Generation of Genetically Modified Mice through the Microinjection of Oocytes

All procedures have been approved by the University of New South Wales Animals Care and Ethics Committee.

1. Preparation of the Transgene (Random Integration)

1. Analytical agarose gel electrophoresis.
   1. Digest the plasmid to excise the transgene using appropriate enzymes (1 h incubation) or fast-digest enzymes (15 to 30 min incubation) in a thermocycler following the manufacturer's recommendations (see Figure 2A and its legend).
   2. Cast a 1% tris-acetate-ethylenediaminetetraacetic acid (EDTA) (TEA) agarose gel, stained with 0.5-1.0 µg/mL ethidium bromide (EtBr).
      NOTE: Use caution when using EtBr; it is a potent mutagen. Please use appropriate personal protective equipment (refer to the material safety data sheet).
   3. Load a 1 kb molecular weight marker.
   4. Load the linearized fragments (i.e., transgene and backbone).
   5. Run the electrophoresis at 100 V for 45 min.
   6. Visualize the gel on an ultraviolet (UV) transilluminator to check that the digestion is complete and to confirm the correct size of the transgene (i.e., 7,338 bp; see Figure 2A).
2. Preparative agarose gel electrophoresis.
   1. Cast a 1% TAE agarose gel stained with a low-toxicity gel stain. Load 8 aliquots (1 µg each) of linearized transgene without a molecular weight marker and run the electrophoresis at 100 V for 45 min. Use a scalpel to excise all 8 bands corresponding to the linearized transgene.
   2. Extract the DNA using a gel extraction kit by following the manufacturer's recommendations (see the Table of Materials).
      1. Melt the gel fragments at 50 °C in 1.5-mL tubes for at least 15 min. Precipitate it with isopropanol and then add the binding buffer.
      2. Combine the entire DNA by pipetting it all into one column. Elute into nuclease-free microinjection buffer (8 mM Tris-HCl and 0.15 mM EDTA). Filter through a 0.22 µm microcentrifuge filter and centrifuge for 60 s at 12,000 x g.
   3. Measure the concentration and the quality of the DNA using a spectrophotometer. Check that the A260/A280 ratio is around 1.8 (i.e., no contamination by proteins) and the A260/A230 ratio is at least 2.0 (i.e., no contamination by organic solvents). Dilute down to 3 ng/µL in nuclease-free microinjection buffer, aliquot (20-50 µL), and freeze at -20 °C.

2. Synthesis of CRISPR Components (Gene Targeting)

1. Cas9 mRNA.
   1. Dilute the Cas9 mRNA (obtained from a commercial source, see the Table of Materials) to a concentration of 1 µg/µL in nuclease-free microinjection buffer.
   2. Aliquot in 2 µL and freeze at -80 °C.
2. Single-guide RNA (SgRNA).
   1. Identify the desired two-target genomic sequences (guides) using a computational design tool allowing minimal potential off-target activity¹⁷.
      1. For gene knockout, systematically select two guides located a few hundred base-pairs (bp) apart, in opposite directions, and include the start codon of the gene of interest.  For homology direct repair, select two overlapping guides (whenever possible), in opposite directions.
         NOTE: As an example, the following guides have recently been successfully co-injected to disrupt the first exon of the TPM4.2 gene:
         Guide 1: 5' CGCCATCCAGTTCGCGCTGC 3';
         Guide 2: 5' CAGAACGATTGAGCTATGGC 3'
   2. Order a set of primers as described in Table 1.
   3. Synthesize a linear DNA template.
      1. Dilute px330¹² plasmid to 10 ng/µL in nuclease-free water.
      2. Prepare the master mix as indicated in Table 1. Add the polymerase at the end and keep the master mix on ice.
      3. Mix and split this into 8 PCR tubes (19 µL/tube). Add 1 µL of px330 per tube and run the PCR under the following conditions: 1 min at 98 °C; 40 cycles of 98 °C for 10 s, 64 °C for 30 s, and 72 °C for 15 s; and a final elongation at 72 °C for 5 min. Hold at 4 °C.
      4. Purify the PCR products using a PCR purification kit (see the Table of Materials), a manifold, and a vacuum source (for faster processing). Apply the maximum vacuum strength (i.e., 8 mbar).
         1. Add 5 volumes (100 µL) of binding buffer to 1 volume of the PCR sample and mix.
         2. Place a (provided) silica membrane spin column in a (provided) 2-mL collection tube for centrifugation, or on the manifold for vacuum processing. To bind DNA, successively apply all 8 samples to the column and vacuum or centrifuge for 60 s at 12,000 x g for each load.
         3. To wash, add 0.75 mL of wash buffer (buffer PE) to the column and vacuum or centrifuge for 60 s at 12,000 x g. Centrifuge the column for 60 s at 12,000 x g to eliminate residual ethanol.
         4. Place the column in a clean 1.5-mL microcentrifuge tube. To elute the DNA, add 30 µL of nuclease-free water to the center of the membrane, let the column stand for 1 min, and centrifuge for 60 s at 12,000 g.
      5. Measure the concentration using a spectrophotometer; typically, the concentration should be 100 ng/µL or higher. Check that the A260/A280 ratio is around 1.8 (i.e., no contamination by proteins) and the A260/A230 ratio is at least 2.0 (i.e., no contamination by organic solvents).
   4. In vitro transcription using a T7 RNA synthesis kit.
      1. Prepare the master mix, as indicated in Table 1, and incubate for 3 h at 37 °C in a thermocycler. Add 28 µL of nuclease-free water and 2 µL of DNase I and incubate for another 15 min at 37 °C in a thermocycler.
   5. RNA purification using spin columns.
      1. Dissolve the powder contained in the provided spin column in 650 µL of nuclease-free microinjection buffer, carefully removing all air bubbles. Cap the tube and hydrate for 5 – 15 min at room temperature.
      2. Remove the blue cap at the bottom and place the column in a 2-mL tube. Centrifuge for 2 min at 750 x g and room temperature. Place the column in a fresh 1.5-mL tube and apply 50 µL of the RNA solution dropwise to the center, without touching the column wall. Spin the column for 2 min at 750 x g.
      3. Measure the RNA concentration using a spectrophotometer (the typical yield of one reaction is 30 – 50 µg). Check that the A260/A280 ratio is around 2.0 (i.e., no contamination by proteins) and the A260/A230 ratio is at least 2.0 (i.e., no contamination by organic solvents). Store the sgRNAs at -80 °C until use.
      4. Assess the quality of the RNA using the 1% TAE gels routinely used for DNA electrophoresis. Run 200-400 ng of the DNA template and the corresponding sgRNA in parallel. The RNA band (≈ 100 bp) should be slightly bigger than the DNA band (see Figure 3A).

3. Donor Template

1. Order commercially synthesized single-stranded oligonucleotides (ssOligos, see the Table of Materials) for point mutations or for the integration of small sequences up to 50 bp; homology arms are typically 60-90 bp long.
2. For larger insertions, use a donor plasmid as a template. Generate a suitable plasmid using either the classic cloning method or de novo synthesis from a commercial source.
   NOTE: A minimum of 800 bp per homology arm is recommended. The size of the backbone has no influence on the efficiency of homology direct repair (HDR).

4. Injection Mix

1. Dilute the Cas9 mRNA to 50 ng/µL and the sgRNAs to 12.5 ng/µL (25 ng/µL in total) using nuclease-free microinjection buffer for gene knockout experiments. Add the donor template (ssOligo or plasmid) at a concentration of 200 ng/µL for genome editing experiments based on homology direct repair.
   NOTE: Dilution volumes can be easily determined using online tools, such as a resuspension calculator¹⁸.
2. Keep each aliquot on ice during the microinjection session and discard afterwards (do not re-freeze the injection mix).

5. Scrotal Vasectomy

NOTE: Two types of vasectomy are commonly performed in mice: abdominal and scrotal. The latter is less invasive and has been previously described¹⁵.

1. Autoclave all stainless steel surgical instruments.
2. Determine the weight of the mouse using a weigh scale and anesthetize the males by intraperitoneal (i.p.) injection with injectable anesthetics (ketamine 100 mg/kg, xylazine 10 mg/kg).
3. Monitor the loss of toe pinch reflex and once the mouse is sedated place it on top of a heating pad.
4. Disinfect the skin around the testes with alternating wipes of a topical antiseptic such as chlorhexidine and 70% ethanol.
5. Push the testes into the scrotal sac and make a skin incision with a scalpel.
6. Visualize the testis, cauda epididymis, and vas deferens.
7. Grab the vas deferens with forceps, hold it, and cauterize on each side of the forceps to remove 3 mm of the vas deferens.
8. Perform the same procedure on the second testis.
9. Close the skin incisions with wound clips and monitor the mouse closely until full recovery.

6. Superovulation (Oocytes Donors) and Time-mating (Pseudo-pregnant Females)

NOTE: The technique to generate a suitable number of fertilized oocytes and plugged foster females for reimplantation has been described elsewhere¹⁵.

1. Inject 10 females i.p. with 5 IU of pregnant mare's serum gonadotropin (PMSG; in 100 µL) at around 12 pm on day 1.
2. Inject the females i.p. with 5 IU of human chorionic gonadotropin (hCG; in 100 µL) 46-48 h later (at around 11 am on day 3).
3. Immediately mate the females with single-housed males overnight.

7. Pronuclear (random-integration) and Cytoplasmic (Gene-targeting) Injections

1. Cull the superovulated mice by cervical dislocation, expose the abdomen, and access the ovaries and oviducts, as previously described¹⁵. Dissect the oviducts and harvest the cumulus-oocyte-complexes (COCs) under a stereomicorscope¹⁵. Place them in a drop of potassium simplex optimization medium supplemented with amino acids (KSOMaa) following the traditional method¹⁵.
2. Purify the oocytes by adding approximatively 1 µL of hyaluronidase (10 mg/mL) to the drop of KSOMaa medium using an aspirator mouth piece and place the dish in a 37 °C / 5% CO₂ incubator for 30 s to 1 min.
3. Wash the oocytes with 4 fresh drops of KSOMaa medium using the aspirator mouth piece and transfer them to a final drop of KSOMaa medium overlaid with mineral oil (around 2 mL). Place the dish in the 37 °C / 5% CO₂ incubator until the oocytes are ready for injection.
4. Pronuclear injection (random integration).
   1. Visualize the eggs under the stereoscopic microscope and transfer approximately 50 eggs to the injection chamber (a drop of M2 medium overlaid with mineral oil) using the aspirator mouth piece.
   2. Transfer the injection chamber to the inverted microscope and inject the fertilized oocytes (two visible pronuclei) with a few picoliters of the injection mix, targeting a pronucleus (any of the pronuclei can be targeted). Visualize a successful injection by observing the swelling of the pronucleus.
5. Cytoplasmic injection (gene targeting).
   1. Transfer approximately 50 eggs to a drop of KSOMaa containing 5 µg/mL Cytochalasin B using the mouth piece and incubate for 5 min in the 37 °C / 5% CO₂ incubator.
   2. Transfer the eggs to the injection chamber using the mouth piece.
   3. Inject few picoliters of the injection mix into the cytoplasm at very low pressure (50-100 hPa), using the compensation pressure of the automated microinjector where possible.
   4. After injection, transfer the oocytes back into a drop of KSOMaa (using the mouth piece) and keep them in the 37 °C/5% CO₂ incubator, until loaded for reimplantation into the oviduct of pseudo-pregnant females.

8. Reimplantation

1. Autoclave all stainless steel surgical instruments.
2. Determine the weight of the mouse using the weigh scale and anesthetize the females by intraperitoneal (i.p.) injection with injectable anesthetics (ketamine 100 mg/kg, xylazine 10 mg/kg).
3. Monitor the loss of toe pinch reflex and once the mouse is sedated place it on top of a heating pad.
4. Clip a large area of the fur around the dorsal midline of the female mouse.
5. Disinfect the exposed skin with alternating wipes of a topical antiseptic such as chlorhexidine and 70% ethanol.
6. Expose the reproductive tract following traditional method¹⁵. Make a 1 cm long skin incision parallel to the dorsal midline, cut the muscle and grab the fat pad with a forceps, then gently pull the ovary out until its attached oviduct and uterus are clearly visible.
7. Fix the fat pad with a vessel clamp. Visualize the oviduct under the stereoscopic microscope and using a pair of micro-scissors make an incision into the wall of the oviduct few millimeters upstream of the ampulla.
8. Under the stereoscopic microscope, load 25 microinjected eggs into the glass capillary connected to the mouth piece (the internal diameter of the capillary should be around 120 µm wide). Introduce the glass capillary into the oviduct and expel until an air bubble is visible inside the ampulla.
9. Gently remove the glass capillary and place the reproductive tract back into the abdomen. Suture the incision with 3-0 non-absorbable surgical sutures, then close with wound clips. Monitor the mouse closely until full recovery.

9. Genotyping Strategies/Sequencing

NOTE: Isolate the genomic DNA from 2-mm tail or ear biopsies, following relevant animal ethics regulations.

1. Fast genomic DNA extraction (random integration).
   1. Lyse the tissue samples (~2 mm) at 95 °C for 1 h in 100 µL of alkaline lysis reagent (25 mM sodium hydroxide and 0.2 mM EDTA, pH = 12).
   2. Add 100 µL of neutralizing reagent (40 mM Tris-HCl, pH = 5).
   3. Centrifuge for 5 min at 12,000 g and 4 °C.
2. High-quality genomic DNA extraction (gene targeting).
   1. Add 500 µL of tail buffer (50 mM Tris, pH = 8; 100 mM EDTA, pH = 8; 100 mM NaCl; 1% SDS, and 0.5 mg/mL proteinase K, freshly added).
   2. Incubate overnight at 55 °C.
   3. Mix for 5 min by inverting (do not vortex).
   4. Add 250 µL of saturated NaCl (6 M) and mix for 5 min by inverting (do not vortex).
   5. Spin for 5-10 min at 12,000 x g and 4 °C and pour the supernatant into new tube.
   6. Add 500 µL of isopropanol and mix for 5 min by inverting (do not vortex).
   7. Spin for 10 min at 12,000 x g and room temperature.
   8. Decant the supernatant (the pellet is invisible and sticks to the tube).
   9. Wash with 1 mL of 70% ethanol.
   10. Spin for 5-10 min at 12,000 x g and room temperature. Remove the supernatant with care, as the white pellet is not sticky anymore and can be lost.
   11. Air dry this for ~1 h.
   12. Add 400 µL of TE buffer (10 mM Tris and 1 mM EDTA, pH = 8).
   13. Dissolve the DNA at 55-60 °C for 2 h.
3. Primer design.
   1. Design primers a minimum of 20 bp long using a computational tool¹⁹.
      NOTE: For random integration, the primers should hybridize with the transgene, generating a distinct fragment of 200-800 bp. For gene targeting, design the primers so that they hybridize with the genomic DNA, generating a distinct fragment a few hundred bp around the cut sites. For large insertions, the primers can sit on the transgene, but confirmation of the targeted integration should subsequently be performed using primer walking or a similar technique.
4. PCR genotyping.
   1. For each genomic modification, design a new PCR protocol and test it empirically. Consider the length of the generated fragment and the melting temperature (Tm) of each pair of primers.
5. Sequencing.
   1. For gene targeting, send PCR products to a Sanger sequencing service provider.