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Overview
Flp Recombinase-Mediated DNA Recombination
Introduction
The following section contains guidelines for maintaining and propagating the pFRT/lacZeo, pcDNA6/TR, and pOG44 vectors. For information about maintaining and propagating the pcDNA5/FRT/TO expression vector, refer to the vector manual.
General Molecular Biology Techniques
For assistance with E. coli transformations, restriction enzyme analysis, DNA biochemistry, and plasmid preparation, refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).
E. coli Strain
Many E. coli strains are suitable for the propagation of the pFRT/lacZeo, pcDNA6/TR, and pOG44 vectors. We recommend that you propagate the vectors in E. coli strains that are recombination deficient (recA) and endonuclease A deficient (endA). For your convenience, TOP10 and DH5a™-T1R E. coli are available as chemically competent or electrocompetent (TOP10 only) cells.
Transformation Method
You may use any method of choice for transformation. Chemical transformation is the most convenient for many researchers. Electroporation is the most efficient and the method of choice for large plasmids.
Maintenance of Plasmids
The pFRT/lacZeo, pcDNA6/TR, and pOG44 vectors contain the ampicillin gene to allow selection of the plasmid using ampicillin.
Note: The pcDNA6/TR plasmid also contains the blasticidin resistance gene to allow selection using blasticidin.
To propagate and maintain the pFRT/lacZeo, pcDNA6/TR, and pOG44 plasmids, we recommend using the following procedure:
Selection of pcDNA6/TR in E. coli Using Blasticidin
To propagate and maintain the pcDNA6/TR plasmid in E. coli using blasticidin selection, follow Steps 1 and 2 of the protocol above. Select transformants on Low Salt LB agar plates containing 100 µg/ml blasticidin.
Note: To facilitate selection of blasticidin-resistant E. coli, the salt concentration of the LB medium must remain low (< 90 mM) and the pH must be 7.0. Failure to lower the salt content of your LB medium will result in non-selection due to inhibition of the drug unless a higher concentration of blasticidin is used.
Preparing a Glycerol Stock
Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long-term storage. It is also a good idea to keep a DNA stock of your plasmid at –20°C.
Introduction
Before you can create a stable Flp-In™ T-REx™ cell line(s) which inducibly expresses your gene of interest, you will first need to generate a stable Flp-In™ T-REx™ host cell line. You will generate the Flp-In™ T-REx™ host cell line by independently transfecting the pFRT/lacZeo and pcDNA6/TR plasmids into your mammalian cell line of interest. Once generated, the Flp-In™ T-REx™ host cell line will exhibit the following features:
Guidelines to generate a Flp-In™ T-REx™ host cell line are provided in this section.
Experimental Outline
The table below outlines the steps necessary to generate a Flp-In™ T-REx™ host cell line. While it is possible to cotransfect pFRT/lacZeo and pcDNA6/TR, we recommend that you first generate a stable cell line containing a single integrated FRT site (from pFRT/lacZeo), and then use this cell line as the host for the pcDNA6/TR plasmid.
Step | Action |
1
|
Transfect the pFRT/lacZeo target site vector into the mammalian cell line of choice and select for Zeocin™-resistant transfectants.
|
2
|
Pick 20 Zeocin™-resistant foci and expand each clone.
|
3
|
Isolate genomic DNA and use Southern blot analysis to test for the number of integrated FRT sites.
|
4
|
Select the single integrants and screen for ß-galactosidase activity.
|
5
|
Select the clone which exhibits the highest ß-galactosidase activity and use this clone as the host for the pcDNA6/TR plasmid.
|
6
|
Transfect the pcDNA6/TR plasmid into the host cell line containing the single integrated FRT site (see Step 5). Select for blasticidin-resistant transfectants.
|
7 | Pick 20 blasticidin-resistant foci and screen for the clone which expresses the highest level of Tet repressor. |
Reminder: Once you have generated your Flp-In™ T-REx™ host cell line, the cells should exhibit the following phenotypes:
The Flp-In™ T-REx™-293 host cell line which contains a single integrated FRT site and stably expresses the Tet repressor is available from Thermo Fisher Scientific. If you wish to inducibly express your gene of interest in 293 cells, you may want to use this cell line as the host and proceed directly to generate your stable expression cell line. Alternatively, several Flp-In™ host cell lines which contain a single integrated FRT site are also available. You may transfect the pcDNA6/TR plasmid into these cell lines to generate Flp-In™ T-REx™ host cell lines.
For more information about the Flp-In™ T-REx™-293 cell line or the Flp-In™ cell lines, refer to our website or call Technical Service.
We have observed down-regulation of the viral CMV promoter and subsequent loss of gene expression when pcDNA5/FRT-based expression constructs are introduced into Flp-In™-3T3 or Flp-In™-BHK cells. We recommend that you DO NOT use 3T3 or BHK cells to generate your Flp-In™ T-REx™ host cell line.
Plasmid Preparation
Plasmid DNA for transfection into eukaryotic cells must be very clean and free from phenol and sodium chloride. Contaminants will kill the cells, and salt will interfere with lipids complexing, decreasing transfection efficiency. We recommend isolating DNA using the S.N.A.P.™ MiniPrep Kit (10-15 mg DNA, Catalog No. K1900-01), the S.N.A.P.™ MidiPrep Kit (10-200 mg DNA, Catalog No. K1910-01) or CsCl gradient centrifugation.
Methods of Transfection
For established cell lines (e.g. HeLa, COS-1), consult original references or the supplier of your cell line for the optimal method of transfection. We recommend that you follow exactly the protocol for your cell line. Pay particular attention to medium requirements, when to pass the cells, and at what dilution to split the cells. Further information is provided in Current Protocols in Molecular Biology (Ausubel et al., 1994). Methods for transfection include calcium phosphate (Chen and Okayama, 1987; Wigler et al., 1977), lipid-mediated (Felgner et al., 1989; Felgner and Ringold, 1989) and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988). We offer the Calcium Phosphate Transfection Kit (Catalog no. K2780-01) and several lipid-based reagents for mammalian cell transfection. For more information on the reagents available, see our website or call Technical Service.
Zeocin™
The pFRT/lacZeo plasmid contains a lacZ-Zeocin™ fusion gene under the control of the SV40 early promoter. Expression of the lacZ-Zeocin™ fusion gene allows selection of stable integrants using Zeocin™ antibiotic. The resulting stable integrants can then be screened by assaying for expression of ß-galactosidase.
Determination of Zeocin™ Sensitivity
To successfully generate a stable cell line containing an integrated FRT site and expressing the ß-galactosidase-Zeocin™ fusion protein, you need to determine the minimum concentration of Zeocin™ required to kill your untransfected mammalian cell line. Typically, concentrations ranging from 50 to 1000 µg/ml Zeocin™ are sufficient to kill most untransfected mammalian cell lines, with the average being 100 to 400 µg/ml. We recommend that you test a range of concentrations (see protocol below) to ensure that you determine the minimum concentration necessary for your cell line. Plate or split a confluent plate so the cells will be approximately 25% confluent. Prepare a set of 7 plates. Allow cells to adhere overnight.
Effect of Zeocin™ on Sensitive and Resistant Cells
Zeocin™'s method of killing is quite different from other antibiotics including hygromycin, G418, and Blasticidin. Cells do not round up and detach from the plate. Sensitive cells may exhibit the following morphological changes upon exposure to Zeocin™:
Eventually, these "cells" will completely break down and only "strings" of protein remain. Zeocin™-resistant cells should continue to divide at regular intervals to form distinct colonies. There should not be any distinct morphological changes in Zeocin™-resistant cells when compared to cells not under selection with Zeocin™.
Transfection Considerations
Once you have determined the appropriate Zeocin™ concentration to use for selection, you are ready to transfect the pFRT/lacZeo plasmid into your mammalian cell line of choice. Before beginning, you will need to consider the following factors:
ß-galactosidase should contain single FRT sites which have integrated into the most transcriptionally active regions.
Because transfection efficiency is dependent upon the nature of your cell line and the amount of DNA transfected, it is possible to generate a cell line containing multiple integrated FRT sites. In theory, cotransfection of your pcDNA5/FRT/TO construct and pOG44 into these cells will allow integration of your gene of interest into multiple genomic loci. We do not recommend generating a Flp-In™ T-REx™ host cell line which contains multiple integrated FRT sites for the following reasons:
Recommendation
As mentioned previously, we recommend that you transfect your mammalian cell line with a limiting amount of pFRT/lacZeo plasmid. We generally use 250 ng to 2 µg of plasmid DNA per 4 x 106 cells for transfection, but the amount of plasmid DNA may vary due to the nature of the cell line, the transfection efficiency of your cells, and the method of transfection used. When transfecting your 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 µg/µl DNA) to optimize transfection conditions for your cell line.
We generally use electroporation to transfect cells, but other methods of transfection are suitable. For a protocol to electroporate cells, refer to Current Protocols in Molecular Biology, Unit 9.3 (Ausubel et al., 1994). Note that if you use calcium phosphate or lipid-mediated transfection methods, the amount of total DNA required for transfection is typically higher than for electroporation (usually between 10 and 20 mg DNA). Depending on the amount of pFRT/lacZeo plasmid that you use for transfection, you may need to supplement your plasmid DNA with carrier DNA (e.g. salmon sperm DNA).
Possible Sites for Linearization of pFRT/lacZeo
To obtain stable transfectants, we recommend that you linearize the pFRT/lacZeo plasmid before transfection. While linearizing the vector may not improve the efficiency of transfection, it increases the chances that the vector does not integrate in a way that disrupts the ATG-FRT-lacZ-Zeocin™ cassette or other elements necessary for expression in mammalian cells. The table below lists unique sites that may be used to linearize your construct prior to transfection. Other restriction sites are possible.
Note: We generally use Sca I to linearize pFRT/lacZeo.
Enzyme | Restriction Site (bp) | Location | Supplier | |
Tth111 I
|
125
|
Backbone
|
Many
| |
Apa I
|
5617
|
Backbone
|
Thermo Fisher Scientific
(Catalog No. 15440-019)
| |
Swa I
|
6075
|
Backbone
|
New England Biolabs, Sigma, Takara
| |
Xmn I
|
6487
|
Ampicillin gene
|
Many
| |
ScaI I
|
6606
|
Ampicillin gene
|
Thermo Fisher Scientific
(Catalog No. 15436-017)
| |
Bsa I
|
7021
|
Ampicillin gene
|
New England Biolabs
| |
Eam1105 I
|
7087
|
Ampicillin gene
|
AGS
*, Fermentas, Takara
| |
Sap I
|
8092
|
Backbone
|
New England Biolabs
|
*Angewandte Gentechnologie Systeme
Selection of Stable pFRT/lacZeo Integrants
Once you have determined the appropriate Zeocin™ concentration to use for selection, you can generate a stable cell line with pFRT/lacZeo.
Isolation of Genomic DNA
Once you have obtained Zeocin™-resistant foci, you will need to expand the cells and isolate genomic DNA. You may use any standard protocol to isolate genomic DNA from your cells. Protocols may be found in Current Protocols in Molecular Biology (Ausubel et al., 1994) or Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989). For easy isolation of genomic DNA, the Easy-DNA™ Kit (Catalog No. K1800-01) is available. Call Technical Service for more information.
Screening Clones by Southern Blot Analysis
You can use Southern blot analysis to determine the number of integrated FRT sites present in each of your Zeocin™-resistant clones. When performing Southern blot analysis, you should consider the following factors:
Introduction
Once you have established your Flp-In™ T-REx™ host cell line, you may cotransfect your pcDNA5/FRT/TO construct and the pOG44 expression plasmid into the host cell line to generate a stable Flp-In™ T-REx™ expression cell line which inducibly expresses your gene of interest. Integration of the pcDNA5/FRT/TO construct into the genome will occur at the FRT site in the Flp-In™ T-REx™ host cells. The pcDNA5/FRT/TO plasmid contains the hygromycin resistance gene to allow selection of stable cell lines (see below). For more information about the pcDNA5/FRT/TO plasmid and cloning your gene of interest into pcDNA5/FRT/TO, refer to the vector manual. For more information about the pOG44 plasmid, see below.
The hygromycin resistance gene in the pcDNA5/FRT/TO vector lacks an ATG initiation codon and a promoter to drive expression of the gene. Transfection of pcDNA5/FRT/TO plasmid alone into a Flp-In™ T-REx™ host cell line will not confer hygromycin resistance to the cells containing the plasmid. The ATG initiation codon and the SV40 promoter required for expression of the hygromycin resistance gene are brought into proximity and frame with the gene only through Flp recombinase-mediated recombination between the FRT sites in the pcDNA5/FRT/TO plasmid and the Flp-In™ T-REx™ host cell line.
If you wish to express your gene of interest in 293 cells, you may want to use the Flp-In™ T-REx™-293 host cell line to establish your expression cell line. For more information, refer to our website or call Technical Service.
It is also possible to cotransfect pcDNA5/FRT/TO and pOG44 into a Flp-In™ host cell line to generate an expression cell line. Flp-In™ host cell lines contain a single integrated FRT site, but do not express the Tet repressor. Cotransfection of pcDNA5/FRT/TO and pOG44 into a Flp-In™ host cell line would allow integration of the pcDNA5/FRT/TO construct into the genome via the FRT sites. However, in this case, the TetO2 sequences in the hybrid CMV/TetO2 promoter of pcDNA5/FRT/TO are inert and the CMV/TetO2 promoter functions to allow constitutive expression of your gene of interest at levels similar to the native CMV promoter.
pOG44 Plasmid
You will cotransfect the pOG44 plasmid and your pcDNA5/FRT/TO construct into your Flp-In™ T-REx™ host cell line to generate stable cell lines that inducibly express your protein of interest. Cotransfection of pOG44 and pcDNA5/FRT/TO allows expression of Flp recombinase and integration of the pcDNA5/FRT/TO plasmid into the genome via the FRT sites. Once the pcDNA5/FRT/TO 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 your pcDNA5/FRT/TO construct. The pOG44 plasmid lacks an antibiotic resistance marker for selection in mammalian cells. Thus, the plasmid and therefore, Flp recombinase expression, will gradually be lost from transfected cells as they are cultured and selected in hygromycin.
Flp Recombinase
The FLP gene was originally isolated from the Saccharomyces cerevisiae 2m plasmid (Broach et al., 1982; Broach and Hicks, 1980). When tested in mammalian cells, the Flp recombinase has been shown to possess optimum recombination activity near 30°C and relatively low activity at 37°C, a result consistent with its physiological role in yeast (Buchholz et al., 1996). The FLP gene in pOG44 is further limited in its activity because it contains a point mutation that encodes a Flp recombinase with a phenylalanine to leucine amino acid substitution at position 70 (Buchholz et al., 1996). The resulting Flp recombinase (flp-F70L) exhibits increased thermolability at 37°C in mammalian cells when compared to the native Flp recombinase (Buchholz et al., 1996). Studies have shown that the Flp recombinase expressed from pOG44 possesses only 10% of the activity at 37°C of the native Flp recombinase (Buchholz et al., 1996).
When generating Flp-In™ T-REx™ expression cell lines, it is important to remember that you are selecting for a relatively rare recombination event since you want recombination and integration of your pcDNA5/FRT/TO construct to occur only through the FRT site and for a limited time. In this case, using a highly inefficient Flp recombinase is beneficial and may decrease the occurrence of other undesirable recombination events.
Reminder: Integration of the pcDNA5/FRT/TO construct into the genome via the FRT sites will result in the following events:
As a result, your Flp-In™ T-REx™ expression cell lines should exhibit the following phenotype:
Positive Control
The pcDNA5/FRT/TO/CAT plasmid is provided as a positive control vector for mammalian cell transfection and expression and may be used to assay for expression levels in your Flp-In™ T-REx™ expression cell line. If you have several different Flp-In™
T-REx™ host cell lines (cell lines containing FRT sites integrated at different genomic loci), you may want to use the pcDNA5/FRT/TO/CAT control vector to compare protein expression levels from the various genomic loci.
For more information about pcDNA5/FRT/TO/CAT, refer to the pcDNA5/FRT/TO vector manual.
Hygromycin B
The pcDNA5/FRT/TO vector contains the E. coli hygromycin resistance gene (HPH) (Gritz and Davies, 1983) for selection of transfectants with the antibiotic, hygromycin B (Palmer et al., 1987). When added to cultured mammalian cells, hygromycin B acts as an aminocyclitol to inhibit protein synthesis by disrupting translocation and promoting mistranslation. Hygromycin B liquid is available separately.
Preparing and Storing Hygromycin B
The hygromycin B available from Thermo Fisher Scientific is supplied as a 100 mg/ml stock solution in autoclaved, deionized water and is filter-sterilized. The solution is brown in color. The stability of hygromycin B is guaranteed for six months, if stored at +4°C. Medium containing hygromycin is stable for up to six weeks.
Determination of Hygromycin Sensitivity
To successfully generate a stable cell line expressing your gene of interest from pcDNA5/FRT/TO, you need to determine the minimum concentration of hygromycin B required to kill your untransfected Flp-In™ T-REx™ host cell line. Typically, concentrations ranging from 10 to 400 mg/ml hygromycin B are sufficient to kill most untransfected mammalian cell lines. We recommend that you test a range of concentrations (0, 10, 50, 100, 200, 400, 600 mg/ml hygromycin B) to ensure that you determine the minimum concentration necessary for your Flp-In™ T-REx™ host cell line.
Tetracycline
Tetracycline (MW = 444.4) is commonly used as a broad spectrum antibiotic and acts to inhibit translation by blocking polypeptide chain elongation in bacteria. In the Flp-In™ T-REx™ System, tetracycline is used as an inducing agent to induce transcription of the gene of interest from the pcDNA5/FRT/TO expression vector. Tetracycline induces transcription by binding to the Tet repressor homodimer and causing the repressor to undergo a conformational change that renders it unable to bind to the Tet operator. The association constant of tetracycline to the Tet repressor is 3 x 109 M-1 (Takahashi et al., 1991). Tetracycline is supplied with the Flp-In™ T-REx™ Core Kit, but may also be obtained separately.
Note: The concentrations of tetracycline used to induce gene expression in the Flp-In™ T-REx™ System are generally not high enough to be toxic to mammalian cells.
Tetracycline-Reduced Serum
When culturing cells in medium containing fetal bovine serum (FBS), note that many lots of FBS contain tetracycline as FBS is generally isolated from cows that have been fed a diet containing tetracycline. If you culture your cells in medium containing FBS that is not reduced in tetracycline, you may observe low basal expression of your 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 CAT from the positive control vector in the absence of additional tetracycline. If your gene of interest produces a toxic protein, you may wish to culture your cells in tetracycline-reduced FBS. For more information, consult the supplier of your serum.
Preparation of Tetracycline
To prepare tetracycline:
Recommendation
Because correct integration of your pcDNA5/FRT/TO construct into the genome is dependent on Flp recombinase, 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 have varied the ratio of pOG44 and pcDNA5/FRT/TO expression plasmid that we cotransfect into mammalian Flp-In™ T-REx™ host cells to optimize the recombination efficiency. We recommend that you cotransfect your Flp-In™ T-REx™ host cell line with a ratio of at least 9:1 (w/w) pOG44:pcDNA5/FRT/TO expression plasmid. 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™ T-REx™ host cell line, be sure to use supercoiled pOG44 and pcDNA5/FRT/TO plasmid DNA. Flp-mediated recombination between the FRT site on pcDNA5/FRT/TO and the integrated FRT site in the Flp-In™ T-REx™ host cell line will only occur if the pcDNA5/FRT/TO plasmid is circularized. The pOG44 plasmid should be circularized to minimize the possibility of the plasmid integrating into the genome.
Selection of Stable Flp-In™ T-REx™ Expression Cell Lines
Once you have determined the appropriate hygromycin concentration to use for selection in your Flp-In™ T-REx™ host cell line, you can generate a stable cell line which inducibly expresses your pcDNA5/FRT/TO construct. Reminder: Following cotransfection, your Flp-In™ T-REx™ expression clones should become sensitive to Zeocin™ therefore, your selection medium should not contain Zeocin™. Remember that your selection medium should contain blasticidin to select for the pcDNA6/TR plasmid.
Polyclonal Selection
If you use a single integrant as your Flp-In™ T-REx™ host cell line, all of the hygromycin-resistant foci that you obtain after cotransfection of pcDNA5/FRT/TO and pOG44 and selection with hygromycin should be isogenic (i.e. pcDNA5/FRT/TO should integrate into the same genomic locus in every clone, therefore, all clones should be identical). Having isogenic clones should allow you to perform “polyclonal” selection and screening of your hygromycin-resistant cells. If you wish, you do not need to pick and screen separate foci for expression of your protein of interest. After hygromycin selection, simply pool the foci and screen the entire population of cells for tetracycline-regulated expression of your protein of interest.
Induction of Gene Expression
Guidelines are provided below to induce expression of your protein of interest with tetracycline. Expression conditions may vary depending on the nature of your protein of interest and on the cell line; therefore, some empirical experimentation may be needed to determine the optimal conditions for inducible expression.
Optimization of Expression
You may want to vary the concentration of tetracycline (0.1 to 1 µg/ml) and time of exposure to tetracycline (8 to 48 hours) to optimize or modulate expression for your cell line.
Other Inducers
You may use doxycycline as an alternative inducing agent in the Flp-In™ T-REx™ System. Doxycycline is similar to tetracycline in its mechanism of action, and exhibits similar dose response and induction characteristics as tetracycline in the Flp-In™ T-REx™ System. Doxycycline has been shown to have a longer half-life than tetracycline (48 hours vs. 24 hours, respectively). Doxycycline may be obtained from Sigma (Cat. No. D9891).
Introduction
The Flp-In™ cell lines stably express the lacZ-Zeocin™ fusion gene and are designed for use with the Flp-In™ System (Catalog nos. K6010-01 and K6010-02). Each cell line contains a single integrated Flp Recombination Target (FRT) site from pFRT/lacZeo or pFRT/lacZeo2 as confirmed by Southern blot analysis. Please see below for information about the generation of the Flp-In™ cell lines. For more information about the Flp-In™ System and its components, please refer to the Flp-In™ System manual, visit our website, or call Technical Service. The Flp-In™ System manual is also available for downloading from our website.
Generation of Flp-In™ expression cell lines requires cotransfection of the Flp-In™ cell line with a Flp-In™ expression vector containing your gene of interest (e.g., pcDNA5/FRT-based vector) and the Flp recombinase expression plasmid, pOG44 (O'Gorman et al., 1991). Flp recombinase mediates insertion of your Flp-In™ expression construct into the genome at the integrated FRT site through site-specific DNA recombination (O'Gorman et al., 1991; Sauer, 1994). Stable cell lines expressing your gene of interest from the Flp-In™ expression vector can be generated by selection using hygromycin B. For more information about FRT sites and Flp recombinase-mediated DNA recombination, please refer to the Flp-In™ System manual.
Parental Cell Lines
The table below provides a brief description of the source of the parental cell line used to generate each Flp-In™ cell line. The parental cell lines were obtained from the American Type Culture Collection (ATCC). The ATCC number for each cell line is included. For further information about the parental cell lines, please refer to the ATCC Web site (www.atcc.org).
Cell Line | Source | ATCC Number | |
293
|
Human embryonic kidney (Graham
et al., 1977)
|
CRL-1573
| |
CV-1
|
African Green Monkey kidney (Kit
et al., 1965)
|
CCL-70
| |
CHO-K1
|
Chinese Hamster ovary (Kao and Puck, 1968)
|
CCL-61
|
Flp-In™-293 and Flp-In™-CV-1 Cell Lines
The Flp-In™-293 and Flp-In™-CV-1 cell lines contain a single integrated FRT site and stably express 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. The Flp-In™ cell lines should be maintained in medium containing Zeocin™. For more information about pFRT/lacZeo and pcDNA5/FRT/CAT, please refer to the Flp-In™ System manual.
Flp-In™-CHO Cell Line
The Flp-In™-CHO cell line contains a single integrated FRT site and stably expresses the lacZ-Zeocin™ fusion gene from the pFRT/lacZeo2 plasmid. Please note that pFRT/lacZeo2 contains a mutated SV40 early promoter (PSV40-D) which is severely abrogated in its activity. The SV40D early promoter in pFRT/lacZeo2 exhibits approximately 60-fold less activity than the wild-type SV40 early promoter in pFRT/lacZeo. Because of the minimal activity of the SV40D 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 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. The Flp-In™-CHO cell line should be maintained in medium containing Zeocin™ (see below). For more information about pFRT/lacZeo2 and the SV40D early promoter, please refer to the pFRT/lacZeo2 manual.
Media for Cell Lines
The table below provides the recommended complete medium, freezing medium, and antibiotic concentration required to maintain and culture each Flp-In™ cell line.
Cell Line | Complete Medium | [Antibiotic] | Freezing Medium | |
Flp-In
™-293
|
DMEM (high glucose)
10% FBS
2 mM L-glutamine
1% Pen-Strep (optional)
|
100 µg/ml Zeocin
™ |
45% complete medium
45% conditioned complete medium
10% DMSO
| |
Flp-In
™-CV-1
|
DMEM (high glucose)
10% FBS
2 mM L-glutamine
1% Pen-Strep (optional)
|
100 µg/ml Zeocin
™ |
45% complete medium
45% conditioned complete medium
10% DMSO
| |
Flp-In
™-CHO
|
Ham’s F12
10% FBS
2 mM L-glutamine
1% Pen-Strep (optional)
|
100 µg/ml Zeocin
™ |
45% complete medium
45% conditioned complete medium
10% DMSO
|
DMEM
Dulbecco’s Modified Eagle Medium (DMEM) is used to culture the Flp-In™-293 and Flp-In™-CV-1 cell lines and can be obtained from Thermo Fisher Scientific (Catalog No. 11965-092).
Ham’s F12
Ham’s F12 is used to culture the Flp-In™-CHO cell line and can be obtained from Thermo Fisher Scientific (Catalog No. 11765-054).
Pen-Strep
We recommend including 1% Penicillin-Streptomycin in the culture medium to prevent bacterial contamination. Penicillin-Streptomycin may be obtained from Thermo Fisher Scientific (Catalog No. 15070-063).
Important Guidelines
Methods: Culturing Flp-In™ Cell Lines
General Cell Handling
Please follow the guidelines below to successfully grow and maintain your cells.
Before Starting
Be sure to have the following solutions and supplies available:
Thawing Cells
The following protocol is designed to help you thaw cells to initiate cell culture. All cell lines are supplied in vials containing 3 x 106 cells in 1 ml of 45% complete medium, 45% conditioned complete medium, and 10% DMSO.
Passaging the Cells
Preparing Freezing Medium
Before freezing your cells, you will need to prepare freezing medium. Since the freezing medium contains conditioned complete medium, you will need to remember to remove and reserve conditioned medium from the cells prior to freezing. Conditioned medium is the medium in which cells have been growing. To obtain conditioned complete medium, perform one of the following steps below:
Freezing medium should be prepared fresh immediately before use.
Freezing the Cells
Before starting, label cryovials and prepare freezing medium (see above). Keep the freezing medium on ice.
Transfection
Transfection Methods
Flp-In™-293 cells and Flp-In™-CV-1 cells are generally amenable to transfection using standard methods including calcium phosphate precipitation (Chen and Okayama, 1987; Wigler et al., 1977), lipid-mediated transfection (Felgner et al., 1989; Felgner and Ringold, 1989), and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988). We typically use calcium phosphate precipitation to transfect Flp-In™-293 and Flp-In™-CV-1 cells. The Calcium Phosphate Transfection Kit (Catalog No. K2780-01) is available from Thermo Fisher Scientific for convenient mammalian cell transfection.
Note: Flp-In™-CHO cells transfect poorly when using the calcium phosphate preci-pitation 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 Thermo Fisher Scientific (Catalog No. 11668-019) to transfect Flp-In™-CHO cells.
Generation of Stable Expression Cell Lines
Stable Flp-In™ expression cell lines can be generated by cotransfection of your Flp-In™ expression construct and the pOG44 plasmid. Stable transfectants are selected using hygromycin B. Before transfection, you may want to test the sensitivity of the Flp-In™ cell line to hygromycin B to more accurately determine the hygromycin B concentration to use for selection. A suggested range of hygromycin B concentrations to use for selection of your Flp-In™ expression vector is listed below. For more information, please refer to the Flp-In™ System manual. Hygromycin B may be obtained from Thermo Fisher Scientific.
Important: Following cotransfection, your Flp-In™ expression clones should become sensitive to Zeocin™ therefore, your selection medium should not contain Zeocin™.
Cell Line (After Transfection with Estimated Hygromycin B
Flp-In™ Expression Vector) Concentration (µg/ml)
Zeocin™
Zeocin™ is a member of the bleomycin/phleomycin family of antibiotics isolated from Streptomyces. Antibiotics in this family are broad spectrum antibiotics that act as strong anti-bacterial and anti-tumor drugs. They show strong toxicity against bacteria, fungi (including yeast), plants, and mammalian cells (Baron et al., 1992; Drocourt et al., 1990; Mulsant et al., 1988; Perez et al., 1989).
The Zeocin™ resistance protein has been isolated and characterized (Calmels et al., 1991; Drocourt et al., 1990). This protein, the product of the Sh ble gene (Streptoalloteichus hindustanus bleomycin gene), is a 13.7 kDa protein that binds Zeocin™ and inhibits its DNA strand cleavage activity. Expression of this protein in eukaryotic and prokaryotic hosts confers resistance to Zeocin™.
Molecular Weight, Formula, and Structure
The formula for Zeocin™ is C60H89N21O21S3 and the molecular weight is 1,535. The diagram below shows the structure of Zeocin™.
Applications of Zeocin™
Zeocin™ is used for selection in mammalian cells (Mulsant et al., 1988); plants (Perez et al., 1989); yeast (Baron et al., 1992); and prokaryotes (Drocourt et al., 1990). Typically, Zeocin™ concentrations ranging from 50 to 1000 µg/ml are used for selection in mammalian cells. Before transfection, we recommend that you first test the sensitivity of your mammalian host cell to Zeocin™ as natural resistance varies among cell lines.
Handling Zeocin™
Ordering Information
Zeocin™ can be purchased from Thermo Fisher Scientific. For your convenience, the drug is prepared in autoclaved, deionized water and available in 1.25 ml aliquots at a concentration of 100 mg/ml. The stability of Zeocin™ is guaranteed for six months, if stored at -20°C.
Amount Catalog no.
1 gram R250-01
5 grams R250-05
Blasticidin
Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells (Takeuchi et al., 1958; Yamaguchi et al., 1965). Resistance is conferred by expression of either one of two blasticidin S deaminase genes: bsd from Aspergillus terreus (Kimura et al., 1994) or bsr from Bacillus cereus (Izumi et al., 1991). These deaminases convert blasticidin S to a non-toxic deaminohydroxy derivative (Izumi et al., 1991).
Molecular Weight, Formula, and Structure
The formula for blasticidin S is C17H26N8O5-HCl, and the molecular weight is 458.9. The diagram below shows the structure of blasticidin.
Handling Blasticidin
Always wear gloves, mask, goggles, and protective clothing (e.g., a laboratory coat) when handling blasticidin. Weigh out blasticidin and prepare solutions in a hood.
Preparing and Storing Stock Solutions
Blasticidin may be obtained separately (Catalog No. R210-01) in 50 mg aliquots. Blasticidin is soluble in water. Sterile water is generally used to prepare stock solutions of 5 to 10 mg/ml.