Chapter 2 Insect Cell Line Development Using Flp-Mediated Cassette Exchange Technology João Vidigal, Fabiana Fernandes, Ana S. Coroadinha, Ana P. Teixeira, and Paula M. Alves Abstract Traditional cell line development is quite laborious and time-consuming as it is based on the random integration of the gene of interest which leads to unpredictable expression behavior. In opposition, recombinase-mediated cassette exchange systems represent a powerful genetic engineering approach, allowing site-specific insertion of recombinant genes into pre-tagged genomic loci with superior expression characteristics, thus bypassing the need for extensive clone screening and shortening the development timelines. Such systems have not been widely implemented in insect cell lines used for the production of recombinant proteins most commonly through the baculovirus expression vector system. Herein, it is provided the protocol for the implementation of a FLP-mediated cassette exchange system in Spodoptera frugiperda Sf 9 cells, in order to grant a flexible cell line for the stable production of recombinant proteins. Key words Insect cell line development, Flipase-mediated cassette exchange, Stable expression of recombinant proteins
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Introduction Insect cells, in particular Sf 9 cells, are widely explored for the production of biologically active recombinant proteins using the baculovirus expression vector system (BEVS) [1–3]. However, significant limitations are associated to this system, namely, (1) transient expression limited by the duration of the infection cycle, after which cells die; (2) lytic nature of infection, affecting the cellular protein processing machinery; (3) added effort to maintain a viral stock; and (4) genetic instability of the baculovirus, reducing its value for large-scale pharmaceutical production [2, 4–6].
Ralf Pörtner (ed.), Animal Cell Biotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 1104, DOI 10.1007/978-1-62703-733-4_2, © Springer Science+Business Media, LLC 2014
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Fig. 1 Timelines for cell line development through random or site-specific integration of the transgene. The first one is a laborious and time-consuming process that takes over 4 months to be concluded, whereas the process that employs the recombinase-mediated cassette exchange strategy is much faster and flexible due to the reuse of pre-tagged high-level expression locus
As an alternative to cope with these drawbacks, stable insect cell lines could be established aiming at continuous baculovirusfree expression [2, 6]. Random integration of the transgene is usually applied for the development of stable expression cell lines, demanding an extensive clone screening to identify high-producer cell clones, which often represent a very small portion of the total population [7, 8]. Moreover, even if a transcriptional hot spot is hit, the random screening process cannot guarantee the recurrence of this desirable genetic location each time the expression of a specific gene is needed. In this respect, the reuse of a previously identified hot spot would reduce the time needed for the development of a production cell line assuring predictability of the expression levels (Fig. 1) [7, 8]. This may be achieved through site-specific recombination using specific recombinase enzymes such as the E. coli P1 phage-derived Cre [9], the Saccharomyces cerevisiaederived Flp [10, 11], and the bacteriophage ΦC31-derived integrase [12]. This technology, codified as recombinase-mediated cassette exchange (RMCE), has been used for cell line development, allowing the transgene integration into a pre-tagged genomic site [13]. RMCE enables multiple reuses of pre-characterized genomic sites [14]. Briefly, a tagging cassette flanked by a pair of incompatible noninteracting sites is stably inserted into the cell genome and isolated clones with different tagged loci are characterized
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in terms of expression. Subsequently, a target cassette bringing the gene of interest, also flanked by identical sites, is exchanged/ inserted into those pre-characterized loci by means of a recombinase [15–17]. In this chapter, the implementation of a flipasebased RMCE system in Sf 9 cells is described.
2 2.1
Materials General
1. Phosphate buffer solution (PBS): 137 mM NaCl, 27 mM KCl, 100 mM Na2HPO4, 2 mM K2HPO4, and pH 7.4. 2. Incubator for cultivation of Sf 9 insect cells at 27 °C. 3. Incubator for cultivation of bacteria cells at 37 °C. 4. Orbital shakers for bacteria and insect cells cultivation. 5. Centrifuge for Falcon and Eppendorf tubes. 6. Flow cytometer (e.g., CyFlow 1 space; Partec GmbH, Münster, Germany). 7. NanoDrop (Thermo Fisher Scientific, Waltham, USA). 8. Thermocycler. 9. Waterbath. 10. Chemidoc ChemiDoc™ XRS + System (Bio-Rad, Hercules, USA).
2.2 Preparation of Vectors
1. Library Efficiency® DH5α™ Competent Cells (Invitrogen, Carlsband, USA). 2. Plasmid DNA purification kits. 3. Terrific Broth (TB) and necessary antibiotics (liquid and agar) for bacteria growth and selection (InvivoGen, San Diego, CA). 4. Bacteria culture-certified disposable plastic ware: Petri dishes and 13 mL Falcon tubes. 5. Shake flasks (1 or 2 L).
2.3
Cell Culture
1. Insect cell line, e.g., Sf 9 cells (ECACC 89070101). 2. SF-900II medium. 3. Grace’s Insect Medium. 4. Cellfectin II Reagent (Invitrogen). 5. Shake flasks (125 mL). 6. Cell culture-certified disposable plastic ware: Petri dishes, 6-well plates, 24-well plates, 96-well plates, pipettes, Falcon tubes (15 and 50 mL), and 1.5 mL Eppendorf tubes. 7. Selective antibiotics for eukaryotes (InvivoGen). 8. Cell concentration and viability analysis: trypan blue, hemacytometer, and microscope.
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9. CryoStor CS10 (Sigma-Aldrich, St Louis, USA). 10. Mr. Frosty (Nalgene Nunc, Penfield, USA). 11. Fetal bovine serum, FBS. 2.4 Molecular Biology Analysis
1. TE buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 8.0. 2. Agarose LE, Analytical Grade (Promega, Madison, USA). 3. RNeasy kit (Qiagen, Courtaboeuf, France) for RNA extraction. 4. First Strand cDNA Synthesis Kit (Roche Diagnostics, Manheim, Germany) for cDNA synthesis. 5. Wizard® Genomic DNA Purification Kit (Promega) for DNA extraction. 6. Proteinase K (Roche Diagnostics). 7. Lysis buffer: 10 mM Tris–HCl, pH 8.5, 5 mM EDTA, 200 mM NaCl, and 0.2 % SDS. 8. PCR Dig Probe Synthesis Kit (Roche Diagnostics) for probe labeling. 9. Grade 3MM Chr Whatman paper (Schleicher and Schuell, Maldstone, England). 10. Solutions for blot transfer: depurination buffer (250 mM HCl), denaturation buffer (0.5 M NaOH, 1.5 M NaCl), and neutralization buffer (0.5 M Tris–HCl pH 7.5, 1.5 M NaCl). Store at room temperature (RT). 11. TurboBlotter (GE Healthcare, Little Chalfont, England). 12. Hybond N + membrane (Amersham, Little Chalfont, England). 13. Transfer buffer: 20× saline-sodium citrate (SSC) buffer (3 M NaCl, 300 mM Sodium Citrate), pH 7.4 with HCl. 14. Dig Easy Hyb (Roche Diagnostics). 15. Dig Wash and Block Buffer Set and Dig Nucleic Acid detection Kits (Roche Diagnostics). 16. Alkaline phosphatase-conjugated Diagnostics).
anti-DIG
Fab
(Roche
17. CDP-star: chemiluminescence substrate for alkaline phosphatase (Roche Diagnostics).
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Methods The use of Flp-RMCE for generation of stable insect cell lines simplifies the process and improves the versatility of the resulting cell line by taking advantage of site-specific recombination. Herein, we present the methodology for the establishment of an Flp-RMCE in insect cell lines (such as Sf 9 cells). The overall system requires the development of three different vectors, specifically tailored to drive the expression in insect cells. The first one (the tagging cassette), aims
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Fig. 2 The FLP-RMCE strategy and implementation in insect cells. Both tag and target cassettes (a and b) harbor a pair of incompatible FRT sites in parallel orientation. Between FRT sites, a strong constitutive insect cell promoter, P1, drives the expression of a reporter gene, Rep 1 (e.g., dsRED or eGFP), and a second constitutive promoter drives the expression of a resistance gene (R1, that can be, for instance, hygromycin, zeocin, or neomycin). An ATG-deleted resistance gene, ΔR2, is present in the tagging cassette downstream to the FRT site. In the target cassette, the P2 promoter drives the expression of an ATG sequence, which allows transcription of the ΔR2 sequence upon cassette exchange. Co-transfecting tagged cells with the target and the Flpe recombinase-expressing plasmid (c) will promote cassette exchange and activation of the resistance to R2. If a reporter fluorescent protein is used, RMCE can be easily monitored by fluorescence microscopy, and selection of cell population which successfully exchanged cassettes can be accelerated by the use of a fluorescentactivated cell sorter
at tagging good expressing genomic spots where the recombination process is going to be mediated. This vector contains two different flipase recognition target (FRT) sites flanking the following regulatory elements: a reporter gene driven by a constitutive insect promoter and a resistance gene driven by a second constitutive promoter (Fig. 2a) (see Note 1). Out of the flanked area, there is an ATG-deleted resistance gene. The second vector (the target cassette, pTarget) harbors the gene of interest and enables the “promoter trap” complementation of the resistance gene lacking the ATG on the tagging cassette (Fig. 2b). As with the previous vector, all important elements are flanked by the same pair of FRT sites: expression of the gene of interest (GOI) is driven by a constitutive insect cell promoter and a second constitutive promoter is placed upstream of the ATG complementary sequence (see Note 1). Finally, the third vector (pFlpe) carries the flipase recombinase gene and an insect constitutive promoter for its expression (Fig. 2c).
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Standard cloning techniques are used to construct the plasmids, which can be further propagated in Escherichia coli DH5α Library Efficient. The DNA used to transfect the cells must be purified (a silica-based anion-exchange DNA purification kit, miniprep kit, or with a maxi-prep kit), according to the manufacturer’s protocol. DNA concentrations must be measured with ultraviolet (UV) spectrometer (NanoDrop) and only DNA with a 260/280 coefficient greater than 1.8 is used to transfect cells. 3.1 Tagging Insect Cell Genome
The first step during the development of a cell line using sitespecific integration is to tag the cell genome randomly and then select for clones tagged in hot spots; this procedure takes over 4 months as described in Fig. 1. The Sf 9 insect cell line is here used as a cell line model for its good expression and growth characteristics; the same procedure can be applied to others insect cell lines. In this case dsRED is used as the reporter gene R1 in the tagging cassette and eGFP is the gene of interest in the target cassette. Routine culture of Sf 9 cells is performed in 125 mL shake flasks with 15 mL of Sf 900II-SFM medium, at 27 °C with an agitation rate of 110 rpm; cells are subcultured every 3–4 days at 0.5 × 106 cell/mL. Cell density and viability are determined by cell counting using a Fuchs–Rosenthal chamber after diluting culture bulk samples in 0.4 % (v/v) trypan blue.
3.1.1 Transfection and Selection (3 Weeks)
Single copy integration of the tagging cassette into the cell genome is desirable to achieve reproducible results in RMCE (see Note 2): 1. Before transfection, seed Sf 9 cells at 0.5 × 106 cell/mL in 10 mL Sf 900II-SFM medium in shake flasks (125 mL total volume) and incubate on an orbital shaker (110 rpm) at 27 °C. 2. Prepare the following two solutions in sterile tubes (see Note 3): Tube A: 1.5 μg of tagging cassette plasmid in 500 μL Grace’s Insect Medium. Tube B: 5 units (40 μL) of Cellfectin II reagent in 500 μL Grace’s Insect Medium. 3. Vortex both solutions and incubate at RT for 30 min. 4. Add the content of tube B to tube A, vortex again, and incubate at RT for 15 min. 5. Add the transfection mixture to the cells. 6. Replace culture medium 24 h post-transfection. 7. At 72 h post-transfection add the R1 selecting agent to the culture (see Note 4). 8. Change the medium every 5–6 days. After approximate 15 days in selection, a cell population expressing the tagging cassette will be selected.
Insect Cell Line Development using RMCE 3.1.2 Limiting Dilution (6–12 Weeks)
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Limiting dilution is applied to obtain cell clones expressing the tagging cassette. 1. Dilute the selected cell population at the density of 10 cell/mL and add 100 μL per well in 96-well plates, in order to obtain one cell per well, and incubate at 27 °C. Conditioned medium (the supernatant of exponentially growing Sf 9 cells) supplemented with 10 % (v/v) fetal bovine serum (FBS) is used to support cell colony growth (see Note 5). 2. After approx. 7 days, mark the wells with single colonies under the microscope. Usually, about 25 % of the wells will have a wellisolated growing colony. As a reporter fluorescence protein is used; best expressing clones can also be identified in this step. 3. At confluence, subcultured each colony to higher surface growing areas until having a 6-well plate confluent. 4. Next step is to identify clones with single copy integration of the tagging cassette. As Southern blot is a time-consuming protocol, it is better to analyze several clones simultaneously. Since cell clones have different growth rates, as soon as cell expansion is enough, extract the required amount of DNA for Southern blot analysis (see below) and in parallel make a small cell bank for each clone. Freeze three vials of each clone with about 5 × 106 cells per vial. Detach cells by pipetting repeatedly. Centrifuge at 200 × g for 10 min and resuspend cell pellet in cryopreservation reagent (CryoStor,). Distribute by three vials, transfer to an isopropanol freezing container (Mr. Frosty) and store at −80 °C (see Note 6).
3.1.3 Southern Blot Analysis by Digoxigenin (DIG)-Labeled Probe (1 Week)
In order to determine the copy number of the pTagging construct within the genome of the cell clones, Southern blot analysis must be performed. A specific labeled DNA probe is used to hybridize with a sequence in the tagging cassette locus. Appropriate restriction enzymes must be selected to digest the DNA and allow to interpret the number and size of the bands upon hybridization.
Purification and Digestion of Genomic DNA from Sf 9 Cells
The following protocol is used for 2–5 × 106 cells (see Note 7): 1. Centrifuge for 5 min at 600 × g to collect the cells. Wash the pellet twice with PBS. 2. Resuspend the pellet in 0.5 mL of lysis buffer and incubate at 60 °C for 5 min. 3. Add 2.5 μL of Proteinase K and 5 μL of RNase A/T1 Mix to the lysates and vortex. Incubate at 60 °C for 1 h. 4. Add 250 μL of 5 M NaCl, vortex, and incubate on ice for 5 min to precipitate protein. 5. Centrifuge for 15 min at 9500 × g. Transfer supernatant to a new tube, add an equal volume of isopropanol, and mix to
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precipitate the DNA. Optional: incubate at −20 °C for up to 60 min to increase the yield of DNA. 6. Centrifuge for 10 min at 9500 × g. Discard the supernatant and wash the pellet with 1.2 mL 70 % cold ethanol. 7. Air-dry the pellet (see Note 8) and dissolve it in nuclease-free water or TE buffer. 8. Usually, 5–10 μg of genomic DNA is digested overnight with excess of enzymes. The use of more than one enzyme is preferred for a more effective digestion. DNA Electrophoresis, Transfer, and Cross-Linking
The probe is obtained according to the instructions of the PCR DIG Probe Synthesis Kit (see Note 9): 1. Digested gDNA samples are loaded on a 0.7 % agarose gel and run for 4 h at 45 V/cm. These conditions are suitable for a migration length of 8–10 cm. 2. Submerge the gel in depurination buffer for at least 30 min at RT. Wash twice with water. 3. Soak the gel in denaturation buffer at RT during 30 min under agitation. Then, soak the gel twice in a neutralization buffer, each during 15 min at RT. 4. This gel is blotted into a Hybond N + nylon membrane using the TurboBlotter apparatus and the grade 3MM Chr Whatman papers, according to the manufacturer’s instructions. Alkaline transfer is accomplished within 1 h. 5. Cut off the membrane in order to give orientation of the blotting. After transfer, stain the gel to check if the DNA has been well transferred. 6. Wash membrane in 2× SSC and let it dry in a sheet of Whatman 3MM paper. Then the DNA is cross-linked to the membrane with UV treatment. At this stage, the membrane can be stored at 4 °C.
Probe Labeling
Hybridization, Washing, and DIG Detection
Dilute 0.01–0.1 μg of the labeled probe. For probe labeling synthesis, a simple PCR is performed, using specific primers to amplify the desired sequence that is required to hybridize the target DNA sequence, by following the PCR Dig Probe Synthesis Kit instructions. The probe aliquots should be stored at −20 °C until further use. 1. Incubate the membrane on a rocker for a minimum of 2 h at 68 °C in a hybridization buffer. 2. Boil probe up to 100 °C and quickly chill on ice for 10 min. 3. Hybridize membrane DNA side down overnight (14–16 h) at 68 °C on a rocker. 4. In the following day, wash membrane two times for 15 min with 2× SSC on a rocker at RT.
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5. Wash two times for 15 min at 65 °C with 0.5× SSC, 0.1 % SDS on a rocker. 6. Incubate the membrane during 30 min at RT in blocking solution; rocking is optional. 7. Wash twice with washing buffer for at least 15 min at RT, with continuous agitation. 8. Proceed to membrane equilibration for 5 min at RT with detection buffer. Then, membrane is carefully placed into saran wrap and rinsed with several drops of CDP-star substrate. Excess of liquid is wiped out and the plastic wrap is sealed. Let it rest for 5 min. To improve chemiluminescent signal, incubate the wrapped membrane for 15–30 min at 37 °C. 9. After this incubation the membrane is exposed on film or seen in ChemiDoc detection machine. 3.1.4 Screening for High and Stable Expression (2–3 Weeks)
Once single copy integration clones are identified, the next step is to screen for high and stable expressing clones. Flow cytometry is used to characterize tagged clones in terms of fluorescence intensity and stability of expression along passages: 1. Exponentially growing cells are harvested and diluted in PBS. 2. Fluorescence intensity from over 10,000 events per sample is analyzed. The most adequate laser for each reporter protein should be used. 3. To analyze if expression of recombinant gene is stable along passages and to discard clones which are more likely to develop genetic instability along the passage, each clone is cultured with and without the selection pressure of the antibiotic.
3.2 Site-Specific Recombination (5 Weeks) 3.2.1 Co-transfection with the Target and Flpe Plasmids
Selected clones will be tested for cassette exchange capacity. In order to minimize multiple copy integration events and favor cassette replacement, a small quantity of target cassette (0.1 μg per 1 × 106) and fivefold excess of Flpe plasmid (0.5 μg per 1 × 106) are used [17–19] (see Note 10): 1. Prepare the inoculum for transfection at a concentration of 0.5 × 106 cells/mL in a 125 mL shake flask with 10 mL of Sf 900II medium as working volume and grow at 27 °C (see Note 11). 2. Prepare the following two solutions in sterile Eppendorf tubes (see Note 3): Tube A: 0.5 μg of target and 2.5 μg of Flpe in 500 μL Grace’s Insect Medium. Tube B: 5 units (40 μL) of Cellfectin II reagent in 500 μL Grace’s Insect Medium. 3. Vortex both Eppendorf tubes and incubate at RT for 30 min.
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Fig. 3 Fluorescence microscopy images of a tagged clone upon co-transfection with the flipase and target constructs, after 72 h (a–c) and 3 weeks (d–f) in R2 selection. Cells were maintained in adherent cultures during selection. In this example, dsRED was used as a reporter protein in the tagging cassette, and eGFP was used as gene of interest in the target cassette. The scale bar corresponds to 100 μm
4. Add the content of tube B to tube A, vortex again, and incubate at RT for 15 min. 5. Add the co-transfection mixture to the tagged cells. 3.2.2 Selection of Cells Which Exchanged Cassettes (3 Weeks)
Upon cassette exchange, resistance to R2 is activated by restoring the missing ATG start codon (Fig. 2). 1. Replace culture medium 24 h post-transfection. 2. At 72 h add the R2 antibiotic (see Note 4). 3. Change culture medium every 5–7 days (see Note 12). After 3 weeks in selection, cell populations with the target cassette inserted in the tagged loci will have been selected (Fig. 3).
3.2.3 Target Population Analysis (1 Week) Genomic PCR
Cassette replacement can be confirmed by performing genomic PCR and RT-PCR of the selected target populations (see Note 13). 1. Purification of genomic DNA from tagged clones and target cell populations is performed as for Southern blot analysis; a total of 100 ng of genomic DNA is used. 2. Perform the PCR analysis using the same primer pair in genomic DNA from tagged clones and target R2-resistant pooled cells after targeting. Primer position (i, j) can be seen in Fig. 2. 3. Visualize the amplified PCR products using agarose gel electrophoresis. A difference in size between the products of the two PCR products will confirm cassette exchange, since the target cassette lacks the R1 gene between the amplified regions (Fig. 4a).
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Fig. 4 Analysis of flipase-mediated cassette exchange performance. (a) PCR of genomic DNA to confirm insertion of the target cassette in the tagged locus. Using primers located at P2 promoter and ΔR2 resistance gene, a smaller fragment is obtained from cells after cassette exchange because the locus lacks the R1 gene. (b) mRNA levels of dsRed and eGFP of clone x before (lanes 1, 3, and 5) and after 3 weeks (lanes 2, 4, and 6) of cassette exchange. (c) Expression stability along passages of one isolated target colony when cultured without antibiotic. eGFP fluorescence intensity analyzed by means of flow cytometry RT-PCR
1. Extraction and purification of mRNA was done using the RNeasy Kit and cDNA is synthesized using the First Strand cDNA Synthesis kit. 2. RT-PCR analysis is performed using cDNA diluted (1/10 and 1/20) as template. 3. RT-PCR analysis of the population in selection will show a decrease in transcripts from the reporter gene and an increase in transcripts from the gene of interest (Fig. 4b).
Expression of Gene of Interest
Analyze expression of the gene of interest along passages in the target population in terms of transcript and/or gene product detection along passages to guarantee that cells maintain expression of the gene of interest (Fig. 4c).
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Notes 1. There are several constitutive promoters that can be employed for recombinant gene expression in insect cells using a RMCE system, such as OpIE1 and OpIE2 promoters from Orgia pseudogata, AcIE1 and AcIE2 promoters from Autographa californica, and Drosophila Actin promoter. 2. DNA concentrations up to 0.3 μg DNA per 1 × 106 cells and generally give single copy integrations [17]. 3. For each 1 × 106 cells, it is used 1 unit of Cellfectin II reagent (8 μL), as described by the manufacturer’s protocol (Invitrogen). 4. Although some concentrations can be found in the literature for different antibiotics in different cell lines, it is recommended to perform a killing curve with different concentrations for proper use of the antibiotics. As an example Hygromycin B is added to this cell line at the final concentration of 200 U/mL. 5. An incubator with controlled humidified air is recommended to incubate the plates in order to avoid evaporation. 6. Thawing Sf 9 insect cells: the cell culture vial can be thawed in hand or in the waterbath and should be performed as quickly as possible. Then cells are washed and centrifuged at 1,200 rpm for 10 min in 10 mL of culture media. After this, the cell pellet is resuspended in appropriate volume Sf-900 II medium for cell culture maintenance. 7. Regarding genomic DNA extraction, the typical yield obtained from 1 × 106 cells is 7–15 μg with good purity (260/280 ratio higher than 1.8). 8. Do not use a vacuum dryer neither let the pellet dry completely, since dried genomic DNA has low solubility. 9. The probe size should have between 500 bp and 1 Kb in length. 10. The efficiency of Flp-mediated cassette exchange is not equal for all tagged clones; FLP-RMCE is not amenable in every locus environment. 11. Not using selection pressure R1 for one passage is preferable for not jeopardizing the cassette exchange process. 12. If viability decreases significantly, it is advisable to perform selection as an adherent culture and to use 10 % FBS supplemented conditioned medium to support growth. 13. It is recommended to check for flipase construct integration. If so, problems regarding the population/clone stability will be expected.
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Acknowledgments We thank Mafalda M. Dias (ITQB-UNL/IBET) for help with clone stability analysis. This work was supported by the Portuguese Fundação para a Ciência e Tecnologia (FCT) through the Projects PTDC/ EBB-EBI/102266/2008 and PTDC/EBB-EBI/102750/2008 and by the European Commission (FP7/2007-2013, grant agreement N° 270089). João Vidigal and Fabiana Fernandes also thank FCT for their PhD fellowships (SFRH/BD/90564/2012 and SFRH/BD/43830/2008, respectively). References 1. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575 2. McCarroll L, King LA (1997) Stable insect cell cultures for recombinant protein production. Curr Opin Biotechnol 8:590–594 3. van Oers MM (2011) Opportunities and challenges for the baculovirus expression system. J Invertebr Pathol 107:3–15 4. Jarvis DL, Fleming JA, Kovacs GR et al (1990) Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells. Biotechnology (N Y) 8: 950–955 5. Van Oers MM, Thomas AA, Moormann RJ et al (2001) Secretory pathway limits the enhanced expression of classical swine fever virus E2 glycoprotein in insect cells. J Biotechnol 86:31–38 6. Harrison RL, Jarvis DL (2007) Transforming lepidopteran insect cells for continuous recombinant protein expression. Methods Mol Biol 388:299–2316 7. Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398 8. Kromenaker SJ, Srienc F (1994) Stability of producer hybridoma cell lines after cell sorting: a case study. Biotechnol Prog 10:299–307 9. Sternberg N, Sauer B, Hoess R et al (1986) Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and for regulation by DNA methylation. J Mol Biol 187:197–212 10. Buchholz F, Angrand PO, Stewart AF (1996) A simple assay to determine the functionality of cre or FLP recombination targets in genomic
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manipulation constructs. Nucleic Acids Res 24:3118–3119 Schaft J, Ashery-Padan R, van der Hoeven F et al (2001) Efficient FLP recombination in mouse ES cells and oocytes. Genesis 31:6–10 Thorpe HM, Smith MC (1998) In vitro sitespecific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A 95:5505–5510 Schlake T, Bode J (1994) Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33:12746–12751 Qiao J, Oumard A, Wegloehner W et al (2009) Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties. J Mol Biol 390:579–594 Turan S, Galla M, Ernst E et al (2011) Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges. J Mol Biol 407:193–221 Coroadinha AS, Schucht R, Gama-Norton L et al (2006) The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange. J Biotechnol 124:457–468 Fernandes F, Vidigal J, Dias MM et al (2012) Flipase-mediated cassette exchange in Sf 9 insect cells for stable gene expression. Biotechnol Bioeng 109:2836–2844 Baer A, Bode J (2001) Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for the targeted integration of transgenes. Curr Opin Biotechnol 12:473–480 Sorrell DA, Robinson CJ, Smith JA et al (2010) Recombinase mediated cassette exchange into genomic targets using an adenovirus vector. Nucleic Acids Res 38:e123
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Introduction Insect cells, in particular Sf 9 cells, are widely explored for the production of biologically active recombinant proteins using the baculovirus expression vector system (BEVS) [1–3]. However, significant limitations are associated to this system, namely, (1) transient expression limited by the duration of the infection cycle, after which cells die; (2) lytic nature of infection, affecting the cellular protein processing machinery; (3) added effort to maintain a viral stock; and (4) genetic instability of the baculovirus, reducing its value for large-scale pharmaceutical production [2, 4–6].
Ralf Pörtner (ed.), Animal Cell Biotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 1104, DOI 10.1007/978-1-62703-733-4_2, © Springer Science+Business Media, LLC 2014
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Fig. 1 Timelines for cell line development through random or site-specific integration of the transgene. The first one is a laborious and time-consuming process that takes over 4 months to be concluded, whereas the process that employs the recombinase-mediated cassette exchange strategy is much faster and flexible due to the reuse of pre-tagged high-level expression locus
As an alternative to cope with these drawbacks, stable insect cell lines could be established aiming at continuous baculovirusfree expression [2, 6]. Random integration of the transgene is usually applied for the development of stable expression cell lines, demanding an extensive clone screening to identify high-producer cell clones, which often represent a very small portion of the total population [7, 8]. Moreover, even if a transcriptional hot spot is hit, the random screening process cannot guarantee the recurrence of this desirable genetic location each time the expression of a specific gene is needed. In this respect, the reuse of a previously identified hot spot would reduce the time needed for the development of a production cell line assuring predictability of the expression levels (Fig. 1) [7, 8]. This may be achieved through site-specific recombination using specific recombinase enzymes such as the E. coli P1 phage-derived Cre [9], the Saccharomyces cerevisiaederived Flp [10, 11], and the bacteriophage ΦC31-derived integrase [12]. This technology, codified as recombinase-mediated cassette exchange (RMCE), has been used for cell line development, allowing the transgene integration into a pre-tagged genomic site [13]. RMCE enables multiple reuses of pre-characterized genomic sites [14]. Briefly, a tagging cassette flanked by a pair of incompatible noninteracting sites is stably inserted into the cell genome and isolated clones with different tagged loci are characterized
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in terms of expression. Subsequently, a target cassette bringing the gene of interest, also flanked by identical sites, is exchanged/ inserted into those pre-characterized loci by means of a recombinase [15–17]. In this chapter, the implementation of a flipasebased RMCE system in Sf 9 cells is described.
2 2.1
Materials General
1. Phosphate buffer solution (PBS): 137 mM NaCl, 27 mM KCl, 100 mM Na2HPO4, 2 mM K2HPO4, and pH 7.4. 2. Incubator for cultivation of Sf 9 insect cells at 27 °C. 3. Incubator for cultivation of bacteria cells at 37 °C. 4. Orbital shakers for bacteria and insect cells cultivation. 5. Centrifuge for Falcon and Eppendorf tubes. 6. Flow cytometer (e.g., CyFlow 1 space; Partec GmbH, Münster, Germany). 7. NanoDrop (Thermo Fisher Scientific, Waltham, USA). 8. Thermocycler. 9. Waterbath. 10. Chemidoc ChemiDoc™ XRS + System (Bio-Rad, Hercules, USA).
2.2 Preparation of Vectors
1. Library Efficiency® DH5α™ Competent Cells (Invitrogen, Carlsband, USA). 2. Plasmid DNA purification kits. 3. Terrific Broth (TB) and necessary antibiotics (liquid and agar) for bacteria growth and selection (InvivoGen, San Diego, CA). 4. Bacteria culture-certified disposable plastic ware: Petri dishes and 13 mL Falcon tubes. 5. Shake flasks (1 or 2 L).
2.3
Cell Culture
1. Insect cell line, e.g., Sf 9 cells (ECACC 89070101). 2. SF-900II medium. 3. Grace’s Insect Medium. 4. Cellfectin II Reagent (Invitrogen). 5. Shake flasks (125 mL). 6. Cell culture-certified disposable plastic ware: Petri dishes, 6-well plates, 24-well plates, 96-well plates, pipettes, Falcon tubes (15 and 50 mL), and 1.5 mL Eppendorf tubes. 7. Selective antibiotics for eukaryotes (InvivoGen). 8. Cell concentration and viability analysis: trypan blue, hemacytometer, and microscope.
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9. CryoStor CS10 (Sigma-Aldrich, St Louis, USA). 10. Mr. Frosty (Nalgene Nunc, Penfield, USA). 11. Fetal bovine serum, FBS. 2.4 Molecular Biology Analysis
1. TE buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 8.0. 2. Agarose LE, Analytical Grade (Promega, Madison, USA). 3. RNeasy kit (Qiagen, Courtaboeuf, France) for RNA extraction. 4. First Strand cDNA Synthesis Kit (Roche Diagnostics, Manheim, Germany) for cDNA synthesis. 5. Wizard® Genomic DNA Purification Kit (Promega) for DNA extraction. 6. Proteinase K (Roche Diagnostics). 7. Lysis buffer: 10 mM Tris–HCl, pH 8.5, 5 mM EDTA, 200 mM NaCl, and 0.2 % SDS. 8. PCR Dig Probe Synthesis Kit (Roche Diagnostics) for probe labeling. 9. Grade 3MM Chr Whatman paper (Schleicher and Schuell, Maldstone, England). 10. Solutions for blot transfer: depurination buffer (250 mM HCl), denaturation buffer (0.5 M NaOH, 1.5 M NaCl), and neutralization buffer (0.5 M Tris–HCl pH 7.5, 1.5 M NaCl). Store at room temperature (RT). 11. TurboBlotter (GE Healthcare, Little Chalfont, England). 12. Hybond N + membrane (Amersham, Little Chalfont, England). 13. Transfer buffer: 20× saline-sodium citrate (SSC) buffer (3 M NaCl, 300 mM Sodium Citrate), pH 7.4 with HCl. 14. Dig Easy Hyb (Roche Diagnostics). 15. Dig Wash and Block Buffer Set and Dig Nucleic Acid detection Kits (Roche Diagnostics). 16. Alkaline phosphatase-conjugated Diagnostics).
anti-DIG
Fab
(Roche
17. CDP-star: chemiluminescence substrate for alkaline phosphatase (Roche Diagnostics).
3
Methods The use of Flp-RMCE for generation of stable insect cell lines simplifies the process and improves the versatility of the resulting cell line by taking advantage of site-specific recombination. Herein, we present the methodology for the establishment of an Flp-RMCE in insect cell lines (such as Sf 9 cells). The overall system requires the development of three different vectors, specifically tailored to drive the expression in insect cells. The first one (the tagging cassette), aims
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Fig. 2 The FLP-RMCE strategy and implementation in insect cells. Both tag and target cassettes (a and b) harbor a pair of incompatible FRT sites in parallel orientation. Between FRT sites, a strong constitutive insect cell promoter, P1, drives the expression of a reporter gene, Rep 1 (e.g., dsRED or eGFP), and a second constitutive promoter drives the expression of a resistance gene (R1, that can be, for instance, hygromycin, zeocin, or neomycin). An ATG-deleted resistance gene, ΔR2, is present in the tagging cassette downstream to the FRT site. In the target cassette, the P2 promoter drives the expression of an ATG sequence, which allows transcription of the ΔR2 sequence upon cassette exchange. Co-transfecting tagged cells with the target and the Flpe recombinase-expressing plasmid (c) will promote cassette exchange and activation of the resistance to R2. If a reporter fluorescent protein is used, RMCE can be easily monitored by fluorescence microscopy, and selection of cell population which successfully exchanged cassettes can be accelerated by the use of a fluorescentactivated cell sorter
at tagging good expressing genomic spots where the recombination process is going to be mediated. This vector contains two different flipase recognition target (FRT) sites flanking the following regulatory elements: a reporter gene driven by a constitutive insect promoter and a resistance gene driven by a second constitutive promoter (Fig. 2a) (see Note 1). Out of the flanked area, there is an ATG-deleted resistance gene. The second vector (the target cassette, pTarget) harbors the gene of interest and enables the “promoter trap” complementation of the resistance gene lacking the ATG on the tagging cassette (Fig. 2b). As with the previous vector, all important elements are flanked by the same pair of FRT sites: expression of the gene of interest (GOI) is driven by a constitutive insect cell promoter and a second constitutive promoter is placed upstream of the ATG complementary sequence (see Note 1). Finally, the third vector (pFlpe) carries the flipase recombinase gene and an insect constitutive promoter for its expression (Fig. 2c).
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Standard cloning techniques are used to construct the plasmids, which can be further propagated in Escherichia coli DH5α Library Efficient. The DNA used to transfect the cells must be purified (a silica-based anion-exchange DNA purification kit, miniprep kit, or with a maxi-prep kit), according to the manufacturer’s protocol. DNA concentrations must be measured with ultraviolet (UV) spectrometer (NanoDrop) and only DNA with a 260/280 coefficient greater than 1.8 is used to transfect cells. 3.1 Tagging Insect Cell Genome
The first step during the development of a cell line using sitespecific integration is to tag the cell genome randomly and then select for clones tagged in hot spots; this procedure takes over 4 months as described in Fig. 1. The Sf 9 insect cell line is here used as a cell line model for its good expression and growth characteristics; the same procedure can be applied to others insect cell lines. In this case dsRED is used as the reporter gene R1 in the tagging cassette and eGFP is the gene of interest in the target cassette. Routine culture of Sf 9 cells is performed in 125 mL shake flasks with 15 mL of Sf 900II-SFM medium, at 27 °C with an agitation rate of 110 rpm; cells are subcultured every 3–4 days at 0.5 × 106 cell/mL. Cell density and viability are determined by cell counting using a Fuchs–Rosenthal chamber after diluting culture bulk samples in 0.4 % (v/v) trypan blue.
3.1.1 Transfection and Selection (3 Weeks)
Single copy integration of the tagging cassette into the cell genome is desirable to achieve reproducible results in RMCE (see Note 2): 1. Before transfection, seed Sf 9 cells at 0.5 × 106 cell/mL in 10 mL Sf 900II-SFM medium in shake flasks (125 mL total volume) and incubate on an orbital shaker (110 rpm) at 27 °C. 2. Prepare the following two solutions in sterile tubes (see Note 3): Tube A: 1.5 μg of tagging cassette plasmid in 500 μL Grace’s Insect Medium. Tube B: 5 units (40 μL) of Cellfectin II reagent in 500 μL Grace’s Insect Medium. 3. Vortex both solutions and incubate at RT for 30 min. 4. Add the content of tube B to tube A, vortex again, and incubate at RT for 15 min. 5. Add the transfection mixture to the cells. 6. Replace culture medium 24 h post-transfection. 7. At 72 h post-transfection add the R1 selecting agent to the culture (see Note 4). 8. Change the medium every 5–6 days. After approximate 15 days in selection, a cell population expressing the tagging cassette will be selected.
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Limiting dilution is applied to obtain cell clones expressing the tagging cassette. 1. Dilute the selected cell population at the density of 10 cell/mL and add 100 μL per well in 96-well plates, in order to obtain one cell per well, and incubate at 27 °C. Conditioned medium (the supernatant of exponentially growing Sf 9 cells) supplemented with 10 % (v/v) fetal bovine serum (FBS) is used to support cell colony growth (see Note 5). 2. After approx. 7 days, mark the wells with single colonies under the microscope. Usually, about 25 % of the wells will have a wellisolated growing colony. As a reporter fluorescence protein is used; best expressing clones can also be identified in this step. 3. At confluence, subcultured each colony to higher surface growing areas until having a 6-well plate confluent. 4. Next step is to identify clones with single copy integration of the tagging cassette. As Southern blot is a time-consuming protocol, it is better to analyze several clones simultaneously. Since cell clones have different growth rates, as soon as cell expansion is enough, extract the required amount of DNA for Southern blot analysis (see below) and in parallel make a small cell bank for each clone. Freeze three vials of each clone with about 5 × 106 cells per vial. Detach cells by pipetting repeatedly. Centrifuge at 200 × g for 10 min and resuspend cell pellet in cryopreservation reagent (CryoStor,). Distribute by three vials, transfer to an isopropanol freezing container (Mr. Frosty) and store at −80 °C (see Note 6).
3.1.3 Southern Blot Analysis by Digoxigenin (DIG)-Labeled Probe (1 Week)
In order to determine the copy number of the pTagging construct within the genome of the cell clones, Southern blot analysis must be performed. A specific labeled DNA probe is used to hybridize with a sequence in the tagging cassette locus. Appropriate restriction enzymes must be selected to digest the DNA and allow to interpret the number and size of the bands upon hybridization.
Purification and Digestion of Genomic DNA from Sf 9 Cells
The following protocol is used for 2–5 × 106 cells (see Note 7): 1. Centrifuge for 5 min at 600 × g to collect the cells. Wash the pellet twice with PBS. 2. Resuspend the pellet in 0.5 mL of lysis buffer and incubate at 60 °C for 5 min. 3. Add 2.5 μL of Proteinase K and 5 μL of RNase A/T1 Mix to the lysates and vortex. Incubate at 60 °C for 1 h. 4. Add 250 μL of 5 M NaCl, vortex, and incubate on ice for 5 min to precipitate protein. 5. Centrifuge for 15 min at 9500 × g. Transfer supernatant to a new tube, add an equal volume of isopropanol, and mix to
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precipitate the DNA. Optional: incubate at −20 °C for up to 60 min to increase the yield of DNA. 6. Centrifuge for 10 min at 9500 × g. Discard the supernatant and wash the pellet with 1.2 mL 70 % cold ethanol. 7. Air-dry the pellet (see Note 8) and dissolve it in nuclease-free water or TE buffer. 8. Usually, 5–10 μg of genomic DNA is digested overnight with excess of enzymes. The use of more than one enzyme is preferred for a more effective digestion. DNA Electrophoresis, Transfer, and Cross-Linking
The probe is obtained according to the instructions of the PCR DIG Probe Synthesis Kit (see Note 9): 1. Digested gDNA samples are loaded on a 0.7 % agarose gel and run for 4 h at 45 V/cm. These conditions are suitable for a migration length of 8–10 cm. 2. Submerge the gel in depurination buffer for at least 30 min at RT. Wash twice with water. 3. Soak the gel in denaturation buffer at RT during 30 min under agitation. Then, soak the gel twice in a neutralization buffer, each during 15 min at RT. 4. This gel is blotted into a Hybond N + nylon membrane using the TurboBlotter apparatus and the grade 3MM Chr Whatman papers, according to the manufacturer’s instructions. Alkaline transfer is accomplished within 1 h. 5. Cut off the membrane in order to give orientation of the blotting. After transfer, stain the gel to check if the DNA has been well transferred. 6. Wash membrane in 2× SSC and let it dry in a sheet of Whatman 3MM paper. Then the DNA is cross-linked to the membrane with UV treatment. At this stage, the membrane can be stored at 4 °C.
Probe Labeling
Hybridization, Washing, and DIG Detection
Dilute 0.01–0.1 μg of the labeled probe. For probe labeling synthesis, a simple PCR is performed, using specific primers to amplify the desired sequence that is required to hybridize the target DNA sequence, by following the PCR Dig Probe Synthesis Kit instructions. The probe aliquots should be stored at −20 °C until further use. 1. Incubate the membrane on a rocker for a minimum of 2 h at 68 °C in a hybridization buffer. 2. Boil probe up to 100 °C and quickly chill on ice for 10 min. 3. Hybridize membrane DNA side down overnight (14–16 h) at 68 °C on a rocker. 4. In the following day, wash membrane two times for 15 min with 2× SSC on a rocker at RT.
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5. Wash two times for 15 min at 65 °C with 0.5× SSC, 0.1 % SDS on a rocker. 6. Incubate the membrane during 30 min at RT in blocking solution; rocking is optional. 7. Wash twice with washing buffer for at least 15 min at RT, with continuous agitation. 8. Proceed to membrane equilibration for 5 min at RT with detection buffer. Then, membrane is carefully placed into saran wrap and rinsed with several drops of CDP-star substrate. Excess of liquid is wiped out and the plastic wrap is sealed. Let it rest for 5 min. To improve chemiluminescent signal, incubate the wrapped membrane for 15–30 min at 37 °C. 9. After this incubation the membrane is exposed on film or seen in ChemiDoc detection machine. 3.1.4 Screening for High and Stable Expression (2–3 Weeks)
Once single copy integration clones are identified, the next step is to screen for high and stable expressing clones. Flow cytometry is used to characterize tagged clones in terms of fluorescence intensity and stability of expression along passages: 1. Exponentially growing cells are harvested and diluted in PBS. 2. Fluorescence intensity from over 10,000 events per sample is analyzed. The most adequate laser for each reporter protein should be used. 3. To analyze if expression of recombinant gene is stable along passages and to discard clones which are more likely to develop genetic instability along the passage, each clone is cultured with and without the selection pressure of the antibiotic.
3.2 Site-Specific Recombination (5 Weeks) 3.2.1 Co-transfection with the Target and Flpe Plasmids
Selected clones will be tested for cassette exchange capacity. In order to minimize multiple copy integration events and favor cassette replacement, a small quantity of target cassette (0.1 μg per 1 × 106) and fivefold excess of Flpe plasmid (0.5 μg per 1 × 106) are used [17–19] (see Note 10): 1. Prepare the inoculum for transfection at a concentration of 0.5 × 106 cells/mL in a 125 mL shake flask with 10 mL of Sf 900II medium as working volume and grow at 27 °C (see Note 11). 2. Prepare the following two solutions in sterile Eppendorf tubes (see Note 3): Tube A: 0.5 μg of target and 2.5 μg of Flpe in 500 μL Grace’s Insect Medium. Tube B: 5 units (40 μL) of Cellfectin II reagent in 500 μL Grace’s Insect Medium. 3. Vortex both Eppendorf tubes and incubate at RT for 30 min.
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Fig. 3 Fluorescence microscopy images of a tagged clone upon co-transfection with the flipase and target constructs, after 72 h (a–c) and 3 weeks (d–f) in R2 selection. Cells were maintained in adherent cultures during selection. In this example, dsRED was used as a reporter protein in the tagging cassette, and eGFP was used as gene of interest in the target cassette. The scale bar corresponds to 100 μm
4. Add the content of tube B to tube A, vortex again, and incubate at RT for 15 min. 5. Add the co-transfection mixture to the tagged cells. 3.2.2 Selection of Cells Which Exchanged Cassettes (3 Weeks)
Upon cassette exchange, resistance to R2 is activated by restoring the missing ATG start codon (Fig. 2). 1. Replace culture medium 24 h post-transfection. 2. At 72 h add the R2 antibiotic (see Note 4). 3. Change culture medium every 5–7 days (see Note 12). After 3 weeks in selection, cell populations with the target cassette inserted in the tagged loci will have been selected (Fig. 3).
3.2.3 Target Population Analysis (1 Week) Genomic PCR
Cassette replacement can be confirmed by performing genomic PCR and RT-PCR of the selected target populations (see Note 13). 1. Purification of genomic DNA from tagged clones and target cell populations is performed as for Southern blot analysis; a total of 100 ng of genomic DNA is used. 2. Perform the PCR analysis using the same primer pair in genomic DNA from tagged clones and target R2-resistant pooled cells after targeting. Primer position (i, j) can be seen in Fig. 2. 3. Visualize the amplified PCR products using agarose gel electrophoresis. A difference in size between the products of the two PCR products will confirm cassette exchange, since the target cassette lacks the R1 gene between the amplified regions (Fig. 4a).
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Fig. 4 Analysis of flipase-mediated cassette exchange performance. (a) PCR of genomic DNA to confirm insertion of the target cassette in the tagged locus. Using primers located at P2 promoter and ΔR2 resistance gene, a smaller fragment is obtained from cells after cassette exchange because the locus lacks the R1 gene. (b) mRNA levels of dsRed and eGFP of clone x before (lanes 1, 3, and 5) and after 3 weeks (lanes 2, 4, and 6) of cassette exchange. (c) Expression stability along passages of one isolated target colony when cultured without antibiotic. eGFP fluorescence intensity analyzed by means of flow cytometry RT-PCR
1. Extraction and purification of mRNA was done using the RNeasy Kit and cDNA is synthesized using the First Strand cDNA Synthesis kit. 2. RT-PCR analysis is performed using cDNA diluted (1/10 and 1/20) as template. 3. RT-PCR analysis of the population in selection will show a decrease in transcripts from the reporter gene and an increase in transcripts from the gene of interest (Fig. 4b).
Expression of Gene of Interest
Analyze expression of the gene of interest along passages in the target population in terms of transcript and/or gene product detection along passages to guarantee that cells maintain expression of the gene of interest (Fig. 4c).
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Notes 1. There are several constitutive promoters that can be employed for recombinant gene expression in insect cells using a RMCE system, such as OpIE1 and OpIE2 promoters from Orgia pseudogata, AcIE1 and AcIE2 promoters from Autographa californica, and Drosophila Actin promoter. 2. DNA concentrations up to 0.3 μg DNA per 1 × 106 cells and generally give single copy integrations [17]. 3. For each 1 × 106 cells, it is used 1 unit of Cellfectin II reagent (8 μL), as described by the manufacturer’s protocol (Invitrogen). 4. Although some concentrations can be found in the literature for different antibiotics in different cell lines, it is recommended to perform a killing curve with different concentrations for proper use of the antibiotics. As an example Hygromycin B is added to this cell line at the final concentration of 200 U/mL. 5. An incubator with controlled humidified air is recommended to incubate the plates in order to avoid evaporation. 6. Thawing Sf 9 insect cells: the cell culture vial can be thawed in hand or in the waterbath and should be performed as quickly as possible. Then cells are washed and centrifuged at 1,200 rpm for 10 min in 10 mL of culture media. After this, the cell pellet is resuspended in appropriate volume Sf-900 II medium for cell culture maintenance. 7. Regarding genomic DNA extraction, the typical yield obtained from 1 × 106 cells is 7–15 μg with good purity (260/280 ratio higher than 1.8). 8. Do not use a vacuum dryer neither let the pellet dry completely, since dried genomic DNA has low solubility. 9. The probe size should have between 500 bp and 1 Kb in length. 10. The efficiency of Flp-mediated cassette exchange is not equal for all tagged clones; FLP-RMCE is not amenable in every locus environment. 11. Not using selection pressure R1 for one passage is preferable for not jeopardizing the cassette exchange process. 12. If viability decreases significantly, it is advisable to perform selection as an adherent culture and to use 10 % FBS supplemented conditioned medium to support growth. 13. It is recommended to check for flipase construct integration. If so, problems regarding the population/clone stability will be expected.
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Acknowledgments We thank Mafalda M. Dias (ITQB-UNL/IBET) for help with clone stability analysis. This work was supported by the Portuguese Fundação para a Ciência e Tecnologia (FCT) through the Projects PTDC/ EBB-EBI/102266/2008 and PTDC/EBB-EBI/102750/2008 and by the European Commission (FP7/2007-2013, grant agreement N° 270089). João Vidigal and Fabiana Fernandes also thank FCT for their PhD fellowships (SFRH/BD/90564/2012 and SFRH/BD/43830/2008, respectively). References 1. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575 2. McCarroll L, King LA (1997) Stable insect cell cultures for recombinant protein production. Curr Opin Biotechnol 8:590–594 3. van Oers MM (2011) Opportunities and challenges for the baculovirus expression system. J Invertebr Pathol 107:3–15 4. Jarvis DL, Fleming JA, Kovacs GR et al (1990) Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells. Biotechnology (N Y) 8: 950–955 5. Van Oers MM, Thomas AA, Moormann RJ et al (2001) Secretory pathway limits the enhanced expression of classical swine fever virus E2 glycoprotein in insect cells. J Biotechnol 86:31–38 6. Harrison RL, Jarvis DL (2007) Transforming lepidopteran insect cells for continuous recombinant protein expression. Methods Mol Biol 388:299–2316 7. Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398 8. Kromenaker SJ, Srienc F (1994) Stability of producer hybridoma cell lines after cell sorting: a case study. Biotechnol Prog 10:299–307 9. Sternberg N, Sauer B, Hoess R et al (1986) Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and for regulation by DNA methylation. J Mol Biol 187:197–212 10. Buchholz F, Angrand PO, Stewart AF (1996) A simple assay to determine the functionality of cre or FLP recombination targets in genomic
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