Transgenesis in Mouse Embryonic Stem Cells

Abstract

Traditionally, transgenic mice are generated by pronuclear injection of exogenous DNA. This technique has several limitations, including limited control over transgene expression, transgene cytotoxicity, ­promiscuity and silencing, and founder mouse sterility. Here we describe two protocols to generate transgenic mice from ES cell clones carrying stably integrated exogenous DNA with inducible transgene expression.

1 Introduction

Transgenesis in mouse oocytes is a widely used technique for the creation of human disease models or to study gene function at the level of the whole animal. A drawback of pronuclear injection of exogenous DNA is that there is limited control over transgene expression, making it necessary to generate and characterize many founder lines to obtain a few with optimal transgene expression. Other frequent problems include transgene cytotoxicity resulting in embryonic lethality, transgene promiscuity, transgene silencing, and founder mouse sterility. Many of these problems can be avoided by generating transgenic mice from ES cell clones carrying stably integrated exogenous DNA. Here, we will describe two applications of this approach, both designed to generate transgenic mice with inducible transgene expression. One approach, which was developed in the laboratory of Corinne Lobe, is based on the Z/EG vector system (1, 2). It allows for induction of tissue-specific transgene expression via Cre recombinase-mediated removal of a “floxed” transcriptional “stop” cassette (Fig. 1a). The second approach, which was developed in our laboratory, is based on a so-called TRIC vector system (for tightly regulatable inducible cDNA). It allows for autonomous doxycycline-­mediated induction of transgene expression (Fig. 1b). Both approaches include an EGFP marker expressed from an internal ribosomal entry site (IRES) sequence that allows for monitoring of transgene expression. First, we will provide an outline of both methods and then a detailed protocol.

Fig. 1.

Schematic representation of the generation of transgenic mice by ES cell-based approaches. (a) Generation of Cre recombinase-inducible transgenics using the Z/EG vector. (b) Generation of doxycycline-inducible transgenics using the TRIC vector.

1.1 Cre Recombinase-Inducible Transgenics

Z/EG is a mammalian expression vector designed to generate ES cell-derived transgenic mice (2). It contains the CAGGS promoter, which encompasses the cytomegalo virus (CMV) immediate enhancer and the chicken β-actin promoter. The CAGGS promoter drives a floxed β-geo-stop cassette, consisting of a β-galactosidase and neomycin-resistance fusion gene and three tandemly arranged polyadenylation sites (Fig. 1a). Downstream of this cassette, the coding sequence of the transgene of interest (TOI) should be cloned. It is recommended that the TOI be marked with an epitope tag to facilitate transgene detection. Upon integration, the transgene is inactive and requires Cre recombinase-mediated removal of the β-geo-stop cassette for induction. EGFP is coexpressed from an IRES as a convenient reporter for TOI expression (Fig. 1a). It is important to linearize the transgenic vector to obtain efficient and proper integration into the genome. Unique ScaI and SfiI restriction sites localized within the backbone of Z/EG are available for this ­purpose. Linearized DNA is then electroporated into ES cells, and clones resistant for G418 are selected and clonally expanded in 96-well plates (Fig. 2). Each clone is then split into three 96-well plates. Cells in plate 1 are infected with an adenovirus that expresses Cre recombinase, which will remove the β-geo-stop cassette and juxtapose the CAGGS promoter with the coding sequence of the TOI and the EGFP marker. ES cell clones that express EGFP are identified by fluorescence microscopy 2–3 days later. The EGFP intensity of the cells serves as an excellent marker for TOI level of expression and is used to preselect clones with low, moderate, or high TOI protein levels. Clones with the appropriate level of expression are then harvested and subjected to Western blot analysis for the epitope tag to confirm proper TOI expression. Cells in plate 2 are fixed and stained for β-galactosidase activity as an independent measure for CAGGS promoter activity. Once cells in plate 3 reach sub-confluency, they are split again into three 96-well plates. Cells in one plate are frozen when sub-confluent, whereas cells in the other two plates are grown to super-confluency for extraction of genomic DNA. This DNA is used to screen for single-copy transgene integration by Southern blotting. This is important to rule out chromosomal instability resulting from recombination between loxP sites at different integration sites. ES clones with suitable transgene expression are reseeded from ­frozen stocks to obtain cells for karyotype analysis and production of chimeric mice through blastocyst injection. Male chimeras from independent ES clones are used to generate hemizygous transgenic mice. These mice still contain the floxed β-geo-stop cassette and need to be crossed to Cre transgenic mice to induce the expression of the TOI and EGFP marker (Fig. 1a). A vast collection of well-characterized transgenic mouse strains that express Cre recombinase under the control of different promoters is available to activate transgene expression in a tissue- or cell type-­specific fashion. Furthermore, several transgenic mouse strains express Cre recombinase in male or female germ cells, allowing for ubiquitous transgene activation. Thus, there is tremendous flexibility with regard to the spatio–temporal induction of TOI expression once the initial transgenic strains have been established. Another major advantage of this approach is that proper transgene expression can be verified and controlled before the animal is created.

Fig. 2.

Schematic overview of the procedure for generating ES cell clones using the Z/EG vector system.

1.2 Doxycycline-Inducible Transgenics

Arguably, the most widely used inducible gene expression system is the Tet-On expression system. One of the disadvantages of this system has been its leakiness, but newly developed reverse tetracycline-controlled transactivator (rtTA) proteins, whose binding to tetracycline-response elements (TREs) is much more dependent on doxycycline, have virtually eliminated this problem. We have designed a novel Tet-On rtTA expression vector, called TRIC, which is tightly regulatable in both cultured cells and mice. In this vector, the CAGGS promoter drives the expression of a tightly regulatable rtTA gene and an IRES-puromycin-resistance gene (Fig. 1b). Immediately downstream of the puromycin-resistance gene are three polyadenylation sites to prevent CAGGS-mediated transcription beyond this gene. The polyadenylation sites are followed by a TRE that binds rtTA exclusively in the presence of doxycycline (Fig. 1b). A short polylinker allows for the insertion of the TOI cDNA behind this element. Downstream of the polylinker is an IRES-EGFP gene that serves as a marker for TOI expression. A major advantage of this vector system is that both the rtTA gene and the TRE-regulated TOI are present in the same vector and will thus co-integrate at the same genomic location, which tremendously simplifies mouse breeding. Again, linearized DNA is electroporated into ES cells, and clones resistant for puromycin are selected and clonally expanded in 96-well plates. Each clone is then split into three 96-well plates (Fig. 3). Cells in plate 1 are grown in the presence of doxycycline, and ES cell clones that express EGFP are identified by fluorescence microscopy 2 days later. Clones with the proper level of expression are harvested for verification of TOI expression by Western blot analysis (using anti-tag antibody). Cells in plate 2 are used to select single-copy transgene integration by Southern blotting, whereas cells in plate 3 are frozen when sub-confluent. ES clones with proper transgene expression are regrown and injected into blastocysts to produce male chimeras. Hemizygous transgenic mice derived from these chimeras are provided with drinking water containing doxycycline to induce transgene expression.

Fig. 3.

Schematic overview of the procedure for generating ES cell clones using the TRIC vector system.

A detailed description of the protocol for generating transgenic mice in ES cells is provided below. The protocol is based on the use of the pZ/EG and pTRIC expression systems, but can be easily adapted for use in other expression systems. This protocol is an adaptation of a previously published protocol (3) and is routinely used in the Transgenics and Gene Knockout Core Facility at Mayo Clinic Rochester.

2 Materials

2.1 Equipment for ES cell culture

• 1. A tissue culture hood with ultraviolet (UV) light, and gas and vacuum (for aspiration).

• 2. A water-jacketed 37°C incubator with 5% CO₂ and 20% O₂ gas and saturated humidity. Clean it thoroughly before the start of the gene-targeting experiment and use it only for the culture of ES cells and SNLH9 feeders for the duration of the experiment.

• 3. A high-capacitance electroporator. A Bio-rad Gene Pulser II is commonly used for this purpose.

• 4. An inverted microscope equipped with 4× (or 5×) and 10× phase-contrast objectives.

• 5. An inverted fluorescent microscope with 20× objective.

• 6. A table-top centrifuge with swing-out rotors for 15-mL tubes and 96-well plates.

• 7. 20- and 200-μl pipettes.

• 8. Multichannel pipette with a 20- to 200-μl volume range.

2.2 Disposables

• 1. The following tissue culture-treated flasks, dishes, and plates are used: 25-cm² (T25), 75-cm² (T75), and 150-cm² (T150) culture flasks (use 5, 10, and 20 mL of medium per flask, respectively); 10-cm dishes; 96-well flat- and U-bottom plates; and 24-well plates.

• 2. Electroporation cuvettes with an electrode gap of 0.4 cm.

• 3. 1-mL cryogenic vials.

• 4. 2-, 5-, 10-, and 25-mL disposable pipettes; 15- and 50-mL tubes; and a 50-mL reservoir for multichannel pipettes.

2.3 Tissue Culture Media and Solutions

2.3.1 Feeder Medium

Prepare this medium by adding the following sterile components to 500 mL of Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose (4,500 mg/L) and low bicarbonate (17 g NaHCO₃ mg/mL) (store at 4°C):

• 1. 50 mL of fetal calf serum (FCS). Long-term serum storage is in 50-mL aliquots at −20°C and short-term storage (for 1–2 months) at 4°C.

• 2. 5 mL of 200 mM l-glutamine. Store at −20°C.

• 3. 5 mL of 100 mM sodium pyruvate. Store at 4°C.

• 4. 5 mL of 10 mM non-essential amino acids. Store at 4°C.

• 5. 0.5 mL of 10 mg/mL gentamycin. Store at 4°C.

• 6. 0.5 mL of 5.5 × 10⁻² M β-mercaptoethanol. Store at 4°C.

2.3.2 ES Medium

Prepare this medium by adding the following sterile components to 500 mL of DMEM containing high glucose (4,500 mg/L) and low bicarbonate (17 g NaHCO₃ mg/mL) (store at 4°C):

• 1. 90 mL of ES-qualified FCS. Long-term serum storage is at −20°C. Short-term storage (for 1–2 months) is at 4°C (see Note 1).

• 2. 30 μl of 10.000.000 U/mL ESGRO (LIF). Store at 4°C (see Note 2).

• 3. 5 mL of 200 mM l-glutamine. Store at −20°C.

• 4. 5 mL of 100 mM sodium pyruvate. Store at 4°C.

• 5. 5 mL of 10 mM non-essential amino acids. Store at 4°C.

• 6. 0.5 mL of 10 mg/mL gentamycin. Store at 4°C.

• 7. 0.5 mL of 5.5 × 10⁻² M β-mercaptoethanol. Store at 4°C.

• 8. Nalgene filter unit (0.22 μm).

ES medium is filtered using a Nalgene filter unit (0.22 μm). Both feeder and ES media should be stored at 4°C, protected from light, and can be used for up to 1 month if l-glutamine is replaced every other week.

2.3.3 Freezing Medium

• 1. Freezing Medium (2×): Supplement 30 mL of DMEM with 50 mL of FCS and 20 mL of dimethylsulfoxide (DMSO).

• 2. Freezing Medium (1×): Supplement 40 mL of DMEM with 50 mL of FCS and 10 mL of DMSO.

Freezing medium is filtered using a Nalgene filter unit (0.22 μm). When stored at 4°C, protected from light, this medium can be used for 1 month.

2.3.4 PBS/0.1% Gelatin

• 1. Add 0.5 g of porcine skin gelatin to 500 mL of PBS without calcium and magnesium. Dissolve in microwave, filter sterilize using a Nalgene filter unit (0.22 μm), and store at room temperature (RT). Gelatin coating is done by incubating tissue culture surfaces with PBS/0.1% gelatin for 10–15 min (longer incubation time is not a problem).

2.3.5 Others

• 1. PBS without calcium and magnesium is used to wash cells. Store at RT.

• 2. TrypLE^TMExpress is used for cell trypsinization. Store at 4°C.

• 3. β-Galactosidase staining kit.

• 4. Doxycycline (for TRIC vectors).

3 Methods

3.1 Preparation of Vector DNA for Electroporation into ES Cells

• 1. Isolate targeting vector DNA from bacteria by alkaline lysis and anion-exchange chromatography.

• 2. Linearize about 20 μg of transgenic vector DNA with an appropriate restriction enzyme according to the instructions of the manufacturer.

• 3. Precipitate linearized DNA with ethanol. Add 1/10 vol. of 3 M sodium acetate (pH 5.2) and 5 μl of 20 mg/mL glycogen. Add 2.5× vol. of 100% ethanol. Mix and centrifuge at 18,400 rcf for 10 min at 4°C. Discard the supernatant being careful not to throw out DNA pellet.

• 4. Rinse with 70% ethanol and centrifuge again for 5 min.

• 5. Aspirate the 70% ethanol in a tissue culture hood and resuspend the DNA pellet at 0.2 μg/mL in sterile PBS. Analyze 0.5 μg of this DNA solution on a 0.8% agarose gel. Store the linearized vector at −20°C until use.

3.2 Preparation of Irradiated Feeder Layers

ES cells can be grown either on primary or immortalized (STO) mouse embryonic fibroblasts. Our laboratory is using TL1.4 ES cells that have been adapted for growth on STO cells. STO feeders that stably express the neomycin and hygromycin phosphotransferase genes (called SNLH9 feeders) are used to generate transgenics with Z/EG vectors, whereas SNLH9 feeders that also express the puromycin-N-acetyl-transferase gene (SNLH9-Puro feeders) are used to create transgenics with TRIC vectors. Preparation of feeders for use as monolayers is as follows:

• 1. About 4–5 days before you plan to start growing ES cells, thaw a vial of frozen SNLH9 (or SNLH9-Puro) cells and seed in a T75 flask. Culture at 37°C.

• 2. When the feeder cells are confluent (usually within 2 days), wash 2× with PBS and add 1.5 mL of TrypLE^TMExpress. After 5 min at 37°C, resuspend the cells in 30 mL of feeder medium and transfer to three T75 flasks.

• 3. When these three cultures become confluent, trypsinize the cells and suspend them in 20 mL of feeder medium. Count the cells and calculate the number of cells per milliliter. Seed the appropriate amounts of feeder cells in four gelatin-coated 10-cm dishes and one gelatin-coated T75 flask (see Table 1). Reseed the residual cells into the original T75 flasks for use later on (expand this culture if necessary).

  Table 1 SNLH9 feeder preparation on various tissue culture surfaces

• 4. The four 10-cm dishes and the T75 flask should be nearly confluent the next day. If so, transfer the cultures to a γ-irradiator (e.g., a ¹³⁷Cesium source) and expose the cells to 3,000 rads.

• 5. The feeders are now ready for use as ES cell feeders. ES cells can be seeded onto these feeders for up to 5 days post-irradiation.

3.3 Preparation of ES Cells for Electroporation

It is important to be sure that your frozen stocks of ES cells are truly pluripotent. In other words, they should have a demonstrated ability to produce germline-transmitting male chimeras following their injection into host C57BL/6 blastocysts. If you obtain a new parental ES cell line from another laboratory (or company), it is recommended that you first prepare a large number of frozen stocks (∼15–30 vials). Use one of these vials to perform blastocyst injections. A suitable ES cell line should produce a high frequency of germline-transmitting chimeric males in your laboratory.

There is no uniform guideline for the culture of ES cells because each independently generated cell line seems to have slightly different growth properties. Usually ES cells need to be diluted when the cultures reach approximately 70–80% confluency. A 70–80% confluent culture is generally diluted three- to sixfold. If diluted three times, the culture will be 70–80% confluent again within 24–36 h. If diluted six times, this will take about 48 h. If one would inadvertently seed ES cells at too low a density, the culture will not reach 70–80% confluency within 3–4 days. Because the formation of large ES cell clusters may trigger ­differentiation, it is advisable to trypsinize the ES cells on the fourth day after seeding. Dilute them only twofold to increase the cell density of the culture.

• 1. Thaw a vial of ES cells (∼10 × 10⁶) in a 37°C water bath. Transfer the ES cell suspension to a 15-mL tube with 5–10 mL of ES medium. Pellet the ES cells at 180 rcf for 5 min.

• 2. Aspirate the supernatant, gently resuspend the cells in 10 mL ES medium, and transfer to the T75 flask with irradiated feeders. Incubate at 37°C.

• 3. When the ES culture is about 70–80% confluent (20–30 × 10⁶ ES cells), aspirate the medium, wash the cell culture three times with 5 mL of PBS (gently swirl the dishes to remove dead cells and traces of ES medium), and add 2 mL of TrypLE^TMExpress. Evenly distribute the TrypLE^TMExpress and incubate at 37°C for 5 min.

• 4. Remove the T75 flask from the incubator and shake it vigorously to generate a single cell suspension. Add 8 mL of ES medium, transfer the suspension to a 15-mL tube, and pellet the ES cells at 180 rcf for 5 min.

• 5. Aspirate the supernatant, gently resuspend the cells in 5 mL of PBS, and pellet the cells again. Repeat this wash once.

• 6. After the last wash, resuspend the cells in PBS such that the total volume is 750 μl. Add 50 μl of the 0.2 μg/mL linearized vector solution (10 μg of total DNA for both Z/EG and TRIC vectors). Transfer the 800-μl cell suspension to an electroporation cuvette with a 0.4-cm gap size. Remove potential air bubbles (see Note 3).

• 7. Place the cuvette in the Bio-Rad Gene Pulser and electroporate at 230 V/500 μF. The time constant should be between 6 and 8 ms. Place the cuvette in the tissue culture hood and let it sit for 5 min (see Note 4).

• 8. In the meantime, fill a 50-mL tube with 40 mL of ES medium. At 5 min after electroporation, gently transfer the ES cells to the 50-mL tube.

• 9. Aspirate the medium from the four 10-cm dishes with irradiated feeders and plate 10 mL of aliquots from the ES cell suspension in the 50-mL tube.

• 10. Exactly 24 h after the electroporation, replace the ES medium on the plates with drug-containing ES medium. The following final drug concentrations are used: G418, 350 μg/mL (for Z/EG vectors) and puromycin, 2 μg/mL (for TRIC vectors). Typically, 8 days after electroporation, ES colonies are ready to be picked. Occasionally, the cells are ready for picking a day earlier or later.

3.4 Picking of Drug-Resistant ES Clones

It takes some experience to identify truly drug-resistant ES colonies rapidly. The shape of the colonies can be dependent on the drug combinations used for ES cell selection. Suitable ES colonies have a uniform appearance with relatively sharp edges due to three-dimensional growth. The center of an ES colony usually has a higher cell density and is somewhat darker in color. ES colonies with a flat “pancake”-like appearance should not be picked because they consist of differentiated ES cells. See Figs. 2 and 3 for a schematic overview of the picking procedure.

• 1. Prepare feeder layers 2–3 days prior to the scheduled picking of the ES cell colonies. For experiments in which Z/EG-based transgenic vectors are used, prepare a total of seven gelatin-coated, 96-well flat-bottom plates with SNLH9 feeders: five plates with 0.1 × 10⁵ feeder cells per well, and two plates with 0.05 × 10⁵ cells per well. For experiments in which TRIC-based transgenic vectors are used, prepare a total of four gelatin-coated, 96-well flat-bottom plates with SNLH9-puro feeders (0.1 × 10⁵ feeder cells per well). For both vector types, prepare two gelatin-coated, 24-well plates with 0.3 × 10⁵ feeders per well (see Note 5).

• 2. Fill a sterile 50-mL reagent reservoir with TrypLE^TMExpress solution and use a multi-channel pipette to add 25 μl of TrypLE^TMExpress in each of the wells of two round-bottom 96-well plates. Prevent evaporation of the TrypLE^TMExpress solution by placing the plates in the 37°C incubator.

• 3. Next, load a sterile 50-mL reagent reservoir with ES selection medium and remove the feeder medium from a 96-well ­flat-bottom plate with 0.1 × 10⁵ irradiated feeders per well. Add 200 μl of ES medium with 350 μg/mL of G418 or 2 μg/mL of puromycin (selection medium) to each of the wells and place the plates back in the 37°C incubator.

• 4. Take a 10-cm dish from the incubator and divide it into four quadrants with a permanent marker (to allow systematic screening of the dish for drug-resistant colonies). Leave the ES selection medium in the dish to preserve the normal morphology of the ES clones during the picking (see Note 6).

• 5. Set an adjustable 20-μl pipette at 2.5–3 μl and apply a standard 200-μl tip. Start scanning the first quadrant for suitable ES colonies. When a suitable colony appears, use the tip of the pipette to interrupt the feeder monolayer around the colony (by “drawing” circles around the colony). Then detach the colony and draw it into the pipette tip with 2.5 μl of ES selection medium (see Note 7). Transfer the colony to the first well of the round-bottom 96-well plate with TrypLE^TM Express solution (pipette up and down at least five times).

• 6. Pick up a total of 48 colonies (assuming that the picking time for 48 colonies is less than 25 min). Change pipette tips between colonies to avoid cross-contamination. After the picking, incubate the plate for 5 min at 37°C.

• 7. Take the trypsinized ES cells and one of the 96-well plates with irradiated feeder monolayers out of the incubator. Disaggregate the ES cells in row A of the round-bottom 96-well plate using a multichannel pipette (pipette up and down at least 10–20 times). Then, transfer the suspended ES cells to row A of the 96-well plate with feeders. Repeat this procedure for the remaining rows. Incubate the plate in a 37°C incubator.

• 8. Add 48 additional colonies to this plate by repeating steps 4–7.

• 9. Usually 2 days after picking, most of the colonies will be 80–90% confluent. The ES cell clones are then ready to be split into three new flat-bottom 96-well plates (0.1 × 10⁵ feeders per well). Separate protocols now apply to Z/EG- and TRIC-based transgenic vectors, which are detailed below.

3.5 Identification of Transgenic ES Clones

3.5.1 Identification of Z/EG-Derived Transgenic ES Clones

• 1. Replace the feeder medium of two 96-well flat-bottom plates containing 0.1 × 10⁵ SNLH9 feeder cells with 250 μl of ES medium (without G418). Replace the feeder medium of one 96-well flat-bottom plate containing 0.1 × 10⁵ SNLH9 feeder cells with 200 μl of ES medium (also without G418). Return all three plates to the 37°C incubator.

• 2. Take the 96-well plate with sub-confluent ES cells from the incubator. Aspirate most of the ES medium using vacuum suction. Then remove the remainder of the medium with a multichannel pipette (use clean tips for each row). Wash ES cells by pipetting 100 μl of PBS gently up and down with a multichannel pipette.

• 3. After all washes have been completed, add 50 μl of TrypLE^TM Express to each well and incubate the plate for 5 min at 37°C.

• 4. In the meantime, take two 96-well flat-bottom plates with 250 μl of ES medium and one with 200 μl of ES medium from the incubator, and align these three plates in the hood.

• 5. Place the 96-well plate containing the ES clones in the hood (Fig. 2). Disaggregate the ES cells in row A using the multichannel pipette by vigorously pipetting the trypsin up and down at least ten times. Then, transfer 50 μl of ES medium from row A of each of the two feeder plates containing 250 μl of ES medium to row A of the plate with the trypsinized ES cells. Dispense 50 μl of ES cell suspension into row A of each of the three 96-well plates with irradiated feeders. When ES cells of all eight rows have been split, incubate all three plates at 37°C. Number the plates 1–3 (Fig. 2).

• 6. Plate #1. Immediately after splitting, add adeno-Cre virus (10 MOI). Refresh the virus at 24 and 48 h. At 54–60 h, remove all but 25 μl of ES medium and measure EGFP expression by fluorescence microscopy. Each clone is scored for EGFP intensity (grade as follows: low, medium, or high) and percentage EGFP-positive ES cells. Select 48 clones with the required level of EGFP expression (low [+], medium [++], and/or high [+++]) and transfer them to a gelatin-coated 24-well plate with 0.3 × 10⁵ irradiated SNLH9 feeder cells (Fig. 2). When ES cultures in these wells become semi-confluent, wash the cells twice with PBS. Add 100 μl of SDS sample (Laemmli) buffer to each well to lyse the cells. Transfer each lysate to an Eppendorf tube and boil for 10 min. These lysates are then used to check for the expression of the TOI by Western blot analysis for the epitope tag.

• 7. Plate #2. ES cells in this plate are stained for β-galactosidase as a measure of CAGGS promoter activity (Fig. 2). This is usually done the day after splitting. We use a β-galactosidase staining kit from Roche. Fixation and staining are carried out according to the manufacturer’s protocol. Each clone is scored for both staining intensity (suggested grading: low, medium, or high) and percentage of β-galactosidase-positive ES cells.

• 8. Plate #3. This plate will be used to prepare both frozen ES cell stocks and genomic DNA for Southern blot analysis of transgene copy number (Fig. 2). For this, the ES clones in plate #3 need to be split into three plates. Usually, the ES cells are ready for splitting 2 days after the initial seeding. The procedure entails essentially a repeat of steps 1–5. The only difference is that one 96-well flat-bottom plate contains 0.1 × 10⁵ SNLH9 cells per well and the other two only 0.05 × 10⁵. The plate with the highest SNLH9 cell density, designated plate #3a, will be frozen at −80°C when the ES cells become 70–80% confluent (usually the next day; the cell freezing step is explained in steps 9 and 10) (Fig. 2). The other two plates, designated plates #3b and #3c, will be used for Southern blot analysis. When the medium turns orange to yellow, refresh the ES medium daily until most ES clones have become super-confluent (usually this takes 4–5 days). To harvest the super-confluent cells, remove the lid, invert the plate, and vigorously tap it into a stack of tissue towels until all residual medium has been removed (do not be afraid to lose ES cells as they will be firmly attached to the plates). No washing with PBS is necessary. Tape the lid to the plate and freeze it for at least 2 h at −80°C (see Note 8). Normally, only one of the two plates will be used for Southern blot analysis. The other plate serves as a backup in case an alternative Southern strategy is needed.

• 9. To freeze ES clones in plate #3a, aspirate most of the ES medium from the wells using vacuum suction. Remove the remainder of the medium with a multichannel pipette (use clean tips for each row). Wash the ES cells by pipetting 100 μl of PBS up and down in the wells using a multichannel pipette. After washing all wells, add 30 μl of TrypLE^TMExpress to each of the wells and incubate the plate for 5 min at 37°C.

• 10. Make a 1:1 mixture of ES medium and FCS (ES medium with ∼50% FCS) and place it in a multichannel reagent reservoir. Then disaggregate the trypsinized ES cells in row A using a multichannel pipette. Pipette vigorously up and down at least 10–20 times. Add 75 μl of medium from a reagent reservoir to the cells in row A and mix. Repeat the procedure for rows B–G.

• 11. Finally, mix the contents of each well with 100 μl of ice-cold 2× freezing medium (pipette vigorously up and down at least five times to dilute the freezing medium). Quickly seal the plate with masking tape, wrap it in several layers of tissue towel, and place it in a closed styrofoam box that is kept in a −80°C freezer. ES cells maintain their viability for up to 2 months but should be placed at −145°C if storage beyond this point is necessary.

3.5.2 Identification of TRIC-Derived Transgenic ES Clones

• 1. Replace the feeder medium of two 96-well flat-bottom plates with irradiated SNLH9-puro feeder cells with 250 μl of ES medium (without puromycin). Replace the feeder medium of one 96-well flat-bottom plate with irradiated SNLH9-puro feeder cells with 200 μl of ES medium (also without puromycin). Return all the three plates to the 37°C incubator.

• 2. Take the 96-well dish with sub-confluent puro-resistant ES clones from the incubator and divide the cells over the three flat-bottom plates with irradiated SNLH9-puro feeder cells by following steps 2–5 of Subheading 3.5.1. Number the plates 1–3 and process each plate as indicated below (Fig. 3).

• 3. Plate #1. At 24 h after splitting, add doxycycline to a final concentration of 0.1 mg/mL to activate TOI and EGFP expression. At 48 h, remove all but 25 μl of ES medium and estimate EGFP expression by fluorescence microscopy (Fig. 3). Each clone is scored for EGFP intensity (grade as follows: low, medium, or high) and percentage of EGFP-positive ES cells (see Note 9). Select 48 clones with proper EGFP expression (low, medium, and/or high) and transfer them to gelatin-coated 24-well plates with 0.3 × 10⁵ irradiated SNLH9 feeder cells. When ES cells in these wells become semi-confluent, wash the cells twice with PBS. Add 100 μl of SDS sample buffer to lyse the cells. Transfer each lysate to an Eppendorf tube and boil for 10 min. The lysates are used to check for expression of the TOI by Western blot analysis for the epitope tag.

• 4. Plate #2. Use this plate for Southern blot analysis (if necessary, one can create a backup plate for Sourthern blot analysis by splitting the cells into two 96-well plates). Refresh the ES medium daily until most of the ES clones have become super-confluent. To harvest the super-confluent cells, remove the lid from the plate, invert the plate, and vigorously tap the plate onto a stack of tissue towels until all residual medium has been removed. Tape the lid to the plate and freeze it for at least 2 h at −80°C (see Note 8).

• 5. Plate #3. Use this plate to prepare frozen ES cell stocks. The freezing procedure is detailed in steps 9–11 of Subheading 3.5.1 (Fig. 3).

3.5.3 Extraction and Restriction Enzyme Digestion of DNA from ES Cells

Allen Bradley and coworkers (4) have developed the following procedure for extraction and restriction enzyme digestion of genomic DNA.

• 1. 96-well plates stored in −80°C freezer are incubated for 5 min at RT.

• 2. Add 50 μl of lysis buffer (10 mM Tris–HCl, pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% sarcosyl, and freshly added 1 mg/mL proteinase K [e.g., Sigma cat. no. P-2308]) to each of the wells, using a multichannel pipette (see Note 10).

• 3. Seal the edges of the 96-well plates with masking tape and incubate them for 3 days at 55°C in a box with water-soaked tissue towels.

• 4. Pre-cool the plates on ice. In the meantime, prepare a ­mixture of 10 mL of ice-cold 100% ethanol and 150 μl of 5 M NaCl. Add 100 μl of this mixture to each well using a multichannel pipette. Allow the plates to sit at RT for at least 3 h. Then screen each well for the presence of precipitated genomic DNA using a microscope with low-power magnification. Precipitated genomic DNA typically has a “web-like” appearance. If DNA is not observable in all wells, continue to incubate the plate at RT (see Note 11).

• 5. Spin the plates at 2,200 rcf for 15 min. Discard the supernatant by careful inversion of the plates. The DNA will adhere to the plastic. Gently add 100 μl of 80% ethanol to each well with a multichannel pipette to wash the precipitated DNA. Invert the plates onto paper towels to discard the wash solution thoroughly. Repeat the ethanol wash three times. After the last wash, add 100 μl of 80% ethanol for 30 min. Either store the plates at −20°C or continue with step 6.

• 6. Invert the plates to remove as much ethanol solution as possible. Leave the plates tilted to air dry for 20 min. Screen the wells for complete evaporation of the ethanol wash solution. If traces of ethanol remain, restriction enzymes will incompletely cut the genomic ES cell DNA.

• 7. While the plates are drying, prepare a restriction digest cocktail. A typical mixture contains the optimal 1× restriction buffer for the enzyme used, 1 mM spermidine, 100 μg/mL acetylated BSA, 50 μg/mL RNase, and 10–15 units enzyme per sample.

• 8. Add 35 μl of restriction digest cocktail per well with a multichannel pipette. Tap the plates to ensure that the cocktail completely covers the surface of each well.

• 9. Seal the plates with masking tape and incubate them overnight in a box with water-soaked tissue towels at the temperature specified by the manufacturer of the enzyme.

• 10. The next day, suspend each DNA solution by pipetting it up and down using a 200-μl pipette. Add 10–15 units of fresh enzyme to each well and incubate for an additional 24 h.

• 11. Take 5 μl of sample from 5–10 random wells and run these on a 0.8% agarose gel to check whether the DNA is properly digested. If so, continue as described in Subheading 3.5.4. If digestion appears incomplete, repeat step 10.

3.5.4 Gel Electrophoresis and Southern Blot Analysis

• 1. Take the plates out of the incubator and add 7 μl of DNA loading buffer to each digest using a multichannel pipette. Place the plates in the refrigerator until electrophoresis (or freeze if longer storage is required).

• 2. Prepare 0.8–1.0% agarose gels in 1× TAE for electrophoresis of the digests. Use an electrophoresis system that has a capacity for many samples.

• 3. Perform electrophoresis at 20–40 V until the diagnostic wild-type and mutant fragments are adequately segregated (usually requires overnight electrophoresis).

• 4. Photograph the gels and mark the positions of DNA marker band with a Pasteur pipette (punch holes in the gel). Using a scalpel, cut the area of gel to be blotted.

• 5. To hydrolyze the DNA, soak the gel pieces in 0.25 M HCl for 40 min. Mix constantly using a shaking platform (e.g., a Belly Dancer).

• 6. While gels are incubating, cut a Hybond N⁺ nylon membrane for each gel piece. Engrave a blot ID in the upper left corner of each membrane (date, gene, and investigator).

• 7. Carefully pour off the 0.25 N HCl (avoid breaking of gels) and incubate in 0.4 M NaOH for 20 min on a shaking platform. Refresh the 0.4 M NaOH solution and incubate for another 20 min.

• 8. Fast, efficient, and reproducible transfer of DNA fragments from gel to nylon membrane is achieved by vacuum blotting (e.g., Stratagene, cat. no. 400330). Transfer for 2–3 h.

• 9. Stop the blotting and rinse the membrane in 3× SSC for about 30 s. Repeat the rinse one more time.

• 10. Hybridization is carried out in roller bottles in a hybridization oven (e.g., Stovall Life Science Inc.) at 65°C. Transfer the membranes to hybridization tubes with the DNA side ­facing the inner tube. Pre-hybridize for 30 min in hybridization buffer at 65°C (hybridization buffer: 30 mL 20 × SSC, 5 mL 100× Denhardt’s solution, 60 mL 15% dextran sulfate, 1 mL 10 mg/mL salmon sperm DNA, and 5 mL 10% SDS).

• 11. While pre-hybridizing the membranes, radioactively label the DNA probe.

• 12. Replace the hybridization buffer once. Add the denatured probe 30 min later and hybridize overnight at 65°C.

• 13. First, wash the membrane twice with 2× SSC/0.1% SDS for 10 min at RT (on a Belly Dancer). Then wash with pre-warmed 2× SSC/0.1% SDS for 5 min at 65°C (with agitation). If necessary, continue to wash with the following pre-warmed solutions: 1× SSC/0.1% SDS, 0.3× SSC/0.1% SDS, and 0.1× SSC/0.1% SDS. Wrap properly washed membranes in plastic, and carefully remove excess liquid and expose the films at −80°C.

• 14. For Z/EG-based transgenesis, it is critical to select transgenic ES clones that have single-copy integrations. Because TRIC-based transgenesis does not involve Cre recombination, it is not essential to select single integrants, although ES clones with single integrations are preferred from a mouse breeding perspective.

3.6 Karyotype Analysis and Long-Term Storage of Transgenic ES Clones

Transgenic ES clones must be thawed and expanded for karyotyping and preparation of frozen stocks. The procedure is the same for both Z/EG- and TRIC-based transgenic ES clones. Selection of transgenic ES clones for reseeding is based on TOI and EGFP expression (as assessed by Western blotting and fluorescence microscopy, respectively) and the transgene copy number (as assessed by Southern blotting).

• 1. Prepare one gelatin-coated 96-well flat-bottom plate, four 24-well plates, and one 12-well plate with the appropriate amounts of feeder cells (see Table 1) 2–3 days prior to the scheduled reseeding of the transgenic ES cell colonies.

• 2. Thaw the plate containing the ES cell clones of interest by letting it float in a 37°C water bath. When the freezing medium in the plate has melted, suspend the cells in the freezing medium and transfer them to a 15-mL tube containing 2 mL of ES medium. Spin at 180 rcf for 5 min.

• 3. Resuspend the pellet in 200 μl of ES medium and transfer the cell suspension to a well in a 96-well plate with irradiated feeders. Incubate at 37°C.

• 4. Monitor the growth of the ES cells closely and split clones that have reached 70–80% confluency. Aspirate the medium, wash the cell culture two times with 200 μl of PBS, add 35 μl of TrypLE^TMExpress, and incubate at 37°C for 5 min.

• 5. Remove the plate from the incubator and thoroughly suspend the trypinized cells to generate a single-cell suspension. Add 100 μl of ES medium and transfer the suspension to a 24-well plate with irradiated feeders. The total volume of ES medium per 24-well should be ∼1 mL.

• 6. Once the ES clones reach 70–80% confluency, transfer each clone to three wells of a 24-well plate with irradiated feeders for further expansion. Use two of these wells to make frozen stocks (see steps 7–9) and one well for karyotyping (see steps 10–13).

• 7. Freezing: when the 24 wells become semi-confluent, rinse two of the wells with PBS, add 200 μl of TrypLE^TMExpress, and incubate for 5 min at 37°C.

• 8. Resuspend the cells and add 1 mL of ES medium. Transfer both cell suspensions to one 15-mL tube and spin for 5 min at 180 rcf.

• 9. Aspirate the medium from the tube and suspend the cell pellet in 2 mL of 1× freezing medium. Divide the cell suspension over two cryogenic vials. Wrap these vials in tissue towels and place them in a styrofoam box in a −80°C freezer. The next day, transfer the vials to liquid nitrogen for long-term storage.

• 10. Karyotyping: when the 24-well designated for karyotyping becomes semi-confluent, transfer the cells to a 12-well plate with irradiated SNLH9 feeders (see Table 1). When the cells become semi-confluent, add 50 μl of Colcemid per mL of medium (10 μg/mL stock) and incubate at 37°C for 4 h.

• 11. Harvest the cells by trypsinization, centrifuge at 180 rcf for 5 min, aspirate the supernatant, and suspend the pellet in 5 mL of pre-warmed hypotonic solution (freshly prepared 0.56 g of KCl in 100 mL of MilliQ water) in a 15-mL tube. Incubate at 37°C for exactly 15 min.

• 12. Add 200 μl of fixative (30 mL of methanol and 10 mL of acetic acid, freshly prepared) and centrifuge at 180 rcf for 5 min. Remove the supernatant. Wash the pellet in 5 mL of fixative, centrifuge at 180 rcf for 5 min, and remove the supernatant. Repeat this wash step once more (if necessary the cells can now be stored at 4°C).

• 13. Resuspend the pellet in 200 μl of fixative. Pipette about 20 μl of suspension and add two drops on a microscope slide from a height of about 2 ft. Prepare two slides per transgenic ES clone. Place the slides in Giemsa staining solution for 15 min. Wash for 2 min with distilled water and three times for 30 s with MilliQ. Let the slides dry at RT and count chromosomes of at least ten metaphases (use a 100× oil objective). ES clones that have 0–20% aneuploid spreads are selected for microinjection into blastocysts.

3.7 Preparation of Transgenic ES Clones for Blastocyst Injection

• 1. Prepare a 24-well plate with the irradiated feeder cells as described in Subheading 3.2. Approximately 2–3 days before the scheduled microinjection, thaw a vial of ES cells in a 37°C water bath. Transfer the ES cell suspension to a 15-mL tube with 4 mL of ES medium and pellet the ES cells at 180 rcf for 5 min.

• 2. Aspirate the supernatant, gently resuspend the cells in 1 mL of ES medium, and transfer to a well of the 24-well plate with irradiated SNLH9 feeders. Incubate at 37°C.

• 3. Once the transgenic clones reach 75–90% confluence (usually within 1–2 days after seeding), trypsinize and reseed them at different dilutions (1:4 and 1:6).

• 4. On the day of blastocyst injection, select a well that is 40–80% confluent. Rinse the well three times with PBS, add 0.5 mL of TrypLE^TM Express, and incubate for 5 min at 37°C.

• 5. Resuspend the cells in 2 mL of ES medium. Transfer 2 mL of the cell suspension to a 15-mL tube and spin at 180 rcf for 5 min (reseed the remaining 0.5 mL of the cell suspension in a well of a 24-well plate with irradiated feeder cells to allow for re-injection of the ES clone, if necessary).

• 6. Aspirate the supernatant and resuspend the cells in 250 μl of ES medium. Place the cells on ice. Use them for microinjection within 1–2 h following trypsinization.

• 7. Typically, 16–24 embryos are injected per transgenic ES clone. Optimally, three to five independent transgenic ES clones will be injected.

4 Notes

• 1. It is important to test serum lots for high ES cell plating efficiency. Companies will send out free aliquots of ES-qualified serum that one can use for selecting the serum with the highest plating efficiency. Test the plating efficiency by seeding 1,000 ES cells onto 10-cm dishes with SNLH9 feeder layers. After 5–7 days, high-quality sera will show a colony-forming efficiency of about 30% or more of the plated cells.

• 2. Although SNLH9 feeders cells should produce sufficient LIF to inhibit the differentiation of ES cells, many investigators using feeders add an extra 1,000 Unit/mL of LIF to the culture medium to assure that the LIF levels remain consistently high.

• 3. The ES cell/DNA mixture can immediately be shocked. Pre-incubations of the ES cell/targeting vector mixture have no beneficial effect on the ultimate targeting frequency.

• 4. Substantial cell lysis will occur during electroporation, which will give rise to a somewhat viscous cell suspension. This is a normal phenomenon.

• 5. Picking 96 ES clones should be sufficient to obtain three to five ES clones that meet all requirements for injection into blastocysts. In instances where more clones need to be obtained, simply pick a total of 192 ES clones.

• 6. Replacing the medium with PBS will round-up ES cells and will make the selection of ES colonies with optimal morphology more difficult.

• 7. The 2.5 μl of ES medium does not interfere with the proper trypsinization of the ES colony.

• 8. Storage at −80°C will crack the cells. This facilitates cell lysis and proteinase K digestion, and increases the DNA yield.

• 9. For unknown reasons, only a relatively small proportion of the ES cells in each well will be EGFP positive. Also, the level of expression between individual cells is quite variable. It is important to note, however, that such mosaicism is not observed at the level of the whole animal. So, it is not a cause for concern.

• 10. Lysis buffer lacking proteinase K (incomplete lysis buffer) can be stored at RT. Prepare a 10 mg/mL stock of proteinase K in MilliQ and freeze it in 500 μl of aliquots. Add 500 μl of proteinase K solution to 5 mL of incomplete lysis buffer. The resulting solution is sufficient for one 96-well plate.

• 11. If the proteinase K digestion is incomplete, it will take longer for the DNA to precipitate.