1 Introduction

Although genetic manipulation of mammalian cells and animals have been achieved successfully by plasmid and viral gene delivery, mammalian artificial chromosomes (MACs), as potential vectors offer significant improvement in gene transfer. MACs provide stable, nonintegrating introduction of large payloads of genetic information, and for this reason several groups generated artificial chromosomes using different approaches (1–12).

We developed a satellite DNA-based artificial chromosome (SATAC) (13–16). This chromosome had some basic advantages compared to previously published MACs. The generation of SATACs was reproducible in a variety of host cells, they were composed of known DNA sequences, and could be purified close to 100% purity by high-speed flow cytometry. Several cell lines were established and also transgenic animals were produced using isolated and purified SATACs (17).

Disadvantage of SATAC-based gene delivery system was that de novo generation of SATACs was necessary for each individual application. To overcome this problem, a mammalian artificial chromosome engineering system (ACE system) was developed (18), based on lambda integrase catalyzed site/specific recombination.

De novo SATAC was generated with multiple lambda integrase specific recognition/acceptor sites (Platform ACE). A plasmid construct pCXLamIntROK (pACE Integrase) provided integrase expression for site-specific loading of exogenous DNA sequences into Platform ACE. For targeting, a vector (ATV) was constructed, containing a lambda integrase recombination site upstream of a promoterless selectable marker gene. In the course of cotransfection of Platform ACE-carrying cell lines with ATV and pACE Integrase, site-specific integration of the ATV molecules took place (see Note 8). The selectable marker gene of ATV acquired a promoter located on the Platform ACE, and cells carrying correctly targeted ACE became resistant to the selective drug (Fig. 1).

Fig. 1.
figure 1_10

Site-specific loading of a transgene onto the Platform ACE. (a) The satellite DNA-based artificial chromosome generated by large-scale amplification contains multiple integration sites. (b) The recombination acceptor sites for the ACE-integrase, attP is located between a selectable marker gene and its promoter. (c) The ACE targeting vector (ATV) carries a gene of interest bordered with insulator sequences, and the attB integrase specific site upstream a promoterless selectable marker gene. (d) Expression of ACE-integrase catalyzes the recombination between attP and attB sites resulting in the site-specific integration of ATV into Platform ACE. Thus, the promoterless selectable marker gene of ATV will be driven by the promoter on Platform ACE and drug resistance will be provided by the integrated marker gene of ATV.

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Selected resistant clones were analysed by PCR using a primer pair specific to sequences of Platform ACE and to the selectable marker gene (Fig. 2).

Fig. 2.
figure 2_10

Detection site-specific integration in targeted cell lines by PCR. PCR was carried out using 193AF as the forward primer specific to Platform ACE and a reverse primer specific to the selectable marker gene of ATV; the templates were genomic DNAs of different transformed clones. Site-specific targeting to Platform ACE was detected in the genome of clones represented in lanes 1, 3, 4, and 5. In lane 2, the PCR showed no site-specific integration of ATV. Lane 6 is the positive, and lane 7 is the negative control of PCR, respectively. The Marker is the 1 kB DNA ladder (Fermentas).

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Conventional, two-color fluorescent in situ hybridization (19) analyses were carried out exclusively on PCR screened, resistant clones. The integrity of ACE and site-specific integration on Platform ACE was also demonstrated (Fig. 3). FISH on metaphase spreads reveals not only the targeted integrations, but allows the detection of the nonspecific integration of the ATV in the genome. In mouse cells, the efficiency of targeting was more than 90%, but in hamster cells it was usually below 50%.

Fig. 3.
figure 3_10

Analysis of targeted cell line by two-color FISH. (a) DIG labeled (red) plasmid DNA probe was used to detect ACE, and biotin labeled transgene-specific PCR sequences (green) was the probe to show the integration of therapeutic gene into ACE. The chromosomes were counterstained with DAPI (blue). (b) The corresponding DAPI stained metaphase spread.

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By the protocol provided here site-specific loading of useful gene(s) onto Platform ACEs at 20–90% integration efficiency was achieved within a reasonably short period of time, i.e. in about 2 months. In addition, the copy number of the introduced gene of interest can be variable on the loaded Platform ACEs, allowing selection of cell lines with the desirable level of transgene expression. Considering that Platform ACE contains multiple acceptor sites, second round targeting of the already engineered ACE can be achieved using the same protocol, with an ATV, carrying a different promoterless selectable marker gene.

This lambda integrase based chromosome engineering system has already been used successfully to generate stable, high MAb expressing CHO cell lines (20, 21) and in a combined artificial chromosome-stem cell gene therapy model experiment (22).

2 Materials

2.1 Culture of Cell Lines

  1. 1.

    CO2 tissue culture incubator (37°C, 5% CO2) (ShelLab).

  2. 2.

    Inverted light microscope Olympus CK30.

  3. 3.

    Bürker counting chamber (Roth).

  4. 4.

    MEM alpha medium (Gibco).

  5. 5.

    DMEM medium (Gibco).

  6. 6.

    FBS (Lonza).

  7. 7.

    D2 Trypsine (Serra).

  8. 8.

    Puromycin (Sigma), the stock solution 11 mg/ml is prepared in triple distilled water, aliquots are kept −20°C.

  9. 9.

    6-Well TC Test plates (Orange).

  10. 10.

    Cell culture dishes (Greiner).

2.2 Purification and Measuring of Plasmid DNAs

  1. 1.

    EndoFree Plasmid Maxi Kit (Qiagen).

  2. 2.

    Nano Drop ND-1000 Spectrophotometer.

  3. 3.

    Sterile TE buffer (10 mM Tris–HCl and 1 mM EDTA at pH 7.5).

  4. 4.

    pCXLamIntROK (pACE Integrase), lambda integrase expression plasmid.

  5. 5.

    pATV targeting vector.

  6. 6.

    1.5-ml test tubes (Eppendorf).

2.3 Cotransfection of Platform ACE Containing Cells

  1. 1.

    PBS (Oxoid).

  2. 2.

    SuperFect Transfection Reagent (Qiagen).

  3. 3.

    Biofuge Pico (Heraeus).

2.4 Selection of Transformants

  1. 1.

    8-channel micropipette (Eppendorf).

  2. 2.

    96-Well Cell Culture Cluster (Costar).

  3. 3.

    24-Well Cell Culture Cluster (Costar).

  4. 4.

    6-Well TC Test plates (Orange).

  5. 5.

    50-ml PS Test tube sterile (Greiner).

  6. 6.

    CO2 Incubator.

2.5 Analyses of Transformants

  1. 1.

    Wizard Genomic DNA purification Kit (Promega).

  2. 2.

    PTC-150 MiniCycler (MJ Research).

  3. 3.

    GoTaq Flexi DNA Polymerase (Promega).

  4. 4.

    193AF primer (5′-ACCCCCTTGCG­CTAA­TGCTCT­G­TTA).

  5. 5.

    1 kB DNA Ladder (Fermentas).

  6. 6.

    Biotin-Nick Translation Mix (Roche).

  7. 7.

    DIG-Nick Translation Mix (Roche).

  8. 8.

    Fluorescence microscope, Olympus Vanox-S or similar.

  9. 9.

    Image analysis system, Quips XL Genetics Workstation system or similar.

  10. 10.

    High sensitivity CCD camera, Photometrics KAF 1400-G2 CCD or similar.

3 Methods

3.1 Culturing of Cell Lines

  1. 1.

    Culture LMTK cell line containing Platform ACE (B19-38) in monolayer culture in plastic culture dishes with DMEM, 10% FBS, streptomycin–penicillin, and supplemented with 5 μg/ml of puromycin. Feed the mouse cells every 3 days.

  2. 2.

    Culture CHO-DG44 derived cells containing the Platform ACE (Y19-13DSFS) in monolayer in MEM alpha medium with 5% FBS, streptomycin–penicillin, and 10 μg/ml puromycin. Feed the hamster cells every 3 days.

Seed the cells at a density of 3  ×  105 cells per well of a 6-well culture dish, from both cell lines 1 day before transfection (see Note 1). Determine the cell concentration using Bürker counting chamber.

3.2 Purification and Measuring of Plasmid DNAs

  1. 1.

    Purify targeting vector (ATV) and pACE Integrase expression plasmid with EndoFree Plasmid Maxi Kit (see Note 2).

  2. 2.

    Measure the DNA concentration with spectrophotometer. On the day of transfection, dilute the plasmid DNAs in TE buffer (pH 7.5) to 0.2 mg/ml concentration.

3.3 Cotransfection of Platform ACE Containing Cells

  1. 1.

    Mix 1 μg of targeting vector (ATV) and 1 μg of pACE Integrase plasmid DNA in a sterile Eppendorf tube in cell growth medium without serum and antibiotics to a total volume of 100 μl (see Note 3). Spin down the tube for a few seconds.

  2. 2.

    Add 10 μl of SuperFect Transfection Reagent to the DNA solution and mix gently by pipetting up and down five times. Incubation of the mixture at room temperature for 10 min allows transfection–complex formation.

  3. 3.

    During this time remove the growth medium from the 60–80% confluent cells and wash the cells once with 2 ml of sterile, prewarmed PBS.

  4. 4.

    Add 600 μl of cell growth medium (containing serum) to the transfection complexes in the Eppendorf tube and mix by pipetting up and down twice. The mix should immediately be added to the cells.

  5. 5.

    Incubate the cells with the transfection complex for 2 h under normal growth conditions, 37°C and 5% CO2. During this time, transient expression of lambda integrase is expected to result in targeting the ATV onto the Platform ACE.

  6. 6.

    After 2 h remove the medium containing the remaining complexes from the cells, and wash the cells three times with 2 ml of PBS (see Note 4).

  7. 7.

    Add fresh cell growth medium containing serum to the cells and incubate under normal growth conditions.

3.4 Selection of Transformants

  1. 1.

    After 24 h, trypsinize the transfected cells, resuspend them in 5 ml of growth medium in a 50-ml sterile tube, and count the cell number using the Bürker chamber.

  2. 2.

    Dilute the cells in growth medium to a density of 50 cells/μl and distribute them into 96-well dishes with the 8-channel pipette; 50 μl of cell suspension is added to each well (see Note 5).

  3. 3.

    48 h after transfection add 150 μl of growth medium into each well supplemented with antibiotic to reach the final selection level of the drug.

  4. 4.

    When the resistant colonies reach a cell number of about 50–60, transfer them into individual wells of a 24-well tissue culture dish. When the cells nearly become confluent, harvest them using trypsin treatment and distribute each clonal suspension into two wells of a 6-well dish.

3.5 Analyses of Transformants

  1. 1.

    From one of the wells of the 6-well dish, purify genomic DNA of the resistant colonies using Wizard Genomic DNA Purification Kit (Promega).

  2. 2.

    In PCR analysis of targeting, use 193AF primer, specific to the Platform ACE and a targeting vector specific primer (Fig. 2) (see Note 6).

  3. 3.

    Freeze one plate of clones giving the site-specific PCR product and culture a twin plate for further analyses.

  4. 4.

    Perform conventional single and two-color FISH (16) on metaphase spreads of selected cell lines. Use plasmid DNA labeled with Biotin-Nick Translation Mix to analyse the presence and integrity of ACEs. Resistant clones that contain other integration sites and/or cytological abnormalities (e.g. double minutes) should be excluded (see Note 7). In two-color FISH experiments, use DIG-labeled plasmid DNA and biotinylated PCR DNA of the integrated gene, to prove the site-specific integration on ACE (Fig. 3).

4 Notes

  1. 1.

    In transfection experiments, use rapidly growing cells. Before transfection experiments, feed cells every day. The seeding number on the day before transfection depends on the cell type. The cells should be at 60–80% confluency on the day of transfection.

  2. 2.

    Plasmid DNAs purified by alkaline lysis method are also suitable for transfection of mouse and Chinese hamster cell lines.

  3. 3.

    Use freshly prepared plasmid DNA in transfection experiments. Using old plasmid DNAs the number of colonies may decrease dramatically.

  4. 4.

    Other transfection reagents like Lipofectamine 2000 (Invitrogen) and ExGen 500 (Fermentas) also proved to be effective in targeting experiments, for both mouse and hamster cell lines. In our hands, SuperFect transfection reagent gave the best results.

  5. 5.

    Do not plate more than 2,500 cells/well of a 96-well plate. Higher number of cells may result in multiple colonies in a single well.

  6. 6.

    In PCR experiments with GoTaq polymerase the MgCl2 concentration should be optimized when a different reverse primer is used.

  7. 7.

    The transgene expression should be achieved from the engineered ACE. When other integration is detected by FISH, the source of transgene expression is uncertain, and therefore, these cell lines must be excluded from further experiments.

  8. 8.

    Successful targeting of more than 110 kB exogenous DNA sequences was carried out onto Platform ACE using the above lambda integrase specific recombination system (Praznovszky et al. unpublished).