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| Genome Modification by Inclusion of loxP Transgene Sequences |
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Kazuaki Ohtsubo and Jamey D. Marth
Department of Cellular and Molecular Medicine and the Howard Hughes Medical Institute
University of California, San Diego, La Jolla, California 92093
This protocol was adapted from "Conditional Mutagenesis of the Genome Using Site-Specific DNA Recombination," Chapter 60, in Gene Transfer: Delivery and Expression of DNA and RNA
(eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.
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| INTRODUCTION |
We describe here a procedure for introducing loxP sites into the mammalian genome. A typical gene-targeting approach to create a conditional null allele is presented, in which the initial placement of loxP sites is not deleterious to allele function. This can be modified to include knock-ins of point mutations, with such mutations flanked by loxP sites that can then be recombined by Cre expression. The choice of sequence or regulatory element to modify is dependent on the experimental design. Consideration must be given to the possible production of truncated and altered gene sequence products, or otherwise aberrantly functioning alleles.
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| RELATED INFORMATION |
The vector to insert loxP sites into the genome is constructed using, for example, the pflox vector to introduce three loxP sites into the targeted transgenic linear genome sequence by homologous recombination (Fig. 1A ). The pflox vector has two juxtaposed selection marker genes, neomycin phosphotransferase (neo) and herpes simplex virus thymidine kinase (tk). The former is used for positive selection for the presence of the integrated parental Type 3 allele (three loxP sites) F[tkneo] (Fig. 1B). The latter is used for subsequent negative selection against recombinant alleles retaining the loxP-flanked neo-tk selectable marker array. The tk gene can be removed, and Cre recombinants that lack the neo gene can be screened for by other means, such as polymerase chain reaction (PCR). The tk gene must be removed prior to its presence in the germ line of male mice; otherwise, spermatogenic failure occurs. The loxP-flanked target gene sequence lacking the marker genes is designated as F (floxed) or a Type 2 allele (two loxP sites) and becomes the precise control genotype (Fig. 1B). Cre recombination among the most distal loxP sites results in a Type 1 or deleted ({Delta}) allele (one loxP site remains), which further lacks the experimentally targeted loxP-flanked sequence (Fig. 1B). One of two possible orientations in the placement of short and long arms of genomic DNA sequence is depicted in Figure 1B. The use of isogenic DNA with linear sequence homology ranging from 4 to 12 kb, indeed, provides the highest frequencies of homologous recombination (te Riele et al. 1992). When using PCR to detect homologous recombinants, a short arm of approximately 1-2 kb is best to achieve a robust sensitivity in the assay. In this regard, it is helpful to produce a control vector bearing the partially integrated structure; this allows for PCR testing of the oligonucleotides chosen (Fig. 1C). Subsequent analyses of loxP presence and position following gene transfer and Cre recombination are facilitated using the loxP probe sequence from the ploxP2 plasmid as a probe for genomic analyses (Fig. 1D).
Figure 1. Modifying genomes by the inclusion of loxP sequences and Cre recombination. (A) The targeting vector is constructed by the insertion of DNA fragments (short, middle, and long) contiguous with the genome into the pflox vector. The middle fragment contains the genetic sequence/element chosen for deletion in the experimental strategy. (Black arrowheads) loxP sites. Restriction enzyme sites present in the pflox vector are indicated: (Bm) BamHI; (H) HindIII; (S) SalI; (Xb) XbaI; (Xh) XhoI; (a-f) unique restriction enzyme sites in genomic DNA. (B) Vector integration into the genome by homologous recombination produces the F[tkneo] allele in the ES cell. The F[tkneo] allele can be converted into Type 1 ({Delta}) or Type 2 (F) alleles by Cre recombinase activity. An alternative allelic structure is possible following crossover during recombination within the middle arm of homology (within large parentheses). This would generate a PCR-positive indication of homologous recombination, depending on primer positioning, but result in the absence of the distal loxP site that flanks the DNA sequence selected for future elimination by Cre recombinase. The frequency of this basically useless internal crossover is almost always low, but may be influenced by the chromatin context and size of the loxP-flanked DNA fragment with respect to the size of the adjacent short-arm genomic DNA sequence. (Small arrowheads) Positions of PCR primers used during screening for homologous recombinant ES cell clones are denoted. (Small parentheses) Restriction enzyme sites destroyed during construction of the targeting vector. Southern blot analysis is accomplished using a genomic probe residing adjacent to sequences transferred by the vector. Examples are provided of restriction enzyme site positions useful for allele analysis by the genomic probe (a-d) and the loxP probe (Lx1-4). (C) Structure of a sample control vector used to develop sensitive specific PCR screening for the F[tkneo] allele. (D) Structure of the isolated (i.e., NotI digest) loxP probe from ploxP2.
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MATERIALS
This procedure requires reagents and equipment for agarose gel electrophoresis and Southern hybridization (see Steps 4 and 25).
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Reagents
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2X Cell-freezing medium, ice cold (for freezing ES cell clones in master plates; see Step 22)
- 40% (v/v/v) Dulbecco's modified Eagle's medium (DMEM)
- 40% (v/v/v) Fetal bovine serum (FBS)
- 20% (v/v/v) Dimethylsulfoxide (DMSO; Sigma-Aldrich D2650)
- DNA target clone for gene transfer
This is typically an isogenic genomic fragment of ~20 kb.
- Embryonic stem (ES) cells
Various mouse ES cell lines are available from academic and commercial sources. Some, such as the R1 line (Nagy et al. 1993), do not require a supportive "feeder-cell" underlay and can be maintained on gelatinized plates in the presence of leukemia inhibitory factor.
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ES cell culture medium
- 500 ml Dulbecco's modified Eagle's medium (DMEM)
- 94 ml Fetal bovine serum (FBS), (15% final concentration)
- 6.2 ml 10 mM MEM nonessential amino acids (0.1 mM final concentration)
- 6.2 ml 100 mM sodium pyruvate (1 mM final concentration)
- 620 µl 55 mM ß-mercaptoethanol (55 µM final concentration)
- 6.2 ml 10,000 units/ml penicillin:10,000 units/ml streptomycin:200 mM L-glutamine (100 units/ml penicillin:100 units/ml streptomycin:2 mM L-glutamine final concentration)
- 62 µl 107 units/ml leukemia inhibitory factor (LIF) (1000 units/ml final concentration)
- Ethanol (70%)
- G418 (200 mg/ml)
- Gelatin (0.1% Type A, from porcine skin; Sigma-Aldrich G2500)
Dissolve 0.5 g of gelatin in 500 ml of tissue-culture-grade H2O. Sterilize by autoclaving.
For preparation of tissue culture dishes and plates; see Equipment.
- Mineral oil, ice-cold (Sigma-Aldrich M8410) (for freezing ES cell clones in master plates; see Step 22)
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NaCl-ethanol (for preparing DNA from replica plates; see Step 22)
- 3 ml NaCl (5M)
- 100 ml ethanol
Dissolve 3 ml of 5 M NaCl in 100 ml of 100% ethanol.
- Oligonucleotides
- Gene-specific primer(s) (must be chosen from target allele of interest)
- TK-specific primer (5'-TTCGAATTCGCCAATGACAAGACGCTG-3')
- PBS (ice cold for Steps 11 and 13)
- PCR kit (Takara Ex Taq polymerase; TAKARA, TAKRR001)
- Plasmid DNA
- pflox vector (variants may be substituted) (Chui et al. 1997)
- ploxP2 (for preparing the loxP-bearing probe by NotI digestion) (Chui et al. 1997)
Other plasmids may be constructed if convenient to include genomic fragments of the surrounding and target DNA sequence. These are used to probe allele structure before and after homologous recombination and after Cre-mediated recombination.
- Proteinase K (1 mg/ml; Sigma-Aldrich P6556) (for quick screening by PCR; see Step 22)
- Restriction endonucleases and buffers (see Steps 1-3)
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Sarkosyl lysis buffer (for preparing DNA from replica plates; see Step 22)
- 10 µM Tris-Cl (pH 7.5)
- 10 µM EDTA
- 10 µM NaCl
- 0.5% Sarkosyl
- 1 mg/ml proteinase K
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TE buffer
- 100 mM Tris-Cl (desired pH)
- 10 mM EDTA (pH 8.0)
Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. Store the buffer at room temperature.
- Trypsin-EDTA (0.05%)
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Equipment
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- Centrifuges
- Adaptors for microtiter plates are optional (see Step 22.xi).
- Dry ice
- Electroporation cuvette (Gene Pulser cuvette, 0.4-cm electrode gap; Bio-Rad 165-2088 or equivalent)
- Electroporator (Gene Pulser II and Capacitance Extender; Bio-Rad or equivalent)
- Hemocytometer
- Ice
- Incubator (37°C, 5% CO2)
- Microcentrifuge
- Microcentrifuge tubes (1.5 ml)
- Micropipettors (P20 and P200)
- Microscope (inverted)
- Parafilm
- PCR machine (GeneAmp PCR System 2700, Applied Biosystems or equivalent)
- PCR tubes (200 µl; Applied Biosystems N8010540)
- Plate-sealing sheet (Seal Plate; Excel scientific STR-SEAL-PLT) (for preparing DNA from replica plates; see Step 22)
- Styrofoam box (sized to fit 96-well plates) (for freezing ES cell clones in master plates; see Step 22)
- Tubes, 15-ml conical (for quick screening by PCR; see Step 22)
- Tubes, 50-ml (see Step 10)
- Tissue-culture dishes (gelatinized, 10 cm; NUNCLON D Surface; NUNC 172958)
Gelatinize tissue-culture dishes with 3 ml of 0.1% gelatin for 1 hour at room temperature, and then remove the excess solution.
- Tissue-culture plates (96 well, flat bottom, gelatinized; NUNCLON D Surface; NUNC 167008)
Gelatinize 96-well plates with 50 µl of 0.1% gelatin for 1 hour at room temperature, and then aspirate the excess solution.
- Tissue-culture plates (96-well, U bottom) (NUNCLON D Surface; NUNC 163320)
- Water bath, boiling (for quick screening by PCR; see Step 22)
- Water baths preset to 37°C and 55°C
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METHOD
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Preparation and Verification of DNA
A control vector can be constructed in parallel that extends the boundary of the F[tkneo] targeting vector to include the chromosomal oligonucleotide primer to be used in PCR detection of homologous recombination of the targeting vector. Use PCR to test for amplification sensitivity and in background of wild-type ES cell genomic DNA.
- Digest the genomic/transgene clone of the gene of interest with appropriate restriction endonucleases.
- Insert DNA fragments into the restriction enzyme sites of the pflox vector using enzymatically produced blunt ends as necessary.
Use XbaI and/or SalI sites for short-arm insertion, the BamHI site for middle-arm insertion, and the HindIII and/or XhoI sites for long-arm insertion (Fig. 1A).
- Linearize the targeting vector (or isolate the targeting construct away from the plasmid vector backbone) with an appropriate restriction endonuclease (e.g., NotI).
- Confirm DNA quantity and quality by Agarose Gel Electrophoresis.
- Store the isolated and linearized DNA targeting vector in nuclease-free TE buffer in 70% ethanol until use.
ES Cell Culture and Gene Transfer by Electroporation
- Thaw and culture R1 or similar ES cells on gelatinized tissue-culture dishes in ES cell culture medium. Change the medium every day until the cells are 70% confluent.
- Change the culture medium 4 hours before electroporation.
- Wash the cells with PBS. Add 1 ml of trypsin-EDTA, and incubate the cells for 5 minutes at 37°C.
- Agitate the plate gently to detach the cells. Add 9 ml of ES culture medium, and pipette gently to resuspend cells and break up clumps.
- Transfer the cell suspension to a 50-ml tube. Centrifuge at 190g for 5 minutes.
- Aspirate the supernatant. Resuspend the cells with 30 ml of ice-cold sterile PBS.
- Count the cells using a hemocytometer.
- Centrifuge at 190g for 5 minutes. Aspirate the supernatant. Resuspend the cells with ice-cold sterile PBS to adjust the cell concentration to 107 cells per 800 µl.
- Transfer the cell suspension to a sterile electroporation cuvette. Mix the cells with 10 µg of the linearized targeting vector (from Step 5). Incubate for 10 minutes on ice.
- Set the electroporator to a voltage of 240 V and a capacitance of 500 µF. Place the cuvette in the holder with electrodes facing the connectors. Deliver the electrical pulse.
- Incubate the cuvette for 10 minutes on ice.
- Plate the cells at varying dilutions (0.5 x 106 to 2 x 106 cells/dish). Incubate for 24 hours.
- To start positive selection, change the medium to ES cell culture medium supplemented with 200 µg/ml (active concentration) of G418. Continue the incubation, changing the culture media every 1-2 days, until G418-resistant colonies grow enough to isolate (~2 mm).
This usually takes 7-10 days.
- Prepare gelatinized flat-bottom 96-well plates containing 150 µl of G418-supplemented ES cell culture medium. Prepare U-bottom 96-well plates containing 70 µl of trypsin/EDTA.
- Replace the culture medium in the dish containing colonies with PBS. Place the dish on the stage of the inverted microscope.
- Use a P20 micropipettor set to 10 µl to pick up the drug-resistant colonies by suction. Transfer them to a trypsin-containing 96-well plate.
- Prepare the cells for additional culture, frozen storage, and/or DNA preparation for PCR.
For freezing ES cell clones in master plates:
- Gently suspend the cells using a P200 micropipettor. Transfer 50 µl of the cell suspension to one 96-well plate (master plate for cell culture and/or freezing). Transfer the remaining cell suspension in the plate containing the trypsin-EDTA (~30 µl) to a gelatinized 96-well flat-bottom plate (the replica plate for DNA preparation), and add 150 µl of culture medium. Return both the master and replica plates to the culture incubator.
For quick screening by PCR without freezing master plates, PCR must be completed on fewer cells and prior to ES cell confluence in culture of the master plates. In these cases, proceed immediately to Step 22.xiv with the replica plate.
- Culture the cells in G418-supplemented ES medium for 3-5 days.
Most of the clones in the master plates will be ready to freeze (~70% confluent); proceed to Step 22.iii with the master plate.
For the highest yield of DNA for PCR analysis, continue to culture the replica plates until they are confluent. Then proceed to Step 22.viii with the replica plate.
- Wash the wells twice with sterile PBS.
- Add 50 µl of trypsin-EDTA to each well. Incubate the cells for 5 minutes at 37°C.
- Add 50 µl of ice-cold 2X freezing medium to each well. Gently suspend the cells by pipetting.
- Add 100 µl of ice-cold mineral oil to each well.
- Seal the plates with Parafilm, place them in a styrofoam box, and transfer them to a -80°C freezer.
For preparing DNA from replica plates:
- Wash the cultured replica plates (from Step 22.ii) with PBS. Add 50 µl of lysis buffer to each well.
- Seal each plate with a plate-sealing sheet. Incubate the plates for 2-3 hours at 55°C.
- Add 150 µl of NaCl-ethanol to each well. Incubate the plates for 30 minutes at room temperature.
- Decant the supernatant by tilting the plate slowly. Alternatively, centrifuge the plate, and lightly aspirate the supernatant.
- Wash the wells three times each with 200 µl of 70% ethanol. Allow the DNA to air-dry at room temperature.
- Add 30 µl of TE buffer to each well. Seal each plate with a plate-sealing sheet. Incubate the plates overnight at 37°C prior to DNA preparation.
For quick screening by PCR without freezing master plates:
- Use a P200 micropipettor to gently resuspend the cells placed in the 96-well replica plate bearing trypsin-EDTA (from Step 22.i).
- Transfer the suspension to a sterile 15-ml conical tube containing at least 5 ml of ES cell culture medium.
- Centrifuge the tube to pellet the cells. Aspirate the medium.
- Wash the cells with 500 µl of PBS. Transfer them to a 1.5-ml microcentrifuge tube, and centrifuge to pellet the cells. Aspirate the supernatant.
- Resuspend the cells in 200 µl of H2O. Immediately place them on dry ice.
- Boil the tubes for 5 minutes. Centrifuge the tubes briefly to remove moisture from the tube walls.
- Add 50 µl of proteinase K to each tube. Incubate the tubes at least 5 hours (or overnight) at 55°C.
- Boil the samples for 5 minutes to heat-inactivate the proteinase K.
- Subject 20 µl of the preparation from either Step 22.xiii or Step 22.xxi to PCR screening.
When PCR sensitivity in detecting the control vector is sufficiently high, combine rows of eight or 12 wells for initial PCR screening.
- Expand ES cells representing PCR-positive clones.
- Analyze clones of interest for the loxP sequence by genomic Southern blotting.
For loxP genomic Southern blotting, use one or more DNA probes outside of the vector sequence used for gene transfer, as well as the loxP probe (isolated as a NotI fragment) (Fig. 1D) to detect the presence of all loxP sites. Alternatively, during gene vector production, include a restriction enzyme site that can be used to distinguish the alleles at this stage.
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ACKNOWLEDGMENTS
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The authors acknowledge support from the National Institutes of Health (DK4247) and the Howard Hughes Medical Institute (to J.D.M.).
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REFERENCES
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Chui, D., Oh-Eda, M., Liao, Y.-F., Panneerselvam, K., Lal, A., Marek, K.W., Freeze, H.H., Moremen, K.W., Fukuda, M.N., and Marth, J.D. 1997. {alpha}-Mannosidase-II deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis. Cell 90: 157-167.
Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., and Roder, J.C. 1993. Derivation of completely cell culture-derived mice from early passage embryonic stem cells. Proc. Natl. Acad. Sci. 90: 8424-8428.
te Riele, H., Maandag, E.R., and Berns, A. 1992. Highly efficient gene targeting in embryonic stems cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. 89: 5128-5132.
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Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult:
Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi), © 2007 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 587-602.
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