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Products for this protocol

• DMEM, with high glucose content (optional; see Step 29)
   
  Immediately before use, add 10 mL of 200 mM glutamine and 10 mL of 5000 µg/mL penicillin/streptomycin solution to 1 L of DMEM.
   
  
• DR50 (optional; see Step 29)
   
  
  • DMEM, with high glucose content
     
    
  • 200 mM glutamine
     
    
  • 5000 µg/mL penicillin/streptomycin solution
     
    
  • Rat Serum (RS)
     
    Inactivate 10 mL of RS for 30 min at 56°C. Prepare DMEM by adding 10 mL of 200 mM glutamine and 10 mL of 5000 µg/mL penicillin/streptomycin solution to 1 L of DMEM. After the RS is inactivated, add 10 mL DMEM to the inactivated RS. (Thus, DR50 is made up of 50% RS and 50% DMEM.) Keep any unused DR50 or RS at 4°C, and replace every 7-10 d to prevent any contamination.
     
    
• DR75 (optional; see Step 29)
   
  
  • DMEM, with high glucose content
     
    
  • 200 mM glutamine
     
    
  • 5000 µg/mL penicillin/streptomycin solution
     
    
  • Rat Serum
     
    Inactivate 10 mL of RS for 30 min at 56°C. Prepare DMEM by adding 10 mL of 200 mM glutamine and 10 mL of 5000 µg/mL penicillin/streptomycin solution to 1 L of DMEM. After the RS is inactivated, add 3.33 mL DMEM to the RS. (Thus, DR75 is made up of 75% RS and 25% DMEM.) Keep any unused DR75 or RS at 4°C, and replace every 7-10 d to prevent any contamination.
     
    
• DNA expression plasmid (2-5 µg/µL, in aqueous solution)
   
  Plasmids should be constructed utilizing a promoter from cytomegalovirus, ß-actin, or the Hmgcr gene to drive widespread non-lineage-restricted expression of transgenes (e.g., eGFP, lacZ or a cDNA for the gene of interest).
   
  
• NaOH (3 M)
   
  
• Mice, pregnant female, 7-7.5 days post-coitum (dpc)
   
  
• Paraffin oil, light (optional; see Step 15)
   
  Light paraffin oil should be cleansed by mixing with PBS to remove water-soluble impurities. Let the emulsion stand until the PBS and the oil are separated into layers, then decant paraffin oil into a clean NUNC cell culture flask.
   
  
• Paraformaldehyde (4%)
   
  
• Sodium pyruvate for PB1
   
  
  • Sodium pyruvate
     
    
  • 0.9% (w/v) NaCl
     
    Dissolve 170 mg of sodium pyruvate in 20 mL of 0.9% (w/v) NaCl. Store it at 4°C for up to 2 wk. Just before adding the solution to PB1, dilute it 1:50 in 0.9% (w/v) NaCl.
     
    
• Phenol red-bicarbonate solution for PB1
   
  
  • 0.5% (w/v) phenol red solution
     
    
  • NaHCO₃
     
    Dissolve 258 mg of NaHCO₃ in 14.8 mL of H₂O. Add 5.2 mL of 0.5% phenol red solution. Store for up to 2 wk at 4°C.
     
    
• Penicillin for PB1
   
  
  • Penicillin
     
    
  • 0.9% NaCl (w/v)
     
    Add 599 mg of penicillin to 10 mL of 0.9% (w/v) NaCl. Store it at -20°C.
     
    

PB1
Reagent	Final concentration
NaCl	8.0 g/L
KCl	0.2 g/L
Na2HPO4 · 12H2O	2.88 g/L
KH2PO4	0.2 g/L
CaCl2 · 2H2O	0.13 g/L
MgCl2 · 6H2O	0.1 g/L
Sodium pyruvate for PB1	0.036 g/L
Phenol red-bicarbonate solution for PB1	0.01 g/L
Penicillin for PB1	0.06 g/L
Glucose	1.0 g/L
Bovine serum albumin	4.0 g/L
Combine the reagents in the order listed. The pH should be between 7.3 and 7.4, and the osmolarity between 286 and 292 mOsm/L. Sterilize the solution by passing it through a 0.22-µm filter. Store it in 50-mL aliquots at 4ºC for up to 2 wk.


 


PBS, Ca2+/Mg2+ -free
Reagent	1X Final Concnetration	10X Stock
NaCl	8 g	80 g
KCl	.2 g	2 g
Na2HPO4	1.44 g	14.4 g
KH2PO4	.24 g	2.4 g
PBS can be made as a 1X solution or as a 10X stock. To prepare 1 L of either 1X or 10X PBS, dissolve the reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H2O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.


 

• Rat serum (RS)
   
  
• Tyrode Ringer's saline, Ca2+/Mg2+-free
   
  
  • NaCl, 8.0 g
     
    
  • KCl, 0.3 g
     
    
  • NaH₂PO₄ · 5H₂O, 0.093 g
     
    
  • KH₂PO₄, 0.025 g
     
    
  • NaHCO₃, 1.0 g
     
    
  • Glucose, 2.0 g
     
    Dissolve in 1 liter of H₂O. Adjust pH to 7.6-7.7.

• Banana plug
   
  
• Banana socket
   
  
• Battery (6V), alkaline
   
  
• Bottle culture apparatus, rotating
   
  
• Bunsen burner
   
  
• Camera, digital (SPOT Advanced, version 3.5.9.1)
   
  
• Capillary tubes, glass, thick-walled, OD = 1.0 mm, ID = 0.60 mm (Leica)
   
  
• Capillary tubes, glass, thin-walled, OD = 1.0 mm, ID = 0.75 mm (Drummond) (optional; see Step 2)
   
  
• Culture bottles, glass, thin-walled (B.T.C. Engineering)
   
  
• Dissecting equipment
   
  
• Drill
   
  
• Electroporator, square-wave (BTX, ECM 830)
   
  
• Gas mixture ([5% CO₂, 5% O₂, 90% N₂] or [5% CO₂, 20% O₂, 75% N₂]; see Step 29)
   
  
• Gas regulator (BOC Gases)
   
  
• Hammer
   
  
• Incubator (37ºC, with 5% CO₂ in air), water-jacketed
   
  
• Injector syringe (de Fonbrune; Alcatel) or oil-tram injector (Eppendorf, 5176 000.025)
   
  
• This oil-filled system is used to apply suction and expulsion actions to the injection micropipette to move the DNA into the host embryo.
   
  
• Microforge (Narishige, MF-900)
   
  The microforge has a glass bead on a heating filament that is used to make injection pipettes of a specific internal diameter. The bead can be heated and cooled quickly, melting the glass capillary wall onto the glass bead, resulting in breakage of the pipette as the glass cools. The microforge is also used to polish the ends of the holding pipettes.
   
  
• Micromanipulation apparatus (Leica Microsystems):
   
  
  • Base plate with fixture points
     
    
  • Instrument holders (11520143)
     
    
  • Manipulators, for left-handed (11520138) or right-handed (11520137) operation
     
    
• Micrometer, ocular
   
  
• Micrometer syringe (Narishige, IM-5B) or air-tram injector (Eppendorf, 5176 000.017)
   
  This oil-filled system is used to apply suction and expulsion actions to the holding pipette.
   
  
• Microscope, dissecting (Leica Microsystems)
   
  
• Needle (20G), syringe
   
  
• Pasteur pipettes (9 inch)
   
  
• Petri dishes (60 and 150 mm)
   
  
• Pipette puller, horizontal (optional; see Step 2)
   
  
• Plasticine modeling clay
   
  
• Positioner (Taurus-R, World Precision Instruments Inc.)
   
  This is used to hold the tungsten wire in place.
   
  
• Rod, wooden
   
  
• Soldering equipment
   
  
• Slides, four-well (Nalgene Nunc International, Lab-Tek Chamber Slides, 177437)
   
  
• Stereomicroscope system, fluorescence (Leica, MZ FLIII) with excitation filter GFP-1 filter set (425 nm)
   
  
• Tape
   
  
• Tubing, plastic
   
  
• Wire (0.2 mm), platinum
   
  
• Wire (0.1 mm), tungsten
   
  

1. Prepare the holding pipettes:
    
   
   1. Using the small flame from the end of a 20-gauge syringe needle connected to a Bunsen burner, melt and pull thick-walled glass capillaries.
       
      
   2. Using a microforge, cut the holding pipette to an internal diameter of 30-60 µm.
       
      
   3. Polish the end of the holding pipette by bringing the tip close to the heated glass bead of the microforge.
       
      This eliminates sharp edges that can tear the embryo when suction is applied.
       
      
   4. Using the small flame from the end of the needle, heat the pipette ~1-2 cm from the tip until it is bent to an angle of ~100º.
       
      
   5. Turn the pipette and heat at a position 1-2 cm proximal to the first bend until the pipette is bent at ~100º.
       
      
2. Prepare the microinjection pipettes:
    
   These are only required for injection of DNA for electroporation of mesoderm or ectoderm (see Steps 13-17).
    
   
   1. Using a horizontal pipette puller, pull thin-walled glass capillary tubes to produce pipettes with a fine tip, long shaft and short shoulder.
       
      Use an ocular micrometer to ensure an inner diameter appropriate for microinjection (10 µm).
       
      
   2. Touch the pipette to the heated glass bead on the microforge filament.
       
      
   3. As the capillary begins to fuse to the bead, turn off the power to the filament.
       
      As the filament cools it retracts, breaking the pipette precisely at the point where the capillary fuses to the bead.
       
      
   4. Using the small flame from the end of the needle, heat the pipette ~1-2 cm from the tip until it is bent to an angle of ~100º.
       
      
   5. Turn the pipette and heat at a position 1-2 cm proximal to the first bend until the pipette is bent at ~100º.
       
      
3. Set up the electroporation apparatus (Fig. 1a ):
    
   
    
   Figure 1. The electroporation setup. (a) Schematic of the electroporation setup. The plate electrode is anchored to a Petri dish and the point electrode is held by a positioner. The embryo is held with a holding pipette in a position such that the electric current will pass through the girth of the cylindrical embryo. (b) The arrangement of instruments around the micromanipulation apparatus.
    
   
   1. Flatten the end of a 3-cm, 0.2-mm-diameter platinum wire with a hammer to form the plate electrode.
       
      
   2. Solder the other end of the platinum wire onto a banana socket.
       
      
   3. Attach the banana socket/platinum plate onto a 150-mm Petri dish with plasticine so the platinum plate is centered on the dish.
       
      For a more secure attachment, drill a hole in the Petri dish and attach the socket with a screw.
       
      
   4. Drill a hole (2-3 mm wide) through the side of a lid from a 60-mm Petri dish. Be sure to position the hole at a height appropriate for the wire now attached to the banana socket to go through.
       
      Petri dish lids are used because the rim has a lower clearance, which gives the angled pipettes better accessibility to the embryos and tissues in the dish.
       
      
   5. Insert the platinum plate through the hole. Place a thin piece of plastic tubing over the platinum wire, leaving the plate exposed.
       
      
   6. Sharpen the end of a 5-7 cm piece of tungsten wire by dipping it in a 3 M NaOH bath connected to a 6V alkaline battery.
       
      
   7. Solder the tungsten wire onto a banana plug.
       
      
   8. Tape the banana plug to the wooden rod.
       
      
   9. Place the wooden rod in the positioner. The tungsten wire of the point electrode may have to be bent into a curve in order to point 90° to the platinum plate. This is determined by the placement of the positioner in relation to the other components of the micromanipulation apparatus.
       
      
   10. Place a drop of Tyrode Ringer's saline over the platinum plate and tungsten wire until both are completely immersed.
        
       
   11. Place a separate drop of PB1 (5-10 µL) in the 60-mm Petri dish lid.
        
       
4. Set up the micromanipulators (Fig. 1b):
    
   
   1. Attach the holding pipette to the instrument holder on the left manipulator.
       
      The holding pipette is controlled using the micrometer syringe on the far right (not shown).
       
      
   2. If performing microinjection of DNA for electroporation of ectoderm or mesoderm, attach an injection pipette to the instrument holder on the right manipulator (if possible set up for microinjection on a separate microscope with manipulators).
       
      The injection pipette is controlled using an injector syringe on the left.
       
      
   3. Bring the holding pipette, plate electrode and point electrode into the field of view on the dissecting microscope.
       
      
   4. Position the point electrode such that it is at a 90° angle to, and centered on, the plate electrode, in the same optical plane. Leave a small gap between the electrodes (Fig. 2a ).
       
      

 
Figure 2. The schematic and outcome of electroporation of the three germ layers. (a) The point electrode is set at right angles to the plate electrode. The point electrode is used for focusing the electric current to a small area of the embryo and the plate electrode provides a broad conducting surface to reduce the voltage required; this achieves a workable current strength without damaging the tissue. In this figure, the embryo is positioned between the electrodes such that the current passes longitudinally through the embryo. As the DNA is negatively charged, it will be driven toward the positive electrode. The point electrode is set as the negative pole for the electroporation of the endoderm and the mesoderm, and as the positive pole for the electroporation of the ectoderm. Arrows indicate the direction of targeting of the DNA into the three germ layers. (b) GFP expressed in the endoderm 3 h after electroporation. The endoderm of a 7.0-dpc embryo was targeted after incubation with a ß-actin-GFP plasmid. (c) GFP expressed in the mesoderm 7 h after electroporation. The mesoderm of an 8.0-dpc embryo was targeted after microinjection of a ß-actin-GFP plasmid into the mesoderm followed by electroporation. (d) LacZ expressed in the neuroectoderm 24 h after electroporation. The ectoderm of a 7.5-dpc embryo was electroporated after microinjection of a Hmgcr-LacZ plasmid into the amniotic cavity.
 

5. Prewarm 100% RS to 37ºC in a four-well slide in the incubator.
    
   
6. Dissect the mouse embryos into PB1. Transfer them to the prewarmed 100% RS in the four-well slide.
    
   
7. Place the slide in the 37ºC upright incubator. Keep collected embryos in the incubator except for the period of manipulation and electroporation, which should be kept to no more than 15 min outside the incubator.
    
   
8. Proceed to Step 9 or Step 13, depending on the germ layers to be electroporated.
    
   
Incubation with DNA for Electroporation of Endoderm
 

9. Using a Pasteur pipette, transfer four to seven embryos to the small drop of PB1 in the 60-mm Petri dish lid.
    
   
10. Add 5-10 µL of concentrated plasmid DNA solution to the PB1 drop containing the embryos to a final concentration of 1-2 µg DNA/µL. Pipette gently to mix the DNA in the PB1 solution.
     
    
11. Incubate the embryos at room temperature for at least 5 min.
     
    
12. Proceed to Step 19.
     
    
Injection of DNA for Electroporation of Mesoderm or Ectoderm
 

13. Using a Pasteur pipette, transfer five to six embryos to a 100-µL drop of PB1 in a 60-mm Petri dish.
     
    
14. Place a 5-10 µL drop of concentrated plasmid DNA solution next to the PB1 drop containing the embryos.
     
    
15. Fill the dish with light paraffin oil to completely cover both drops.
     
    
16. Aspirate a small volume of the plasmid DNA solution into an injection micropipette.
     
    
17. Inject the DNA solution:
     
    
    1. To target the ectoderm, inject ~0.5 µL of the DNA solution into the proamniotic cavity of the embryo.
        
       
    2. To target the mesoderm, inject ~0.1 µL of the solution into the space between the ectoderm and endoderm.
        
       Because the mesoderm is a relatively spacious layer, the DNA should be injected close to the endoderm layer, so electroporation will target the majority of the mesoderm cells further away from the endoderm.
        
       
18. Proceed to Step 19.
     
    
Electroporation of Gastrula-Stage Germ Layers
 

19. Connect the plate electrode and point electrode to the square-wave electroporator (Fig. 2a): As the DNA is negatively charged, it will be driven toward the positive electrode. Depending on the setting of the polarity of the electrodes, the position of the embryo and where the DNA is delivered in the preparatory step, the appropriate germ layer can be targeted.
     
    
    1. For electroporation of the endoderm and mesoderm, set the plate electrode as the positive pole and the point electrode as the negative pole.
        
       
    2. For electroporation of the ectoderm, set the plate electrode as the negative pole and the point electrode as the positive pole.
        
       Electroporation of the ectoderm is done by reversing the polarity of the electrodes relative to the settings for the mesoderm or the endoderm, so that the DNA will be driven toward the luminal surface of the ectoderm from within the proamniotic or amniotic cavity.
        
       
20. Using a Pasteur pipette, carefully transfer one embryo at a time to the drop of Tyrode Ringer's saline in the electroporation dish. For electroporation of the endoderm, fill the pipette with Tyrode Ringer's saline first so that the embryo and only a small amount of DNA solution are picked up with the embryo by capillary action. Also, release the embryo into the electroporation drop with minimal carryover of the DNA solution.
     
    
21. Using the holding pipette, hold the embryo by applying negative pressure via the micrometer syringe. Hold the embryo at a point away from the embryo proper whenever feasible (e.g., by the ectoplacental cone or extraembryonic region) and at the best orientation relative to the point electrode for targeting the electroporation.
     
    
22. Suspend the embryo between the platinum plate and tungsten wire without touching either of the electrodes (Fig. 2a). It is essential that the embryo is properly positioned between the electrodes and that the point electrode is as close as possible to the site of electroporation without causing burn damage to the tissue. Air bubbles will emit from the tungsten wire indicating electric current has been generated. It is necessary to leave enough space for the large air bubbles to escape from the electrode without touching or moving the embryo.
     
    
23. Use the square-wave electroporator to deliver five pulses at 15V, 50 msec each, with a 1-sec gap between each pulse.
     
    
24. Release the embryo from the holding pipette by applying positive pressure via the micrometer syringe.
     
    
25. Using a Pasteur pipette, transfer the embryo to a fresh drop of PB1 for a brief rinse.
     
    
26. Transfer the embryo to a new well of culture medium in the four-well slide. Return the slide to the incubator.
     
    
27. Repeat Steps 20-26 for each embryo to be electroporated.
     
    
Culture and Analysis of Electroporated Embryos
 

28. Culture the electroporated embryos in the four-well slide in 100% RS in the 37ºC incubator for 3 h. Initial static culture improves the viability of the embryos and allows recovery from any damage caused by electroporation.
     
    
    1. If a GFP fluorescent construct is used, photographs of the electroporated embryos can be taken at this point; see Step 30 for details.
        
       At least 3 h of static culture are required before photos are taken to allow sufficient time for the production of GFP encoded by the construct. See Troubleshooting.
        
       
29. Place embryos in a rotating culture system with 2-3 mL of 100% RS. Incubate in an atmosphere of 5% CO₂, 5% O₂, and 90% N₂ for 24 h at 37ºC.
     
    Details for the technique for culturing whole post-implantation mouse embryos can be found in Roller Culture of Post-implantation Embryos. Culture media and gas requirements will differ with the age of the embryo. 100% RS is used for grafted embryos aged 7-7.5-dpc in 5% O₂, 5% CO₂, and 90% N₂ for a culture period of 24 h. If culturing the 7-7.5-dpc embryos for 48 h, the serum and gas requirements change to those of the 8-8.5-dpc embryo after 24 h of culturing. Embryos aged 8-8.5 dpc are cultured in DR75 and 5% CO₂, 20% O₂, and 75% N₂. DR50 with 5% O₂ has been used successfully in other labs.
     
    
30. Analyze the distribution of the electroporated cells and their descendants (see Troubleshooting):
     
    
    1. Transfer the embryos to 37ºC PB1.
        
       
    2. Image the cultured embryos on a fluorescence stereomicroscope with GFP excitation filter (if using a construct encoding for GFP).
        
       
    3. Take photographs using a SPOT advanced digital camera. Capture both a bright-field and fluorescent image for each embryo.
        
       
    4. Use Adobe Photoshop 7.0 to merge the two images, in order to show the position of fluorescent cells in the embryonic structure (Fig. 2b and Fig. 2c).
        
       
    5. Stain the embryos (if marked by a lacZ transgene; see Staining Whole Mouse Embryos for ß Galactosidase (lacZ) Activity) (Fig. 2d) and/or fix them in 4% paraformaldehyde. Store at -4ºC. Distribution of electroporated cells can be analyzed in fixed embryos by whole-mount in situ or on serial sections of the embryo if the cells are marked by a lacZ transgene.