Flp-In System: For Generating Constitutive Expression Cell Lines

Overview

The Invitrogen™ Flp-In™ System allows integration and expression of your gene of interest in mammalian cells at a specific genomic location. The Flp-In System involves introduction of a Flp Recombination Target (FRT) site into the genome of the mammalian cell line of choice. An expression vector containing your gene of interest is then integrated into the genome via Flp recombinase-mediated DNA recombination at the FRT site (O'Gorman et al., 1991). The major components of the Flp-In™ System include:

• A Invitrogen™ Flp-In™ target site vector, pFRT/lacZeo, for generation of a host cell line containing an integrated FRT site
• 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 the human cytomegalovirus (CMV) immediate-early enhancer/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 Invitrogen™ Flp-In™ host cell line, expresses CAT

Advantages of the Flp-I System

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

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

 
 
Description of the Flp-In System

The Flp-In System streamlines the generation of stable 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(s) of interest into a specific site in the genome of mammalian cells.
 
In the Flp-In System, three different vectors are used to generate isogenic stable mammalian cells lines expressing your gene(s) of interest. The first major component of the Flp-In™ System is the pFRT/lacZeo target site vector that is used to generate a Flp-In host cell line. The 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 Invitrogen™ lacZ-Zeocin™ fusion gene. The FRT site serves as the binding and cleavage site for the Flp recombinase. The pFRT/lacZeo plasmid is transfected into the mammalian cell line of interest and cells are selected for Zeocin resistance. Zeocin-resistant clones are screened to identify those containing a single integrated FRT site. The resulting Flp-In host cell line contains an integrated FRT site and expresses the lacZ-Zeocin™ fusion gene (see the figure below). Note: Integration of the pFRT/lacZeo plasmid into the genome is random.
 
The second major component of the Flp-In System is the pcDNA5/FRT expression vector into which the gene of interest will be cloned. Expression of the gene of interest is controlled by the human CMV 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.
 
The third major component of the Flp-In System is the pOG44 plasmid which 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 vector containing your gene of interest are cotransfected into the Flp-In™ 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) such that the pcDNA5/FRT construct is inserted into the genome at the integrated FRT site (see the figure below). Insertion of pcDNA5/FRT 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. Thus, stable Flp-In expression cell lines can be selected for hygromycin resistance, Zeocin sensitivity, lack of ß-galactosidase activity, and expression of the recombinant protein of interest.
 
 
Diagram of the Flp-In System

The figure below illustrates the major features of the Flp-In System as described. For a brief description about FRT sites and the mechanism of Flp-mediated recombination, see below and published reviews (Craig, 1988; Sauer, 1994).




Flp Recombinase-Mediated DNA Recombination

In the Flp-In System, integration of your pcDNA5/FRT 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).



Experimental Outline

To create a stable Flp-In cell line expressing your gene of interest at a site-specific genomic locus, you will perform the following steps:

1. Transfect the Flp-In target site vector, pFRT/lacZeo, into the mammalian cell line of choice to generate your Flp-In™ host cell line(s) (see figure below).


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


3. Co-transfect your pcDNA5/FRT construct and the Flp recombinase expression vector, pOG44, into your Flp-In™ host cell line to generate your Flp-In expression cell line (see figure below).


4. 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 host cell line with pOG44 to demonstrate that the system is working properly.
 

Introduction

The Flp-In System protocol  is supplied with the kits listed below. The Core System includes vectors and primers for sequencing. The Complete System includes the Core System plus selection agents. See below for a detailed description of the contents of each Flp-In™ System.
 
Shipping/Storage

The Invitrogen™ Flp-In™ Core System is shipped at room temperature. Store at -20°C.
The Invitrogen™ Flp-In™ Complete System is shipped in 2 boxes. Store as described below:

• Box 1 contains vectors, primers, and hygromycin and is shipped at room temperature. Upon receipt, remove the vectors and primers and store at -20°C. The bottle of hygromycin B liquid should be stored at +4°C protected from exposure to light.
• Box 2 contains Zeocin™ and is shipped on blue ice. Store at -20°C protected from exposure to light. 

Expression Vectors
Each Flp-In Complete and Core System also includes an expression vector for cloning your gene of interest and a corresponding positive control vector containing the CAT gene as described below. Refer to the vector manual for specific information pertaining to the expression vector. Store at -20°C.
 
Vector Amount
pcDNA5/FRT
20 mg, lyophilized in TE, pH 8.0
 
pcDNA5/FRT/CAT
20 mg, lyophilized in TE, pH 8.0
 

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, 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 Invitrogen. For more information, call Technical Service.


4. Prepare a glycerol stock of each plasmid for long-term storage.

Introduction

Before you can create a stable Flp-In cell line(s) expressing your gene of interest, you will first need to generate a stable mammalian cell line containing an integrated FRT site (Flp-In™ host cell line). The following section provides guidelines and instructions to generate stable Flp-In host cell lines by transfection using the pFRT/lacZeo plasmid. For a map and a description of the features of pFRT/lacZeo, refer to Vector Maps.
 
Several Flp-In host cell lines which stably express the lacZ-Zeocin fusion gene from pFRT/lacZeo or pFRT/lacZeo2 and which contain a single integrated FRT site are available from Invitrogen (see table below). If you wish to express your gene of interest in one of the cell lines listed below, you may want to use one of Invitrogen’s Flp-In cell lines as the host to establish your stable expression cell line. For more information, refer to our World Wide Web site  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 Invitrogen™ Flp-In™-3T3 or Invitrogen™ Flp-In™-BHK cells. If you will be cloning your gene of interest into a pcDNA5-FRT-based expression construct, we recommend that you do not use 3T3 or BHK cells to create your Flp-In host cell line.

Alternatively, if you prefer to use 3T3 or BHK cells to create your Flp-In host cell line, we recommend that you clone your gene of interest into a pEF5/FRT-based expression plasmid (e.g.,  pEF5/FRT/V5-D-TOPO or pEF5/FRT/V5-DEST). Loss of gene expression due to down-regulation of the promoter is not observed in these cell lines when using pEF5/FRT-based expression constructs. For more information about the pEF5/FRT/V5-D-TOPO or pEF5/FRT/V5-DEST vectors, refer to our Web site  or call Technical Service.
 
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 lipid complexing, decreasing transfection efficiency. We recommend isolating DNA using the S.N.A.P. MiniPrep Kit (10-15 µg DNA, Catalog no. K1900-01), the Invitrogen™ S.N.A.P.™ MidiPrep Kit (10-200 µg 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). Invitrogen offers the Calcium Phosphate Transfection Kit (Catalog no. K2780-01) and Invitrogen™ Lipofectamine™ 2000 Reagent (Catalog no. 11668-027) for mammalian cell transfection. For more information, refer to our World Wide Web site 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 b-galactosidase. For more information about preparing and handling Zeocin, See Reagents and Solutions.
 
The pFRT/lacZeo2 plasmid contains a lacZ-Zeocin fusion gene under the control of a truncated SV40 promoter and is available separately from Invitrogen. The minimal activity of the promoter allows for isolation of clones that have FRT sites integrated in the most transcriptionally active genomic loci. For more information, refer to our Web site  or call Technical Service.
 
Determination of Zeocin Sensitivity

To successfully generate a stable cell line containing an integrated FRT site and expressing the LacZ-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.

See Reagents and Solutions instructions on how to prepare and store Zeocin.

1. 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.


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


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


4. 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 to generate the Flp-In host cell line and 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 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 (see Recommendation below).
• 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 ß-galactosidase levels. Those clones expressing the highest levels of 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.

 
If you want to increase the expression levels of your gene of interest in the cell line of choice, you may wish to generate a Flp-In host cell line containing multiple integrated FRT sites. In theory, cotransfection of your pcDNA5/FRT construct and pOG44 into these cells will allow integration of your gene of interest into multiple genomic loci. Note that 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 µg 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 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 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 b-galactosidase activity. Select those clones which exhibit the highest levels of ß-galactosidase expression (if desired) to use as your Flp-In host cell line(s).


9. Once you have obtained a stable Flp-In host cell line, you can use this cell line to isolate a stable cell line expressing your gene of interest from the pcDNA5/FRT plasmid (see the next section). Note: The Flp-In host cell line should be maintained in medium containing the appropriate amount of Zeocin™ until generation of your Flp-In expression cell line.

 
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 from Invitrogen. 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/lacZeo. We generally use Hind II

Introduction

Once you have established your Flp-In host cell line, you may cotransfect your pcDNA5/FRT construct and the pOG44 expression plasmid into the host cell line to generate a stable Flp-In expression cell line. Integration of the pcDNA5/FRT construct into the genome will occur at the FRT site in the Flp-In host cells. The pcDNA5/FRT plasmid contains the hygromycin resistance gene to allow selection of stable cell lines (see Important note below). For more information about the pcDNA5/FRT plasmid and generating the pcDNA5/FRT expression construct, refer to the vector manual. For more information about the pOG44 plasmid, see below.
 
The hygromycin resistance gene in the pcDNA5/FRT vector lacks an ATG initiation codon and a promoter to drive expression of the gene. Transfection of pcDNA5/FRT plasmid alone into a Flp-In 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 plasmid and the Flp-In™ host cell line.
 
Recommendation

If you wish to express your gene of interest in one of the cell lines listed in the table below, you may want to use one of Invitrogen’s Flp-In host cell lines. For more information, refer to our Web site or call Technical Service.

Note:    If you are generating Flp-In expression cell lines using the Invitrogen™ Flp-In™-3T3 or Invitrogen™ Flp-In™-BHK cell line, we recommend that you clone your gene of interest into a pEF5/FRT-based expression plasmid (e.g. pEF5/FRT/V5-D-TOPO® or pEF5/FRT/V5-DEST). 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.
 
pOG44 Plasmid

You will cotransfect the pOG44 plasmid and your pcDNA5/FRT construct into your Flp-In host cell line to generate stable cell lines that express your protein of interest. Cotransfection of pOG44 and pcDNA5/FRT allows expression of Flp recombinase and integration of the pcDNA5/FRT plasmid into the genome via the FRT sites. Once the pcDNA5/FRT construct has integrated into the genome, the Flp recombinase is no longer required. In fact, the continued presence of Flp recombinase would be detrimental to the cells because it could mediate excision of your pcDNA5/FRT 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 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 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 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 promoter, your gene of interest, and the BGH polyadenylation signal upstream of the lacZ-Zeocin fusion gene
• Disruption of the functional lacZ-Zeoci transcriptional unit caused by loss of the SV40 early promoter and the ATG initiation codon and insertion of the cassette containing the CMV promoter, gene of interest, and the BGH polyadenylation signal

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

• Hygromycin resistance
• Zeocin sensitivity
• Lack of ß-galactosidase activity
• Expression of the gene of interest

Positive Control

The pcDNA5/FRT/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™ expression cell line. If you have several different Flp-In™ host cell lines (cell lines containing FRT sites integrated at different genomic loci), you may want to use the pcDNA5/FRT/CAT control vector to compare protein expression levels from the various genomic loci. For more information about pcDNA5/FRT/CAT, refer to the pcDNA5/FRT vector manual.
 
 
Hygromycin B

The pcDNA5/FRT 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 supplied with the Flp-In Complete System and is also available separately from Invitrogen.

• 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 included with the Flp-In Complete System 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, you need to determine the minimum concentration of hygromycin B required to kill your untransfected Flp-In™ host cell line. Typically, concentrations ranging from 10 to 400 ug/ml hygromycin B are sufficient to kill most untransfected mammalian cell lines. We recommend that you test a range of concentrations (see protocol below) to ensure that you determine the minimum concentration necessary for your Flp-In host cell line.

1. 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.
2. The next day, substitute culture medium with medium containing varying concentrations of hygromycin B ( 0, 10, 50, 100, 200, 400, 600 ug/ml hygromycin B).
3. Replenish the selective media every 3-4 days, and observe the percentage of surviving cells.
4. Note the percentage of surviving cells at regular intervals to determine the appropriate concentration of hygromycin that kills the cells within 1-2 weeks after addition of hygromycin.

Recommendation

Because correct integration of your pcDNA5/FRT 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 expression plasmid that we cotransfect into mammalian Flp-In host cells to optimize the recombination efficiency. We recommend that you cotransfect your Flp-In host cell line with a ratio of at least 9:1 (w/w) pOG44:pcDNA5/FRT 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 host cell line, be sure to use supercoiled pOG44 and pcDNA5/FRT plasmid DNA. Flp-mediated recombination between the FRT site on pcDNA5/FRT and the integrated FRT site in the Flp-In host cell line will only occur if the pcDNA5/FRT plasmid is circularized. The pOG44 plasmid should be circularized to minimize the possibility of the plasmid integrating into the genome.
 
 
Your gene of interest will be expressed from pcDNA5/FRT under the control of the human CMV promoter. Once you have generated the Flp-In expression cell line, note that your recombinant protein should be expressed constitutively.
 
 
Selection of Stable Flp-In Expression Cell Lines

Once you have determined the appropriate hygromycin concentration to use for selection in your Flp-In host cell line, you can generate a stable cell line expressing your pcDNA5/FRT construct. Reminder: Following cotransfection, your Flp-In expression clones should become sensitive to Zeocin therefore, your selection medium should not contain Zeocin.

1. Cotransfect your mammalian Flp-In host cells with a 9:1 ratio of pOG44:pcDNA5/FRT plasmid DNA using the desired protocol. Remember to include a plate of untransfected cells as a negative control and the pcDNA5/FRT/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 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 construct has integrated into the FRT site by testing each clone for Zeocin sensitivity and lack of ß-galactosidase activity.
8. Select those clones that are hygromycin-resistant, Zeocin-sensitive, and LacZ–, and assay for expression of your gene of interest.

 
Polyclonal Selection                                                                                                                                                               

If you use a single integrant as your Flp-In host cell line, all of the hygromycin-resistant foci that you obtain after cotransfection of pcDNA5/FRT and pOG44 and selection with hygromycin should, in theory, be isogenic (i.e., pcDNA5/FRT 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 expression of your protein of interest.
 
 
Assay for CAT Protein

The CAT protein expressed from the pcDNA5/FRT/CAT control plasmid is approximately 32 kDa in size. You may assay for CAT expression using your method of choice. For Western blot analysis, you may use CAT Antiserum available from Invitrogen for detection. Other commercial kits are available for assaying CAT expression

Introduction

The Flp-In cell lines stably express the lacZ-Zeocin fusion gene and are designed for use with the Flp-InSystem (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 World Wide Web site , or call Technical Service. The Flp-In System manual is also available for downloading from our Web site.
 
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-I 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 (PSV40D) 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 Invitrogen (Catalog no. 11965-092).
 
Ham’s F12

Ham’s F12 is used to culture the Flp-I-CHO cell line and can be obtained from Invitrogen (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 Invitrogen (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 from Invitrogen, 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.  Repeat Steps 1-7 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.

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 x 10⁶ 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 Invitrogen for convenient mammalian cell transfection.
 
Note:   Invitrogen™ 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 Invitrogen™ LIPOFECTAMINE™ 2000 Reagent available from Invitrogen (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 obt

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 Invitrogen™ 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, Zeoci 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 Invitrogen. 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 Zeoci 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 from Invitrogen (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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