Generation of Isogenic Expression Cell Lines using Flp-In™ T-Rex™ System

 Overview

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. To generate these cell lines, the Flp-In™ T-REx™ System involves the following major steps:

1. Independent integration of two plasmids into the genome of the mammalian cell line of choice to generate a Flp-In™ T-REx™ host cell line


2. Integration of an expression vector containing your gene of interest under the control of a tetracycline-inducible promoter into the genome via Flp recombinase-mediated DNA recombination at the FRT site (O'Gorman et al., 1991)


3. Induction of the gene of interest by the addition of tetracycline

            a.   A plasmid containing a Flp Recombination Target (FRT) site
            b.   A plasmid expressing the Tet repressor

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Major Components of the System

The major components of the Flp-In™ T-REX™ System include:

• A Flp-In™ target site vector, pFRT/lacZeo, for generation of a Flp-In™ host cell line containing an integrated FRT site
• A Tet repressor expression plasmid, pcDNA6/TR, for expression of the Tet repressor under the control of the human CMV promoter
• 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 your gene of interest under the control of a tetracycline-regulated CMV/TetO2 promoter
• A Flp recombinase expression plasmid, pOG44, for expression of the Flp recombinase under the control of the human CMV promoter
• A control expression plasmid containing the chloramphenicol acetyl transferase (CAT) gene, which when cotransfected with pOG44 into your Flp-In™ T-REx™ host cell line, expresses CAT upon induction with tetracycline

 
Advantages of the Flp-In™ T-REx™ System

Use of the Flp-In™ T-REx™ System to generate stable expression cell lines provides a number of advantages as described below:

• Once the Flp-In™ T-REx™ host cell line containing an integrated FRT site has been created, subsequent generation of Flp-In™ T-REx™ cell lines expressing the gene(s) of interest is rapid and efficient.
• The Flp-In™ T-REx™ System allows the generation of isogenic, inducible stable cell lines.
• The Flp-In™ T-REx™ System permits polyclonal selection of stable expression cell lines.

 
 
Generating Flp-In™ T-REx™ Host Cell Lines

The Flp-In™ T-REx™ System streamlines the generation of stable, inducible mammalian expression cell lines by taking advantage of a Saccharomyces cerevisiae-derived DNA recombination system. This DNA recombination system uses a recombinase (Flp) and site-specific recombination (Craig, 1988; Sauer, 1994) to facilitate integration of the gene of interest into a specific site in the genome of mammalian cells.

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To generate a Flp-In™ T-REx™ host cell line, you will sequentially transfect two plasmids into the mammalian cell line of choice:

1. pFRT/lacZeo target site vector: The pFRT/lacZeo vector contains a lacZ-Zeocin™ fusion gene whose expression is controlled by the SV40 early promoter.  A FRT site has been inserted just downstream of the ATG initiation codon of the lacZ-Zeocin™ fusion gene. The FRT site serves as the binding and cleavage site for the Flp recombinase. After transfection with pFRT/lacZeo, cells are selected with Zeocin™. Zeocin™-resistant clones are screened to identify those containing a single integrated FRT site. The resulting Flp-In™ host cell line contains a single integrated FRT site and expresses the lacZ-Zeocin™ fusion gene (see the figure below).


2. pcDNA6/TR: The pcDNA6/TR plasmid constitutively expresses the Tet repressor under the control of the human CMV promoter (see the figure below). 

Note:   Integration of the pFRT/lacZeo and pcDNA6/TR plasmids into the genome is random and occurs independently.
 
 
Generating Inducible Expression Cell Lines

Once you have generated a Flp-In™ T-REx™ host cell line, you will integrate the pcDNA5/FRT/TO expression vector containing your gene of interest into the cells via Flp recombinase-mediated DNA recombination at the FRT site (O'Gorman et al., 1991). This step requires the following plasmids:

1. pcDNA5/FRT/TO expression vector: The pcDNA5/FRT/TO plasmid contains your gene of interest under the control of a tetracycline-regulated, hybrid human cytomegalovirus (CMV)/TetO2 promoter. The vector also contains the hygromycin resistance gene with a FRT site embedded in the 5' coding region. The hygromycin resistance gene lacks a promoter and the ATG initiation codon.


2. pOG44: The pOG44 plasmid constitutively expresses the Flp recombinase (Broach et al., 1982; Broach and Hicks, 1980; Buchholz et al., 1996) under the control of the human CMV promoter.

The pOG44 plasmid and the pcDNA5/FRT/TO vector containing your gene of interest are cotransfected into the Flp-In™ T-REx™ host cell line. Upon cotransfection, the Flp recombinase expressed from pOG44 mediates a homologous recombination event between the FRT sites (integrated into the genome and on pcDNA5/FRT/TO) such that the pcDNA5/FRT/TO construct is inserted into the genome at the integrated FRT site (see the figure below). Insertion of pcDNA5/FRT/TO into the genome at the FRT site brings the SV40 promoter and the ATG initiation codon (from pFRT/lacZeo) into proximity and frame with the hygromycin resistance gene, and inactivates the lacZ-Zeocin™ fusion gene (see the figure below). Thus, stable Flp-In™ T-REx™ expression cell lines can be selected for the following phenotypes:

• Blasticidin resistance
• Hygromycin resistance
• Zeocin™ sensitivity
• Lack of ß-galactosidase activity

Once the pcDNA5/FRT/TO vector has been stably integrated into the genome at the FRT site, expression of the recombinant protein of interest can be induced by the addition of tetracycline. For more information about the mechanism of tetracycline-induced expression of the gene of interest, see below.
 
Diagram of the Flp-In™ T-REx™ System

The figure below illustrates the major features of the Flp-In™ T-REx™ System as described earlier.

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Flp Recombinase-Mediated DNA Recombination

In the Flp-In™ T-REx™ System, integration of your pcDNA5/FRT/TO expression construct into the genome occurs via Flp recombinase-mediated intermolecular DNA recombination. The hallmarks of Flp-mediated recombination are listed below.

• Recombination occurs between specific FRT sites (see below) on the interacting DNA molecules
• Recombination is conservative and requires no DNA synthesis; the FRT sites are preserved following recombination and there is minimal opportunity for introduction of mutations at the recombination site
• Strand exchange requires only the small 34 bp minimal FRT site (see below)

For more information about the Flp recombinase and conservative site-specific recombination, refer to published reviews (Craig, 1988; Sauer, 1994).

Note:   If your cell line contains multiple integrated FRT sites, Flp-mediated intramolecular recombination may also occur. Intramolecular recombination may result in:

• Excision of the intervening DNA if the FRT sites are directly repeated (i.e. integration of multiple FRT sites on the same DNA strand)
• DNA inversion if the sites are in opposing orientations
• Deletion of genomic sequences

FRT Sites

As described above, Flp recombinase-mediated recombination occurs between specific FRT sites. The FRT site, originally isolated from Saccharomyces cerevisiae, serves as a binding site for Flp recombinase and has been well-characterized (Gronostajski and Sadowski, 1985; Jayaram, 1985; Sauer, 1994; Senecoff et al., 1985). The minimal FRT site consists of a 34 bp sequence containing two 13 bp imperfect inverted repeats separated by an 8 bp spacer that includes an Xba I restriction site (see figure below). An additional 13 bp repeat is found in most FRT sites, but is not required for cleavage (Andrews et al., 1985). While Flp recombinase binds to all three of the 13 bp repeats, strand cleavage actually occurs at the boundaries of the 8 bp spacer region (see figure below for cleavage sites (CS)) (Andrews et al., 1985; Senecoff et al., 1985).




Tetracycline Regulation in the Flp-In™ T-REx™ System

The Flp-In™ T-REx™ System uses regulatory elements from the E. coli Tn10-encoded tetracycline (Tet) resistance operon (Hillen and Berens, 1994; Hillen et al., 1983) to allow tetracycline-regulated expression of your gene of interest from pcDNA5/FRT/TO. The mechanism of tetracycline regulation in the system is based on the binding of tetracycline to the Tet repressor and derepression of the promoter controlling expression of the gene of interest (Yao et al., 1998). In the system, expression of your gene of interest is repressed in the absence of tetracycline and induced in the presence of tetracycline (Yao et al., 1998).

Expression of your gene of interest from the pcDNA5/FRT/TO vector is controlled by the human cytomegalovirus (CMV) promoter into which 2 tandem copies of the tet operator 2 (TetO2) sequence have been inserted. The TetO2 sequences consist of 2 copies of the 19 nucleotide sequence:

5´-TCCCTATCAGTGATAGAGA-3´
separated by a 2 base pair spacer (Hillen and Berens, 1994; Hillen et al., 1983). Each 19 nucleotide TetO2 sequence serves as the binding site for 2 molecules of the Tet repressor.
 
 
Mechanism of Repression/Derepression

In the absence of tetracycline, the Tet repressor (expressed from the pcDNA6/TR plasmid) forms a homodimer that binds with extremely high affinity to each TetO2 sequence in the promoter of the pcDNA5/FRT/TO vector (Hillen and Berens, 1994). The 2 TetO2 sites in the promoter of pcDNA5/FRT/TO serve as binding sites for 4 molecules (or 2 homodimers) of the Tet repressor (see figure below). The affinity of the Tet repressor for the tet operator is KB = 2 x 1011 M-1 (as measured under physiological conditions), where KB is the binding constant (Hillen and Berens, 1994). Binding of the Tet repressor homodimers to the TetO2 sequences represses transcription of your gene of interest. Upon addition, tetracycline binds with high affinity to each Tet repressor homodimer in a 1:1 stoichiometry and causes a conformational change in the repressor that renders it unable to bind to the Tet operator. The association constant, KA, of tetracycline for the Tet repressor is 3 x 109 M-1 (Hillen and Berens, 1994). The Tet repressor:tetracycline complex then dissociates from the Tet operator and allows induction of transcription from the gene of interest (see figure below).
 
Diagram of Tetracycline Regulation
The figure below illustrates the mechanism of tetracycline-regulated repression and derepression of the gene of interest in the Flp-In™ T-REx™ System.

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Experimental Outline

To create a stable Flp-In™ T-REx™ cell line expressing your gene of interest at a site-specific genomic locus, you will perform the steps listed below (see below for a diagram).

1. Transfect the Flp-In™ target site vector, pFRT/lacZeo into the mammalian cell line of choice and screen for integrants containing a single FRT site.


2. Transfect the Tet repressor expression plasmid, pcDNA6/TR, into a single site integrant (from Step 1) to generate the Flp-In™ T-REx™ host cell line.


3. Clone your gene of interest into the pcDNA5/FRT/TO expression vector.


4. Co-transfect your pcDNA5/FRT/TO construct and the Flp recombinase expression vector, pOG44, into your Flp-In™ T-REx™ host cell line to generate your Flp-In™ T-REx™ expression cell line.


5. Induce expression of the gene of interest with tetracycline.


6. Assay for expression of your recombinant protein of interest.

Note:   The positive control vector containing the CAT gene can be cotransfected into your Flp-In™ T-REx™ host cell line with pOG44 to demonstrate that the system is working properly.
 
Diagram of the Experimental Outline

The figure below illustrates the major steps necessary to generate a Flp-In™ T-REx™ expression cell line.
 

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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:

1. Resuspend each vector in 20 µl sterile water to prepare a 1 µg/µl stock solution. Store the stock solution at -20°C.


2. Use the stock solution to transform a recA, endA E. coli strain like TOP10, DH5a™-T1R, JM109, or equivalent.


3. Select transformants on LB agar plates containing 50 to 100 µg/ml ampicillin. For fast and easy microwaveable preparation of Low Salt LB agar containing ampicillin, imMedia™ Amp Agar (Catalog No. Q601-20) is available from Thermo Fisher Scientific. For more information, call Technical Service.


4. Prepare a glycerol stock from each transformant containing plasmid for long-term storage.

 

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.

1. Streak the original colony out on an LB plate containing 50 µg/ml ampicillin. Incubate the plate at 37°C overnight.


2. Isolate a single colony and inoculate into 1–2 ml of LB containing 50 µg/ml ampicillin.


3. Grow the culture to mid-log phase (OD600 = 0.5–0.7).


4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial.


5. Store at -80°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:

• Contains a single integrated FRT site (introduced by transfection of the pFRT/lacZeo plasmid)
• Stably expresses the Tet repressor (introduced by transfection of the pcDNA6/TR plasmid)

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.

Reminder:   Once you have generated your Flp-In™ T-REx™ host cell line, the cells should exhibit the following phenotypes:

• Zeocin™ resistance
• ß-galactosidase activity
• Blasticidin resistance
• Expression of the Tet repressor

 
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.

1. The next day, substitute culture medium with medium containing varying concentrations of Zeocin™ ( 0, 50, 100, 250, 500, 750, and 1000 mg/ml Zeocin™).


2. Replenish the selective media every 3-4 days, and observe the percentage of surviving cells.


3. Note the percentage of surviving cells at regular intervals to determine the appropriate concentration of Zeocin™ that kills the cells within 1-2 weeks after addition of Zeocin™.

 
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™:

• Vast increase in size (similar to the effects of cytomegalovirus infecting permissive cells)
• Abnormal cell shape
• Presence of large empty vesicles in the cytoplasm (breakdown of the endoplasmic reticulum and Golgi apparatus, or other scaffolding proteins)
• Breakdown of plasma and nuclear membrane (appearance of many holes in these membranes)

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:

• Insertion of the FRT site into the genome: Integration of the pFRT/lacZeo plasmid containing the FRT site into the genome will occur randomly. Subsequent integration of the pcDNA5/FRT/TO expression plasmid containing your gene of interest will occur through Flp recombinase-mediated recombination at the genomic FRT site.
• Transfection efficiency of your cell line: The aim of most users will be to create stable cell lines containing a single integrated FRT site (“single integrants” see Note below). The probability of obtaining stable integrants containing a single FRT site or multiple FRT sites will depend upon the transfection efficiency of your cell line and the amount of DNA transfected. To increase the likelihood of obtaining single integrants, you will need to lower the transfection efficiency by limiting the amount of plasmid DNA that you transfect 
• Selection of foci: You will select for stable transfectants by plating cells in medium containing Zeocin™. Zeocin™-resistant foci can then be screened by Southern blot analysis to identify single integrants. To increase the chances of obtaining single integrants, we recommend that you pick foci from plates that have been transfected with the least amount of plasmid DNA.
• 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.

ß-galactosidase should contain single FRT sites which have integrated into the most transcriptionally active regions.

• 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, note that you may need to use lower concentrations of Zeocin™ to obtain single integrants.

 
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:

• Because expression of your gene of interest is based upon a repression/derepression mechanism, the amount of Tet repressor produced in your Flp-In™ T-REx™ host cell line may not be sufficient to repress basal transcription of your gene if the expression plasmid can integrate into multiple genomic loci.
• Proper tetracycline regulated expression of your gene of interest may be more difficult to achieve.
• The presence of multiple integrated FRT sites in the genome may increase the occurrence of chromosomal rearrangements or unexpected recombination events in your host cell line.

 
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.

*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.

1. Transfect mammalian cells with the appropriate amount of pFRT/lacZeo using the desired protocol. Remember to include a plate of untransfected cells as a negative control.


2. 24 hours after transfection, wash the cells and add fresh medium to the cells.


3. 48 hours after transfection, split the cells into fresh medium. Split the cells such that they are no more than 25% confluent. If the cells are too dense, the antibiotic will not kill the cells. Antibiotics work best on actively dividing cells.


4. Incubate the cells at 37°C for 2-3 hours until they have attached to the culture dish.


5. Remove the medium and add fresh medium containing Zeocin™ at the pre-determined concentration required for your cell line.


6. Feed the cells with selective medium every 3-4 days until foci can be identified.


7. Pick at least 20 Zeocin™-resistant foci and expand each clone to test for the number of integrated FRT sites. Isolate genomic DNA and use Southern blot analysis to distinguish between single and multiple integrants (see below). Select the single integrants and proceed to the next step.


8. Screen the single integrants for ß-galactosidase activity. Select those clones which exhibit the highest levels of ß-galactosidase expression to use as your host for the pcDNA6/TR plasmid.


9. Once you have obtained a stable Flp-In™ cell line, you can use this cell line to isolate a stable cell line expressing the Tet repressor from the pcDNA6/TR plasmid. Note: Remember to maintain the Flp-In™ host cell line in medium containing the appropriate amount of Zeocin™.

 
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:

• Probe: We recommend that you use a fragment of the lacZ gene (100 to 500 bp) as the probe to screen your samples. Mammalian cells do not contain an endogenous lacZ gene, therefore, a lacZ probe should allow you to identify those clones which contain pFRT/lacZeo DNA. To label the probe, we generally use a standard random priming kit (e.g. Ambion, DECAprime II™ Kit, Catalog No. 1455). Other random priming kits are suitable.
• Restriction digest: When choosing a restriction enzyme to digest the genomic DNA, we recommend choosing an enzyme that cuts at a single known site outside of the lacZ gene in the pFRT/lacZeo vector. Hybridization of the lacZ probe to digested DNA should then allow you to detect a single band containing the lacZ gene from pFRT/lac

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:

• Insertion of the hygromycin resistance gene downstream of the SV40 early promoter and the ATG initiation codon (provided by pFRT/lacZeo)
• Insertion of the plasmid containing the CMV/TetO2 promoter, your gene of interest, and the BGH polyadenylation signal upstream of the lacZ-Zeocin™ fusion gene
• Disruption of the functional lacZ-Zeocin™ transcriptional unit caused by loss of the SV40 early promoter and the ATG initiation codon and insertion of the cassette containing the CMV/TetO2 promoter, gene of interest, and the BGH polyadenylation signal

As a result, your Flp-In™ T-REx™ expression cell lines should exhibit the following phenotype:

• Hygromycin resistance
• Zeocin™ sensitivity
• Lack of ß-galactosidase activity
• Blasticidin resistance
• Tetracycline-regulated expression of the gene of interest

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.
 

• Hygromycin B is light sensitive. Store the liquid stock solution at +4°C protected from exposure to light.
• Hygromycin B is toxic. Do not ingest solutions containing the drug.
• Wear gloves, a laboratory coat, and safety glasses or goggles when handling hygromycin B and hygromycin B-containing solutions.

 
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.

• Tetracycline is light sensitive. Store the powdered drug at +4°C in the dark. Prepare medium containing tetracycline immediately before use.
• Tetracycline is toxic. Do not ingest or inhale the powder or solutions containing the drug.
• Wear gloves, a laboratory coat, and safety glasses or goggles when handling tetracycline and tetracycline-containing solutions.

Preparation of Tetracycline

To prepare tetracycline:

1. Weigh out 10 mg of tetracycline (provided in the Flp-In™ T-REx™ Core Kit) and transfer to a sterile 15 ml conical polypropylene tube.


2. Resuspend 10 mg of tetracycline in 10 ml of 100% ethanol. This will provide you with 1 mg/ml stock solution of tetracycline that is yellow in color.


3. Store the stock solution at -20°C protected from exposure to light.

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.

1. Cotransfect your mammalian Flp-In™ T-REx™ host cells with a 9:1 ratio of pOG44:pcDNA5/FRT/TO plasmid DNA (see above) using the desired protocol. Remember to include a plate of untransfected cells as a negative control and the pcDNA5/FRT/TO/CAT plasmid as a positive control.


2. 24 hours after transfection, wash the cells and add fresh medium to the cells.


3. 48 hours after transfection, split the cells into fresh medium. Split the cells such that they are no more than 25% confluent. If the cells are too dense, the antibiotic will not kill the cells. Antibiotics work best on actively dividing cells.


4. Incubate the cells at 37°C for 2-3 hours until they have attached to the culture dish.


5. Remove the medium and add fresh medium containing hygromycin (and blasticidin) at the pre-determined concentration required for your cell line.


6. Feed the cells with selective medium every 3-4 days until foci can be identified.


7. Pick 5-20 hygromycin-resistant foci and expand the cells. Verify that the pcDNA5/FRT/TO construct has integrated into the FRT site by testing each clone for Zeocin™ sensitivity and lack of ß-galactosidase activity. Note: You may also screen a polyclonal population of cells, if desired.


8. Select those clones that are hygromycin-resistant, Zeocin™-sensitive, and LacZ– , and proceed to the next page to assay for tetracycline-regulated expression of your gene of interest.

 
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.

• Plate your Flp-In™ T-REx™ expression cell line in medium containing blasticidin and hygromycin.
• To induce expression of the gene of interest, remove medium and add fresh medium containing the appropriate concentration of tetracycline to the cells. In general, we recommend that you add tetracycline to a final concentration of 1 µg/ml (5 µl of a 1 mg/ml stock per 5 ml of medium) to the cells and incubate the cells for 24 hours at 37ºC.
• Harvest the cells and assay for expression of your gene.

 
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).

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.

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

• 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.
• If cells are split at a 1:5 to 1:10 dilution, they will generally reach 80-90% confluence in 3-4 days.

 
Methods: Culturing Flp-In™ Cell Lines
 
General Cell Handling

Please follow the guidelines below to successfully grow and maintain your cells.
 

• All solutions and equipment that come in contact with the cells must be sterile. Always use proper sterile technique and work in a laminar flow hood.
• Before starting experiments, be sure to have cells established and also have some frozen stocks on hand. We recommend that you always use early-passage cells for your experiments. Upon receipt of the cells, grow and freeze multiple vials of the particular cell line to ensure that you have any adequate supply of early-passage cells.
• Cells should be at the appropriate confluence (approximately 60%) and >90% viability prior to transfection.
• For general maintenance of cells, pass all cell lines when they are 80-90% confluent (3-4 days if split at a 1:5 to 1:10 dilution).
• Use trypan blue exclusion to determine cell viability. Log phase cultures should be >90% viable.

 
Before Starting

Be sure to have the following solutions and supplies available:

• 15 ml sterile, conical tubes
• 5, 10, and 25 ml sterile pipettes
• Cryovials
• Phosphate-Buffered Saline (PBS)
• 0.4% Trypan blue in PBS
• Hemacytometer
• Tissue culture grade 200 mM L-glutamine
• Fetal Bovine Serum (FBS)
• Appropriate complete medium
• Freezing Medium
• Table-top centrifuge
• 75 cm2 flasks, 175 cm2 flasks and other appropriately-sized tissue culture flasks or plates
• Trypsin/versene (EDTA) solution or other trypsin solution

 
 
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.

1. Remove the vial of cells from the liquid nitrogen and thaw quickly at 37°C.
2. Just before the cells are completely thawed, decontaminate the outside of the vial with 70% ethanol, and transfer the cells to a T-75 flask containing 12 ml of complete medium without Zeocin™.
3. Incubate the flask at 37°C for 2-4 hours to allow the cells to attach to the bottom of the flask.
4. Aspirate off the medium and replace with 12 ml of fresh, complete medium without Zeocin™.
5. Incubate cells overnight at 37°C.
6. The next day, aspirate off the medium and replace with fresh, complete medium containing Zeocin™ (at the recommended concentration listed above).
7. Incubate the cells and check them daily until the cells are 80-90% confluent (2-7 days).
8. Proceed to Passaging the Cells, below.

Passaging the Cells

1. When cells are ~80-90% confluent, remove all medium from the flask.
2. Wash cells once with 10 ml PBS to remove excess medium and serum. Serum contains inhibitors of trypsin.
3. Add 5 ml of trypsin/versene (EDTA) solution to the monolayer and incubate 1 to 5 minutes at room temperature until cells detach. Check the cells under a microscope and confirm that most of the cells have detached. If cells are still attached, incubate a little longer until most of the cells have detached.
4. Once the cells have detached, briefly pipet the solution up and down to break up clumps of cells.
5. Add 5 ml of complete medium to stop trypsinization.
6. To maintain cells in 75 cm2 flasks, transfer 1 ml of the 10 ml cell suspension from Step 5 to a new 75 cm2 flask and add 15 ml fresh, complete containing Zeocin™. Note: If you want the cells to reach confluency sooner, split the cells at a lower dilution (i.e. 1:4).
7. To expand cells, add 28 ml of fresh, complete medium containing Zeocin™ to each of three 175 cm2 flasks, then transfer 2 ml of the cell suspension to each flask to obtain a total volume of 30 ml.
8. Incubate flasks in a humidified, 37°C, 5% CO₂ incubator.
9. Repeat Steps 1-10 as necessary to maintain or expand 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:

• Remove and reserve the medium from the cells on the day before you plan to freeze them (see Freezing the Cells, Step 1, below). Refeed the cells with fresh complete medium containing Zeocin™. Store the conditioned medium in a 50 ml sterile, conical centrifuge tube at +4°C until use.
• Remove and reserve the medium from the cells just prior to freezing (see Freezing the Cells, Step 2, below). Transfer the conditioned medium to a 50 ml sterile, conical centrifuge tube and place the tube on ice.

Freezing medium should be prepared fresh immediately before use.

1. In a sterile, conical centrifuge tube, mix together the following reagents for every 1 ml of freezing medium needed:
   Fresh complete medium                                     0.45 ml
   Conditioned complete medium                            0.45 ml
   DMSO                                                                 0.10 ml
    
    
2. Place the tube on ice. Discard any remaining freezing medium after use.

 
 
Freezing the Cells
Before starting, label cryovials and prepare freezing medium (see above). Keep the freezing medium on ice.

1. Change medium the day before you plan to freeze the cells. Remove and reserve the conditioned medium, if desired. Add fresh complete medium containing the appropriate concentration of Zeocin™ to the cells.
2. When cells are ~80% confluent in a 175 cm2 flask, remove and reserve the medium (if needed to make up freezing medium). Wash the cells once with 10 ml PBS.
3. Add 5 ml of trypsin/versene (EDTA) solution and incubate for 1 to 5 minutes until cells detach.
4. Once cells have detached, briefly pipet solution up and down to break up clumps of cells.
5. Add 5 ml of complete medium to stop trypsinization. Count the cells.
6. Pellet cells at 250 x g for 5 minutes in a table top centrifuge at room temperature and carefully aspirate off the medium.
7. Resuspend the cells at a density of at least 3 x10⁶ cells/ml in chilled freezing medium (45% complete medium, 45% conditioned complete medium, and 10% DMSO).
8. Place vials in a styrofoam microcentrifuge rack and aliquot 1 ml of cells per vial. Once vials are capped, place a second styrofoam rack on top of the vials to provide additional insulation. Transfer vials to -20°C for 2 hours.
9. Transfer vials to a -70 or -80°C freezer and hold overnight.
10. Transfer vials to liquid nitrogen for long-term storage.

 
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™

• Store Zeocin™ at -20°C and thaw on ice before use.
• Zeocin™ is light sensitive. Store drug, plates, and medium containing drug in the dark.
• Wear gloves, a laboratory coat, and safety glasses or goggles when handling solutions containing Zeocin™.
• Zeocin™ is toxic. Do not ingest or inhale solutions containing the drug.

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.

• Dissolve blasticidin in sterile water and filter-sterilize the solution.
• Aliquot in small volumes suitable for one time use (see next to last point below) and freeze at -20°C for long-term storage or store at +4°C for short-term storage.
• Aqueous stock solutions are stable for 1-2 weeks at +4°C and 6-8 weeks at -20°C.
• pH of the aqueous solution should be 7.5 to prevent inactivation of blasticidin.
• Do not subject stock solutions to freeze/thaw cycles (do not store in a frost-free freezer).
• Upon thawing, use what you need and store the thawed stock solution at +4°C for up to 2 weeks.
• Medium containing blasticidin may be stored at +4°C for up to 2 weeks.

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