates. Cells are grown in
medium containing Zeocin for 7 days. The medium containing Zeocin is removed and replaced with fresh
medium without Zeocin. After 3 days, the cells are assayed for 尾-galactosidase activity using the 尾-Gal
Staining Kit from Invitrogen. Each cell line must exhibit greater than 95% cells expressing 尾-galactosidase.
None of the genetically modified cell lines that we sell have been virus tested. Only the parental cells coming
out of Grand Island have been virus-tested.
Flp-In T-REx- 293 Cell Line
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The cell line is tested independently and certified to be free of mycoplasma.
Prior to freezing, cells are greater than 95% viable. Forty-eight hours after thawing, cells are greater than 90%
viable.
The cell line is functionally tested by plating 1 x 105 cells into three 6-well plates and performing the following
tests:
(1) To test for an intact integrated FRT site, one 6-well plate is selected 24 hours post-seeding and assayed for
B-galactosidase activity using the B-Gal Staining Kit from Invitrogen. The cell line must exhibit greater than
95% cells expressing B-galactosidase.
(2) To test for tetracycline-regulated expression, cells in the two remaining 6-well plates are transiently
transfected with either pcDNA5/FRT/TO/CAT (inducible expression plasmid, pcDNA3.1/Zeo/CAT
(constitutive expression plasmid), or no DNA (negative control). 24 hours post-transfection, tetracycline is
added to the cells transfected with pcDNA5/FRT/TO/CAT to a final concentration of 1 碌g/ml. Cells are
induced for 24 hours, then assayed for CAT expression . The cells transfected with pcDNA5/FRT/TO/CAT
must exhibit greater than 20-fold induction of CAT expression as compared to cells transfected with
pcDNA3.1/Zeo/CAT.
PROTOCOL AND APPLICATION NOTES
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Flp-In vectors
pFRT/lacZeo
pFRT/lacZeo2
pOG44
pcDNA5/FRT
pcDNA5/FRT/V5-His TOPO
pSec/Tag/FRT/V5-His TOPO
pEF5/FRT/V5-D-TOPO and pEF5/FRT/V5-DEST
pcDNA6/FRT/V5-DEST
pcDNA5/FRT/TO, pcDNA5/FRT/TO-TOPO, pcDNA5/FRT/TO-E Echo-Adapted
pcDNA6/TR
pcDNA5/FRT/CAT, pcDNA5/FRT/TO/CAT, pcDNA5/FRT/TO-E/Uni-CAT, pcDNA5/FRT/V5-
His/CAT
Generation of Flp-In Host Cell lines
Suggestions to increase the likelihood of obtaining single integrants
Strategy for optimizing chances of obtaining single integration events of pFRT/lacZeo in CHO cells
Flp-In Cell lines
General Guidelines when using Flp-In cell lines
Flp-In-CV-1 cells
Flp-In-BHK cells
Flp-In-3T3 cells
Flp-In-Jurkat cells
Flp-In-CHO cells
Flp-In-293 cells
Flp-In T-REx- 293 Cells
Generation of Flp-In Expression Cell Lines
Maximum size of insert that can be used
Selection with Hygromycin
Transfection protocol for Flp-In Jurkat cells
Transfection protocol for Flp-In -CHO cells
Efficiency of Flp-In integration versus random integration
Stability of Flp-In cell lines containing the gene of interest
Notes related to Expression in Flp-In Cell lines
Qualitative and quantitative data to demonstrate that isolated foci from transfection into Flp-in cell
lines will express a reporter at near equivalent levels
Comparison of reporter activities between different Flp-In cell lines
Background bands on Western Blots with the anti-V5 Antibody
Custom Flp-In Host, and Expression Cell Lines Service
Flp-In vectors
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pFRT/lacZeo
pFRT/lacZeo2
pOG44
pcDNA5/FRT
pcDNA5/FRT/V5-His TOPO
pSec/Tag/FRT/V5-His TOPO
pEF5/FRT/V5-D-TOPO and pEF5/FRT/V5-DEST
pcDNA6/FRT/V5-DEST
pcDNA5/FRT/TO, pcDNA5/FRT/TO-TOPO, pcDNA5/FRT/TO-E Echo-Adapted
pcDNA6/TR
pcDNA5/FRT/CAT, pcDNA5/FRT/TO/CAT, pcDNA5/FRT/TO-E/Uni-CAT, pcDNA5/FRT/V5-His/CAT
pFRT/lacZeo
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pFRT/lacZeo is a 8.1 kb FRT-site vector.
Contains the SV40 early promoter and origin for high-level constitutive expression of the lacZ-Zeocin fusion
gene in mammalian cells.
Neither the lacZ gene nor the Zeocin resistance gene contains its native ATG initiation codon. The ATG
initiation codon is placed directly upstream of a FRT site and allows expression of the lacZ-Zeocin fusion gene
in cells
pFRT/lacZeo2
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pFRT/lacZeo2 is a 6.5 kb FRT-site vector.
Differs from pFRT/lacZeo in the SV40 enhancer region, which is truncated in pFRT/lacZeo2. The SV40 early
promoter contains a 99 bp deletion from the 5鈥?-end when compared to the wild-type SV40 early promoter.
This truncated SV40 early promoter (PSV40) exhibits approximately 60-fold less activity when compared to the
wild-type SV40 early promoter (in pFRT/lacZeo).
Stable transformants contain a single FRT site, which has integrated into a highly transcriptionally active
genomic locus.
Approximately 4-8 Zeocin-resistant clones are observed when transfecting pFRT/lacZeo2 and approximately
25-50 Zeocin-resistant clones when transfecting pFRT/lacZeo into 1-5 x 106 mammalian cells.
In order to increase the number of Zeocin-resistant clones, we recommend increasing the number of cells that
are transfected by increasing the number of plates. Do not increase the amount of plasmid DNA that is
transfected.
pOG44
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pOG44 is a 5.8 kb Flp recombinase expression vector.
Contains the human cytomegalovirus (CMV) immediate-early enhancer/promoter for high-level constitutive
expression of the Flp recombinase in a wide range of mammalian cells.
Contains a synthetic intron to enhance expression of the FLP gene.
Contains the FLP gene encoding the Flp recombinase to mediate integration of the expression plasmid into the
genome.
The expression levels of Flp recombinase in the cell will determine the efficiency of the recombination
reaction. Flp recombinase levels must be sufficiently high to mediate recombination at the FRT sites (single
recombination event) and overcome the low intrinsic activity of the enzyme. We recommend that you co-
transfect you Flp-In host cell line with a ratio of at least 9:1 (w/w) pOG44:pcDNA5/FRT plasmid. Please note
that this ratio may vary depending on the nature of the cell line. You may want to determine this ratio
empirically for your cell line.
When transfecting your Flp-In host cell line, be sure to use supercoiled pOG44 and expression vector plasmid
DNA. Flp-mediated recombination between the FRT site on the expression vector and the integrated FRT site
in the Flp-In host cell line will only occur if the expression plasmid is circularized. The pOG44 plasmid should
be circularized to minimize the possibility of the plasmid integrating into the genome.
Once the pcDNA5/FRT construct has integrated into the genome, the Flp recombinase is no longer required.
The continued presence of Flp recombinase would actually be detrimental to the cells because it could mediate
excision of the pcDNA5/FRT construct. For this reason, the pOG44 plasmid lacks an antibiotic resistance
marker for selection in mammalian cells. When generating stable expression cell lines, the pOG44 plasmid
and, therefore, Flp recombinase expression, will gradually be lost from transfected cells as they are cultured
and selected.
pcDNA5/FRT
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It is a 5.1 kb expression vector.
Contains the human cytomegalovirus (CMV) immediate-early enhancer/promoter for high-level constitutive
expression of the gene of interest in a wide range of mammalian cells.
Contains the Hygromycin resistance gene for selection of integrants.
We do not believe that there is an upper size limit for integration using pcDNA5/FRT, so everything in the
plasmid will be moved into the genome.
There are two Sal I sites in this vector. One recognition sequence is from 2878-2883, the other is from 5067-3
and spans the beginning/end of the plasmid (total size is 5069). Therefore, the second Sal1 site only shows up
on the restriction map on the circular plasmid and not the linear plasmid.
The pcDNA5/FRT vector contains a single FRT site immediately upstream of the hygromycin resistance gene
for Flp recombinase-mediated integration and selection of the pcDNA5/FRT plasmid following co-transfection
of the vector (with pOG44) into Flp-In mammalian host cells. The FRT site serves as both the recognition and
cleavage site for the Flp recombinase and allows recombination to occur immediately adjacent to the
hygromycin resistance gene.
The hygromycin resistance gene in pcDNA5/FRT lacks a promoter and an ATG initiation codon; therefore,
transfection of the pcDNA5/FRT plasmid alone into mammalian host cells will not confer hygromycin
resistance to the cells. The SV40 promoter and ATG initiation codon required for expression of the
hygromycin resistance gene are integrated into the genome (in the Flp-In host cell line) and are only brought
into the correct proximity and frame with the hygromycin resistance gene through Flp recombinase-mediated
integration of pcDNA5/FRT at the FRT site.
We have maintained a number of Flp-In cell lines over four months and have not observed pcDNA5/FRT
recombining out, whether or not under hygromycin selection.
pcDNA5/FRT/V5-His TOPO
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Contains the CMV promoter for high-level constitutive expression of your gene of interest.
Contains the Hygromycin resistance gene for selection of integrants.
Contains a C-terminal V5-His tag for easy detection with an Anti-V5-Antibody and rapid purification on
ProBond resin.
Design of the PCR primers to clone your DNA sequences of interest is critical for expression. This is a C-
terminal fusion vector, which does not contain an ATG initiation codon. If there is no initiating ATG codon or
optimal sequences for translation initiation (Kozak sequences) in the DNA to be amplified, then these features
need to be incorporated into your forward primer. Example: Kozak consensus sequence is ANNATGG.
pSec/Tag/FRT/V5-His TOPO
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Contains the CMV promoter for high-level constitutive expression of your gene of interest.
Contains the Hygromycin resistance gene for selection of integrants.
Contains a C-terminal V5-His tag for easy detection with an Anti-V5-Antibody and rapid purification on
ProBond resin.
Contains a secretion signal from the V-J2-C region of the mouse Ig kappa-chain for efficient secretion of
recombinant proteins.
Design PCR primers to clone your gene of interest in frame with the N-terminal Ig kappa -chain secretion
signal and the C-terminal peptide containing the V5 epitope and the polyhistidine (6xHis) tag (if desired).
The control protein PSA from the pSecTag vector runs between the 36 and 50kD markers and is estimated to
be 43-47kD.
pEF5/FRT/V5-D-TOPO and pEF5/FRT/V5-DEST
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Contains the pEF mammalian promoter.
Contains a C-terminal V5-His tag for easy detection with an Anti-V5-Antibody and rapid purification on
ProBond resin
The EF promoter is expressed in many mammalian cell lines and is functional in some cell lines where the CMV
promoter is not active or gives inconsistent expression. The pEF promoter has been tested in the Flp-In-BHK and
3T3 cell lines, and in both of these cell lines it works much better than the CMV promoter.
pcDNA6/FRT/V5-DEST
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Uses Blasticidin instead of Hygromycin as the selective antibiotic, which offers better selection
Does not contain a BGH Poly A sequence .
pcDNA5/FRT/TO, pcDNA5/FRT/TO-TOPO, pcDNA5/FRT/TO-E Echo-Adapted
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This is a 5.1 kb expression vector
Contains a hybrid promoter consisting of the human cytomegalovirus immediate-early (CMV) promoter and
tetracycline operator 2 (TetO2) sites for high-level tetracycline-regulated expression in a wide range of
mammalian cells.
The TetO sites are 3' of transcription initiation at pCMV (downstream). TetO2 sequences consist of 2 copies
of the 19 nucleotide sequence: 5麓-TCCCTATCAGTGATAGAGA-3麓 separated by a 2 base pair spacer. It
serves as binding sites for 4 Tet repressor molecules (comprising two Tet repressor homodimers).
Contains a single FRT site immediately upstream of the hygromycin resistance gene for Flp recombinase-
mediated integration and selection of the pcDNA5/FRT/TO plasmid.
Used in co-transfection with pOG44 into Flp-In T-REx mammalian host cells.
TOPO adapted vector with T-overhangs for TA cloning
Echo-adapted vector has loxP site following MCS.
Possible sites for Linearization of pcDNA5/FRT/TO
Enzyme Restriction Location Supplier
Site (bp)
Mun I 162 Upstream of CMV Many
promoter
Nru I 209 Upstream of CMV Many
promoter
Sap I 3206 Backbone New England Biolabs
Eam1105 4215 Ampicillin gene Angewandte Gentechnologie
I Systeme, Fermentas, Takara
Fsp I 4437 Ampicillin gene Many
Ssp I 5019 Backbone Many
pcDNA6/TR
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This is a 6.7 kb vector expresses high levels of the tetracycline (Tet) repressor under CMV promoter.
High-level stable (bsd) and transient expression of the Tet repressor can be carried out in most mammalian
cells.
It includes the rabbit beta-globin intron II to enhance expression of the TetR gene
There are extra 33 coding bases present (11 amino acids) in tetR after position 2304 before reaching the STOP
codon. The Tet Repressor ORF that is present on pcDNA6/TR actually runs from 1684-2337. However, the Tet
Repressor gene itself (as determined by alignment with the published TetR sequence) goes from 1684-2304.
Therefore, an additional 11 amino acids are present on the C-terminus of TetR that is expressed from
pcDNA6/TR. These extra amino acids don't have an affect on the function of the TetR repressor.
Antibodies to TetR are not currently available from Invitrogen. We don鈥檛 recommend using antibodies to
screen for functional clones. Functional testing by using the control plasmid is recommended.
Typical yield for pcDNA6/TR based on Invitrogen's in-house results from three plasmid preps is between 1-1.4
ug/ml.
Removal of the blasticidin gene - BbrPI will remove the EM7-Blastcidin cassette. Alternatively, MscI + CelII
(double digest) will remove the Blasticidin gene only. If using MscI + CelII, you need to be aware of
positioning the initiator sequences appropriately to get the correct reading frame.
Possible sites for linearization of pcDNA6/TR
Enzyme Restriction Location Supplier
Site (bp)
Bst1107 4470 Backbone Angewandte Gentechnologie
I Systeme, Fermentas, Takara
Sap I 4733 Backbone New England Biolabs
BspLU11 4849 Backbone Boehringer-Mannheim
I
Eam1105 5739 Ampicillin gene Angewandte Gentechnologie
I Systeme, Fermentas, Takara
Fsp I 5961 Ampicillin gene Many
pcDNA5/FRT/CAT, pcDNA5/FRT/TO/CAT, pcDNA5/FRT/TO-E/Uni-CAT, pcDNA5/FRT/V5-His/CAT
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positive control expression construct.
Assay expression via anti-CAT or CAT assay.
The CAT protein expressed from the control plasmid is approximately 32 kDa in size. CAT expression can be
assayed by ELISA, western blot analysis, fluorometric, or radioactive assays.
A Commercial kit to assay for CAT protein is available from Invitrogen (Catalog no. F-2900).
Generation of Flp-In Host Cell lines
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Suggestions to increase the likelihood of obtaining single integrants
Strategy for optimizing chances of obtaining single integration events of pFRT/lacZeo in CHO cells
Flp-In system not highly recommended for B lymphocyte applications
Suggestions to increase the likelihood of obtaining single integrants
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1. Lower transfection efficiency by limiting the amount of transfected plasmid DNA. We typically use 250 ng to
2 碌g of plasmid DNA per 4 x 106 cells for transfection. However the amount of plasmid DNA may vary due to
the nature of the cell line, the transfection efficiency of the cells, and the method of transfection used. When
transfecting the mammalian cell line of choice, we suggest that you try a range of plasmid DNA concentrations
(e.g. 0.25, 0.5, 1, 2, 5 碌g/ml DNA) to optimize transfection conditions for the cell line. To increase the
chances of obtaining single integrants, pick foci from plates that have been transfected with the least amount of
plasmid DNA. From our experience, when different cell lines were transfected with ranges of DNA between
500-1000 ng per 1 million cells we typically observe cell lines that have one or two integration events. The
best way to determine single FRT sites is a Southern blot.
2. Chromosomal position effects: Because integration of the pFRT/lacZeo plasmid into the genome occurs
randomly, expression levels of the lacZ-Zeocin fusion gene will be dependent on the transcriptional activity of
the surrounding sequences at the integration site (i.e. chromosomal position effect). Once you have obtained
single integrants, you may want to screen the Zeocin-resistant clones for those expressing the highest B-
galactosidase levels. Those clones expressing the highest levels of B-galactosidase should contain single FRT
sites, which have integrated into the most transcriptionally active regions.
3. Antibiotic concentration: Single integrants will express only a single copy of the lacZ-Zeocin fusion gene and
therefore, may be more sensitive to Zeocin selection than multiple integrants. If you have previously used your
mammalian cell line for transfection and Zeocin selection, please note that you may need to use lower
concentrations of Zeocin to obtain single integrants.
Strategy for optimizing chances of obtaining single integration events of pFRT/lacZeo in CHO cells:
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Use as little DNA as possible. A very high transfection efficiency is not recommended, therefore do not use
lipids as the delivery choice.
For Electroporation we suggest trying 250 ng, 500 ng and 1000 ng per 3x106 cells (this keeps the DNA/cell
ratio low to minimize the amount of DNA available to each cell). Have cells in serum-free media, on ice for
10 min. For CHO cells we typically use 330V, 25mA, 25W, then back to ice for 10 min before plating into
100mm dishes.
Keep the concentration of zeocin as low as possible (100ug/ml is good) otherwise high Zeo expressers usually
carrying multiple copies will be selected for. Plate a "no DNA" control so you know that the Zeo selection is
complete. Select foci from the 250 ng plate if possible.
For CHO cells, the frequency of multiple vs. single integrants is around 50% each. Using linear DNA usually
gives us fewer stable colonies but not necessarily more single integrants.
Flp-In system not highly recommended for B lymphocyte applications
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(back to Protocol and Application Notes)
(back to Generation of Flp-In Host Cell Lines)
CMV promoter strength will be poor in B cells. SV40 promoter also tends to be weak so it鈥檚 likely that there won鈥檛
be good enough expression of the lacZeo fusion to allow resistant cells to grow out. This should be tested as part of
the optimization procedure. If one cannot detect lacZ from pFRTlacZeo, then that means SV40 promoter would
need to be changed out for some other promoter. EF-1alpha and UbC are appropriate.
Transfection efficiencies are notably poor for lymphocytes, in particular B lymphocytes. This will further impact
the success of using the system in B lymphocytes. Transfection optimization is strongly recommended.
Flp-In Cell lines
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General Guidelines when using Flp-In cell lines
Flp-In-CV-1 cells
Flp-In-BHK cells
Flp-In-3T3 cells
Flp-In-Jurkat cells
Flp-In-CHO cells
Flp-In-293 cells
Flp-In T-REx- 293 Cells
General Guidelines when using Flp-In cell lines
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FBS does not need to be heat inactivated for use with these cell lines.
Cell lines should be maintained in medium containing Zeocin at the concentrations listed in the Flp-In Cell
Lines manual
Passaging adherent cells (e.g. Flp-In-293, Flp-In-CV-1, Flp-In-CHO, Flp-In-3T3, Flp-In BHK) at a 1:5 to 1:10
dilution will result in 80-90% confluence within 3-4 days.
The recommended trypsin to use is Cat. No. 25300.
Our studies have indicated that there is no observable difference in cell viability whether or not conditioned
media is used in the freezing medium.
Complete medium is defined as medium supplemented with FBS.
Conditioned complete medium is medium that has been used by the cells. During growth, the cells release
growth factors into the medium that also contains debris from dead cells. Rather than aspirating and discarding
the medium, this medium along with an equal amount of fresh medium, plus 10% DMSO can be used to make
up the freezing medium.
Information on the parental cell lines can be found in the Flp-In Cell Lines manual. The different functional
characteristics (glycosylation, kinase, phosphatase, protease) of the Flp-In cell lines have not been investigated.
These cell lines originated from ATCC stocks and have gone through a known number of passages prior to
being sold. They have not changed with respect to growth, morphology and transfectability compared to the
parent line, and they are certified mycoplasma-free.
Flp-In-CV-1 cells
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This cell line contains a single integrated FRT site and stably expresses the lacZ-Zeocin fusion gene from the
pFRT/lacZeo plasmid under the control of the SV40 early promoter. The location of the FRT site in each Flp-
In cell line has not been mapped, but is presumed to have integrated into a transcriptionally active genomic
locus as determined by generation of a Flp-In expression cell line containing the pcDNA5/FRT/CAT control
plasmid.
This Flp-In cell line should be maintained in medium containing 100 ug/ml Zeocin.
Flp-In-BHK cells
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(back to Flp-In Cell lines)
This cell line contains a single integrated FRT site and stably expresses the lacZ-Zeocin fusion gene from the
pFRT/lacZeo plasmid under the control of the SV40 early promoter. The location of the FRT site in each Flp-
In cell line has not been mapped, but is presumed to have integrated into a transcriptionally active genomic
locus as determined by generation of a Flp-In expression cell line containing the pcDNA5/FRT/CAT control
plasmid.
This Flp-In cell line should be maintained in medium containing 100 ug/ml Zeocin.
We have observed a loss of gene expression from CMV promoters when pcDNA5/FRT-based vectors are
transfected into this cell line. This is not observed with pEF5/FRT expression constructs therefore this line of
vectors is recommended in the 3T3 and BHK cell lines.
Flp-In-3T3 cells
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(back to Flp-In Cell lines)
This cell line contains a single integrated FRT site and stably expresses the lacZ-Zeocin fusion gene from the
pFRT/lacZeo plasmid under the control of the SV40 early promoter. The location of the FRT site in each Flp-
In cell line has not been mapped, but is presumed to have integrated into a transcriptionally active genomic
locus as determined by generation of a Flp-In expression cell line containing the pcDNA5/FRT/CAT control
plasmid.
This Flp-In cell line should be maintained in medium containing 100 ug/ml Zeocin.
We have observed a loss of gene expression from CMV promoters when pcDNA5/FRT-based vectors are
transfected into this cell line. This is not observed with pEF5/FRT expression constructs therefore this line of
vectors is recommended in the 3T3 and BHK cell lines.
The CMV promoter in 3T3 cells is variable, depending on where it gets integrated in the chromosome. We
know for a fact that the FRT site in the Flp-In 3T3 cell line is at a chromosomal location where CMV (or
CMV/TO) is downregulated quite rapidly (occurs during the 2 weeks of hygromycin selection post Flp-In
transfection). We have examined expression levels upto 3 weeks, which was very poor and many clones did
not have detectable expression.
Flp-In-Jurkat cells
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(back to Flp-In Cell lines)
This cell line contains a single integrated FRT site and stably expresses the lacZ-Zeocin fusion gene from the
pFRT/lacZeo plasmid under the control of the SV40 early promoter. The location of the FRT site in each Flp-
In cell line has not been mapped, but is presumed to have integrated into a transcriptionally active genomic
locus as determined by generation of a Flp-In expression cell line containing the pcDNA5/FRT/CAT control
plasmid.
This Flp-In cell line should be maintained in medium containing 100 ug/ml Zeocin.
Flp-In-Jurkat cells will demonstrate optimal growth characteristics if maintained at a cell density between 1 x
10e5 cells/ml and 1 x 10e6 cells/ml.
When maintaining Flp-In-Jurkat cells in suspension culture, do not allow the medium to turn yellow; this
indicates that cells have reached too high a density or that the medium is depleted of nutrients. If this occurs,
either add fresh complete media to the cells or passage them.
Flp-In-CHO cells
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(back to Flp-In Cell lines)
This cell line contains a single integrated FRT site and stably expresses the lacZ-Zeocin fusion gene from the
pFRT/lacZeo2 plasmid. The pFRT/lacZeo2 contains a mutated SV40 early promoter, which is severely
abrogated in its activity and exhibits approximately 60-fold less activity than the wild-type SV40 early
promoter in pFRT/lacZeo. Because of the minimal activity of the SV40鈭? promoter, we expect that stable
transfectants expressing the lacZ-Zeocin gene from pFRT/lacZeo2 should contain FRT sites, which have
integrated into the most transcriptionally active genomic loci.
The Flp-In -CHO cell line should be maintained in medium containing 100 ug/ml Zeocin.
The exact location of the FRT site in the Flp-In -CHO cell line has not been mapped, but has been
demonstrated to have integrated into a highly transcriptionally active genomic locus as determined by
generation of a Flp-In expression cell line containing the pcDNA5/FRT/luc (luciferase-expressing) control
plasmid.
CHO cells are tested for single integrated FRT site. Numerous zeocin-resistant colonies were isolated and
expanded for B-gal staining and Southern Blotting. To determine if a single integration occurred, a Southern
blot analysis was performed by digesting genomic DNA with NcoI and hybridizing the DNA with a P-32
labeled probe detecting the lacZ gene. Colonies which showed a single band were then tested for B-gal
activity. The highest expresser was chosen. For the clone that was selected, the single band for the NcoI
Southern digest product was around 6 kb.
Reversion of transfected Flp-in CHO cells to non-transfected state: Transfected Flp-in CHO cells have been
reported to revert to non-transfected state presumably via site-specific recombination between the two integrated Flp
recombinase target sites during mitosis (Quest Vol 2(2), Pg 9, 2005). Increasing the Hygromycin concentration did not
kill the revertants, instead it seemed to facilitate or even cause the reversion. Reduction of hygromycin concentration to
250 ug/ml resulted in culture stabilization within just a few days and the number of revertants dropped to less than 1%
of the population.
Flp-In CHO cell morphology: There will always be variable morphology in immortalized cell lines CHO cells. Cell
density, phase of the cell cycle, etc. influence how each cell in the population looks, even though the Flp-In CHO came
from a single cell colony. There is no visible difference between wild type CHO-K1 (ATCC #CCL-61) and the Flp-In
CHO cells, and their growth characteristics are identical. Some cells will look fibroblast-like and some will look more
rounded just as for wild type CHO. As the cultures become more confluent, all cells will begin to resemble each other.
If there is a real concern about morphology, we recommend continuing to culture the cells between 40% and 100%
confluency. Generally CHO cells do not like to get over-confluent and tend to stay rounded up if over-confluent.
Flp-In-293 cells
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This cell line contains a single integrated FRT site and stably expresses the lacZ-Zeocin fusion gene from the
pFRT/lacZeo plasmid under the control of the SV40 early promoter. The location of the FRT site in each Flp-
In cell line has not been mapped, but is presumed to have integrated into a transcriptionally active genomic
locus as determined by generation of a Flp-In expression cell line containing the pcDNA5/FRT/CAT control
plasmid.
This Flp-In cell line should be maintained in medium containing 100 ug/ml Zeocin.
We typically observe 50-100 or more Flp-In expression stables when transfecting 5x105 Flp-In 293.
Flp-In T-REx- 293 Cells
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The Flp-In T-REx -293 cell line stably expresses the Tet repressor from pcDNA6/TR and contains a single
integrated Flp Recombination Target (FRT) site from pFRT/lacZeo as confirmed by Southern blot analysis.
FBS does not need to be heat inactivated for use with this cell line.
The recommended trypsin to use is Cat. No. 25300.
The cell line should be maintained in medium containing Zeocin 100 ug/ml and blasticidin 15 ug/ml
If cells are split at a 1:5 to 1:10 dilution, they will generally reach 80-90% confluence in 3-4 days.
Generation of Flp-In Expression Cell Lines
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Maximum size of insert that can be used
Selection with Hygromycin
Transfection protocol for Flp-In Jurkat cells
Transfection protocol for Flp-In -CHO cells
Efficiency of Flp-In integration versus random integration
Stability of Flp-In cell lines containing the gene of interest
Maximum size of insert that can be used:
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(back to Generation of Flp-In Expression Cell Lines
We don鈥檛 believe that the Flp-In system has any size limitation with just a single recombination event. There have been
reports of BAC DNA (>150 kb) being successfully Flp-ed-In with no problem. In theory it should be possible to insert
two genes with an IRES sequence in between although this has not been tested in-house.
Selection with Hygromycin
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Since integration at the FRT site should disrupt lacZ, in theory, all hygromycin-resistant foci should be white
instead of blue after beta-gal staining. However, blue foci have been observed; they usually disappear after
longer incubations with hygromycin. Either the foci picked are not really from a single cell or the lacZ-Zeo is a
very stable protein and it is still around during the selection.
In some of our Flp-In cell lines (esp. CHO and 293) we often see some residual lacZ activity after the gene of interest
has integrated into the FRT site. Since the FRT sites are at very transcriptionally active locations in the genome, there
may be some residual expression of LacZ from this locus.
Transfection protocol for Flp-In Jurkat cells:
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Cell density should be ~8 x 105 per ml
Use 1.5 ml per well of 6-well plate
Cells should be healthy and actively growing for efficient transfection.
To each well to be transfected:
1. Dilute 8 ul Lipofectamine 2000 in 250 ul OptiMEM, incubate 5 min. room temp
2. Dilute 3 ug total DNA (1:9 ratio of pEF5/FRT/GOI:pOG44) in 250 ul OptiMEM
3. Combine diluted lipid with diluted DNA (500 ul final), mix gently (no vortexing), incubate 20 min. room temp
4. Add lipid:DNA mixture dropwise to cells (final volume now 2 ml), swirl plate gently to mix
5. Incubate 24 hours (37 degrees C, 5% CO2)
6. Change media to fresh media
7. Incubate 24 hours
8. Add hygromycin 200 ug/ml final to select
Selection will take approximately 2 weeks; cells will need to be passaged into fresh hygromycin media every couple
days or so (make sure the cells are healthy, not too sparse or they slow way down) and be sure to run a control plate
(pEF5/FRT/GOI transfected without pOG44) and a mock transfected plate so you can see when the selection is
complete.
Transfection protocol information for Flp-In -CHO cells:
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The Flp-In CHO cell line is unique in that the FRT site is located in a very transcriptionally active region of the
chromosome. Others and we have observed a low level of lacZeo expression post-Flp-In, which means that
there is a potential to obtain both Hygromycin and Zeocin resistant clones.
Numerous Southern blots have been performed to verify a single FRT site to obtain a single copy of the GOI
post-Flp-In, so we hypothesize that there is some type of "read-thru" transcription to the lacZeo ORF post-Flp-
In that generates some lacZ staining and may also yield zeocin resistance. Hence we believe that the typical
blue-to-white switch following Flp-In is not indicative of a successful Flp-In in the Flp-In CHO cells.
Control transfections especially one without pOG44 are important to be certain that the only surviving colonies
are ones in which the FRT construct has integrated at the FRT site
CHO cells do require high levels of hygromycin to complete the selection. But more importantly, they must be
kept at low cell densities through the entire selection process or they will not die. If they are confluent, they
will not be affected by hygromycin at all. Hygromycin concentration of 500-600 ug/ml should be sufficient.
These cells transfect poorly when using the calcium phosphate precipitation method. We recommend using
lipid-mediated transfection to introduce the pcDNA5/FRT-based construct containing your gene of interest into
Flp-In -CHO cells. We routinely use LIPOFECTAMINE 2000 Reagent available from Invitrogen (Catalog no.
11668-019) to transfect Flp-In -CHO cells and typically achieve 80% or better transient transfection. Stable
Flp-In efficiencies are similar to making a traditional stable cell line with hygromycin and range from 1% to
10%.
CHO cells are not very sensitive to hygromycin; we typically need to select with hygromycin for 2 weeks or
more before picking resistant colonies.
It is extremely important that both "mock"-transfected and FRT construct transfection without pOG44
followed by hygromycin selection must be done in parallel to the Flp-In transfection so that you know when
the selection is complete. Many surviving cells may be observed on these control plates after just one week of
selection in hygromycin. There should be no survivors in >600 ug/ml.
We typically plate cells at approximately 20% confluency directly into media with hygromycin as opposed to
letting them set down for one day first. This tends to really affect the untransfected cells fast and doesn't give
them a chance to up-regulate the multi-drug resistance genes (MDR genes) that allow them to survive in
hygromycin.
To keep the cells at low density, here are some suggestions. Selection should be complete within 3 weeks:
Day 1: Transfect cells with pOG44 and the Flp-In expression vector (9:1 ratio) in one well of 6-well plate (be
sure to include untransfected controls)
Day 2: Feed cells with fresh media
Day 3: Trypsinize each well and transfer the entire well into a 100 mm plate (10 cm plate) each in the
presence of hygromycin. Final volume in the plate should be 10 ml with final hygromycin concentration of
500 ug/ml. Having hygromycin around with the freshly trypsinized cells significantly helps the process. By
moving to a larger plate the cells get spread apart so that hygromycin selection is optimal.
Following days: Aspirate old media and replace with fresh media containing 500 ug/ml hygromycin every 2-3
days. Untransfected cells should be dead within 2 weeks or so. Cells in the transfected plate should form
easily detectable colonies during this time.
Efficiency of Flp-In integration versus random integration
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The efficiency of Flp-In targeted integration compared to random integration when generating stable cell lines
depends on several factors including transfection efficiency. The exact percentage of transfected cells that will
go through a flp-induced recombination event is not known. It is known that the efficiency of flp-induced
recombination is approximately 100-fold more efficient than random integration. Depending on different
factors such as cell type the number can range from 10-1000 more efficient.
Using a pcDNA-based vector we routinely get 30-100 drug-resistant colonies when we generate stable clones
in NIH3T3 cells with good transfection efficiemcy. So for the Flp-In, you could expect to see 500-10,000
resistant colonies. This is only a general ball park figure, and these numbers are based purely on extrapolation
and not direct experiments. We typically observe 50-100 and sometimes more, Flp-In expression stables when
transfecting 5x105 Flp-In 293 cells.
We have done some side-by-side comparisons in CV-1 cells of a regular stable transfection (pcDNA5/CAT)
vs. a Flp-dependent transfection (pcDNA5/FRT/CAT). In both cases we get about the same number of
hygromycin resistant colonies. Comparisons of the individual clones indicated that the regular stable
transfection showed gene expression in only 2 out of 19 clones picked and the expression was very low. On
the other hand the Flp-dependent transfection showed gene expression in all 19 out of 19 clones with equal
levels of expression in every clone.
Using pcDNA5/FRTduring transfection to generate Flp-In expression cell lines can, in theory, produce the Flp-
In-recombinant as well as a second site integrant. However since the flp integration is more efficient than than
random integration, this is not a common event. Limiting the amount of DNA in the transfection should lessen
the chance of second site insertions. Since 1 million cells are being transfected with 1 ug of pcDNA5/FRT (in
combination with pOG44), the most common event should be integration at the FRT site. However, second site
integration could happen and can be tested by transfecting the expression cell line with pOG44 and selecting
back on Zeocin (effectively removing the Flp-In-recombined plasmid). If Zeocin resistant clones are now
expressing the gene of interest then there is a second site integration. We have identified one potential second
site integrant on screening over 200 clones.
Random integration of the pcDNA5/FRT vector is generally not a concern. This is because random integrants
of the expression vector will not restore function of the hygromycin resistance gene. Recombination between
the FRT sites in the genome and on the Flp-In expression vector place the SV40 promoter and ATG in line
with the hygromycin resistance gene, ensuring the generation of isogenic cells.
Stability of Flp-In cell lines containing the gene of interest
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In house, a polyclonal (pooled foci) as well as a single foci clone containing both pFRT/lacZeo with
pcDNA5/FRT/CAT flipped in has been maintained for 6 months wherin expression from the CAT gene can
still be detected.
The CAT protein expressed from the pcDNA5/FRT/CAT control plasmid is approximately 32 kDa in size.
CAT expression can be assayed by ELISA, western blot analysis, fluorometric, or radioactive assays.
A Commercial kit (FAST CAT Chloramphenicol Acetyltransferase Assay Kit) to assay for CAT protein is
available from Invitrogen (Catalog no. F-2900).
Expression in Flp-In Cell lines
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Use of Tetracycline-Tested Gibco FBS in Flp-in T-Rex expression system
Qualitative and quantitative data to demonstrate that isolated foci from transfection into Flp-in cell lines will
express a reporter at near equivalent levels
Comparison of reporter activities between different Flp-In cell lines
Background bands on Western Blots with the anti-V5 Antibody
Use of Tetracycline-Tested GIBCO FBS in Flp-in T-Rex expression system
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When culturing cells in medium containing fetal bovine serum (FBS), please note that almost all lots of FBS
contain tetracycline. This is due to the fact that FBS is generally isolated from cows that have been fed a diet
containing tetracycline. If cells are cultured in medium containing FBS that is not reduced in tetracycline, there
may be low basal expression of the gene of interest in the absence of tetracycline.
We have cultured our mammalian cells in medium containing FBS that may not be reduced in tetracycline, and
have observed undetectable to very low basal expression of beta galactosidase from the positive control vector in
the absence of additional tetracycline. If the gene of interest produces a toxic protein, consider tetracycline-reduced
or tetracycline-free FBS.
Invitrogen scientists have developed and validated an assay using reverse-phase HPLC with fluorescence for
detecting the presence of tetracycline in FBS. Using this assay, as little as 19.7 ng/ml of tetracycline can be
detected (Quest, Vol 2 (2), Pg 29, 2005).
Binding constant for Tet-repressor protein with tetracycline is 3nM. If tetracycline is at or just below 19.7 ng/ml in
serum (our detection limit), that is equivalent to 4nM when serum concentration is 10% in media. That would
easily give basal expression if the expression assay is sensitive enough. If it is above 19.7 ng/ml (because it wasn鈥檛
tested) it is pretty likely that basal expression will be detected.
GIBCO Certified and Qualified FBS lots are regularly tested for tetracycline (Quest, Vol 2 (2), Pg 29, 2005). When
using Invitrogen鈥檚 Flp-in T-Rex system, one may request a lot of tetracycline-reduced Certified or Qualified FBS
from GIBCO. To be tetracycline reduced, these lots have to have below 19.7ng/ml tetracycline (the assay detection
limit). Even so, because of binding constant and limits of detection, it is still possible to get basal level expression
from tetracycline-reduced FBS.
Qualitative and quantitative data to demonstrate that isolated foci from transfection into Flp-in cell lines will
express a reporter at near equivalent levels
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(back to Expression in Flp-In Cell lines)
Figure 1: The Flp-In 293 Cell Line was transfected with pcDNA5/FRT/CAT and pOG44. Cells were selected with 500
ug/ml of hygromycin and individual foci expanded. Cells were harvested and 20 ug of cell lysate was loaded onto a Novex
gel, transferred to nitrocellulose, and detected with anti-V5 antibody. M indicates mock transfection. Lanes 1-7 correspond
to individual Flp-In clones
Figure 2: Flp-In Jurkat cells were co-transfected with pEF5/FRT/V5/CAT and pOG44. Cells were selected with 200 ug/ml
of hygromycin and individual foci expanded. Seven individual clones (lanes 2-8) were selected for western blot analysis
using the anti-V5 antibody.
Lane 1 is Mock-transfected Flp-In Jurkat control. The CAT-V5 protein is indicated.
The * band is a background V5 band present in Jurkat cells, and can serve as a loading control.
Flp-In CHO luciferase clones
100000
90000
80000
70000
60000
RLU/碌g
50000
40000
30000
20000
10000
0
M 1 2 3 4 5 6 7 8 9 10 11 12
CLONE #
Figure 3: Flp-In CHO cells were co-transfected with pcDNA5/FRT/luc + pOG44 (ratio 1:9) and cells were selected
with 600 碌g/ml hygromycin for ~3 weeks. Twelve individual clones were picked and luciferase activity was measured
(in duplicate) for each clone and plotted as relative light units (RLU) per microgram total protein.
Comparison of reporter activities between different Flp-In cell lines
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Our in-house experiments examining expression of the Luciferase gene in Flp-In-CHO, -293, and 鈥揅V1 cells
demonstrated that the Flp-In-CHO and -293 showed similar luciferase expression, but Flp-In-CV-1 had
expression that was about 20-fold lower. This is not at all surprising for the following reasons:
1. They are radically different cell types.
2. The pFRT/lacZeo has been integrated at different locations in the chromosomes of each of these cell
types. The Flp-In CHO were purposely selected for integration at a transcriptionally hot chromosomal
location, so you would expect high lacZ expression. The 293 cells are well known for their ability to
transactivate promoters, so again high lacZ expression is expected.
Other than the information mentioned above, Invitrogen R&D has never made comparisons between different
cell lines for any other reporter including GFP or B-gal. We believe that data from these comparisons does not
necessarily provide useful information. There are several reasons for this namely the difference in gene
promoters, half-lives of proteins, and the integration site of the pFRT/laxZeo, thus making it difficult to make
valid comparisons between cell types. For example, the CMV promoter has different activity in, for example,
293 cells vs. HeLa cells vs. CV-1 cells. The CMV promoter is typically much stronger in 293 cells due to the
endogenous expression of adenovirus E1A. Also, the initial site of integration of the pFRT/lacZeo in a cell
line can give different levels of expression in each clone. When we create a Flp-In cell line here, we look at a
variety of clones for: a) single integration site and b) maximum expression of lacZeo. So even within one cell
type a variety of expression levels can be observed. However once a single clone is chosen, the expression
levels of the gene of interest will always be the same.
Background bands on Western Blots with the anti-V5 Antibody
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Occasionally one or two background bands whose identity is not known are observed on Western Blots with the anti-V5
antibody that typically run ~65 kD. We believe that this observation is not specific to the Flp-In cells but is dependent
on how long the blot was exposed to film. High-level transient expressions (e.g. CMV in 293 cells) typically only need
a few seconds of exposure to film for detection of the V5 protein. In stable cells or Flp-In cell lines, where only one
copy of the gene is expressed, the western blots need to be exposed for longer times (sometimes up to 3 to 5 minutes)
and this is when the background band(s) becomes visible. It is strongly recommended to run an untransfected control to
be certain of the real band and the background bands.
Custom Flp-In Host, and Expression Cell Lines Service
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Flp-In Host Cell Line Service: For customers who need their favorite cell line transformed into a Flp-In Host Cell
Line, so as to stably express the GOI in it.
Flp-In Expression Cell Line Service: For customers who need an Flp-In Expression cell line expressing their GOI(s)
in an existing Flp-In Host Cell Line (off the shelf or created using the Flp-In Host Cell Line Service).
For additional information contact Custom Services at:
Email: custom.services@invitrogen.com
Telephone: 800 955 6288 x45682
PRODUCT DOCUMENTATION
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Brochures Citations Cell lines
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Manuals MSDS Newsletters
Vector Data
PRODUCT NAME AND CATALOG NUMBERS
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Name Size Part Number Catalog Number
Constitutive
Expression
Flp-in pcDNA5/FRT 1 Kit N/A K601001
Complete Kit
Flp-in pcDNA5/FRT 1 Kit 440310 K601002
Core Kit
pEF5/FRT/V5 20 rxns N/A K603501
Directional TOPO
Expression Kit
pEF5/FRT/V5-DEST 6 ug 430110 V602020
Gateway Vector Kit
pcDNA5/FRT/V5-His 20 rxns N/A K602001
TOPO TA Expression
Ki
Kit
pSecTag/FRT/V5-His 20 rxns N/A K602501
TOPO TA Expression
Kit
pcDNA5/FRT 20 ug 430314 V601020
pFRT/lacZeo 20 ug 353020 V601520
pFRT/lacZeo2 20 ug 351189 V602220
pOG44 20 ug 353018 V600520
Inducible Expression
Flp-In T-REx Core Kit 1 Kit N/A K650001
pcDNA5/FRT/TO- 20 rxn N/A K651020
TOPO TA Expression
Kit
pcDNA5/FRT/TO-E 1 Kit N/A ET47001
Echo-Adapted
Expression Vector Kit
pcDNA5/FRT/TO 20 ug 430313 V652020
pcDNA6/TR 20 ug 351592 V102520
Cell Lines
3 x 106 cells
Flp-In 293 510021 R75007
3 x 106 cells
Flp-In CHO 510025 R75807
3 x 106 cells
Flp-In CV-1 510022 R75207
3 x 106 cells
Flp-In BHK 510032 R76007
3 x 106 cells
Flp-In 3T3 510033 R76107
3 x 106 cells
Flp-In Jurkat 510034 R76207
3 x 106 cells
Flp-In T-REx- 293 Cell 510026 R78007
Line
COMPONENTS
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Flp-in pcDNA5/FRT Complete Kit (K601001)
Name Size Part Number Catalog Number
Flp-In Core Kit 1 Kit 440310 K601002
Zeocin 1 gm 450430 R25001
Hygromycin 20 ml 10687010 10687010
Flp-in pcDNA5/FRT Core (K601001; part no. 440310)
Name Size Part Number Catalog Number
pcDNA5/FRT 20 ug 353014 V601020
pcDNA5/FRT/CAT 20 ug 353016 N/A
pOG44 20 ug 353018 V600520
pFRT/lacZeo 20 ug 353020 V601520
CMV FWD Primer 2 ug 400115 N/A
BGH REV Primer 2 ug 400226 N/A
pEF5/FRT/V5 Directional TOPO Expression Kit (K603501)
Name Size Part Number Catalog Number
pEF5/FRT/V5 D- 20 rxn 450226 N/A
TOPO
One-shot TOP10 20 rxn 440301 C404003
chemically competent
E.coli
pcDNA5/FRT/V5-His TOPO TA Expression Kit (K602001)
Name Size Part Number Catalog Number
pcDNA5/FRT/V5-His- 20 rxn 450093 N/A
TOPO TA
One-shot TOP10 20 rxn 440301 C404003
chemically competent
E.coli
pSecTag/FRT/V5-His TOPO TA Expression Kit (K602501)
Name Size Part Number Catalog Number
pSecTag/FRT/V5-His- 20 rxn 450094 N/A
TOPO TA
One-shot TOP10 20 rxn 440301 C404003
chemically competent
E.coli
Flp-In T-REx Core Kit (K650001, part no 440311)
Name Size Part Number Catalog Number
Tetracycline 5 gm 550205 N/A
pcDNA5/FRT/TO 20 ug 351191 V652020
pcDNA5/FRT/TO/CAT 20 ug 351193 N/A
pcDNA6/TR 20 ug 351592 V102520
pOG44 20 ug 353018 V600520
pFRT/lacZeo 20 ug 353020 V601520
CMV FWD Primer 2 ug 400115 N/A
BGH REV Primer 2 ug 400226 N/A
pcDNA5/FRT/TO-TOPO TA Expression Kit (K651020)
Name Size Part Number Catalog Number
pcDNA5/FRT/TO 20 rxn 450143 N/A
TOPO-TA
One-shot TOP10 20 rxn 440301 C404003
chemically competent
E.coli
Product Amount Catalog
no.
Lipofectamine 2000 0.75 ml 11668027
1.5 ml 11668019
Calcium Phosphate Transfection Kit 75 reactions K278001
CAT antiserum 50 ul R90225
FAST CAT Chloramphenicol Acetyltransferase 1 Kit F2900
Assay Kit
Geneticin 1 gm 11811023
5 gm 11811031
Zeocin 1 gm R25001
5 gm R25005
Hygromycin 1g R22005
Blasticidin 50 mg R21001
Beta-gal Staining Kit 1 Kit K146501
Beta-gal Assay Kit 100 reactions K145501
Dulbecco鈥檚 Modified Eagle Medium (DMEM) 500 ml 11965092
Ham鈥檚 F-12 500 ml 11765054
RPMI Medium 1640 500 ml 11875093
Fetal Bovine Serum 500 ml 16000044
Donor Calf Serum 500 ml 16030074
200 mM L-Glutamine 100 ml 25030081
Penicillin-Streptomycin 100 ml 15070063
Trypsin-EDTA 100 ml 25300054
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