Microinjection of Mouse Zygotes

Materials

Equipment

  • Microinjection setup

Reagents

  • Fertilized mouse embryos 0.5 dpc
  • KSOM, M16 or other embryo culture medium

Procedure

  1. Use an embryo transfer pipette to transfer a group of fertilized oocytes into the injection chamber. The number of zygotes to be moved into the microinjection drop should be determined by the skills of the injector and quality of the setup. Do not attempt to work with more zygotes than can be injected within 20-30 minutes.
  2. Examine the zygotes under high power, making sure that two pronuclei are visible and that the morphology is good. Discard all zygotes that appear abnormal.
  3. To ensure that the injection pipette is not closed at the tip or clogged, place the tip of the injection pipette close to (but not touching) a zygote in the same horizontal plane as the midplane of the zygote (i.e., in the same focal plane, on high power). Apply pressure using the regulator of the injector.
    If the pipette is open, a stream of DNA will move the zygote away from the tip of the injection pipette.
    If the pipette is closed or clogged, flush DNA with high power through the injection pipette by using the “Clear” function on the FemtoJet. Repeat the test. If the tip is still not open, tip it carefully on the holding pipette and so break up to a larger tip diameter. If the diameter becomes too large, or the tip is still not open, discard the pipette and use a new one.
  4. To prepare a zygote for injection, place the tip of the holding pipette next to the zygote and suck it onto the end of the pipette by applying a negative pressure to the pressure control unit. Focus the microscope to locate the pronuclei.
    A pronucleus can be most easily injected if it is located in the zygote hemisphere closest to the injection pipette. The pronucleus should also be as close as possible to the central axis of the holding pipette; if it is far from this axis, the zygote will tend to rotate when the injection pipette is pushed toward the pronucleus.  If it is necessary to reorient the zygote to place the pronucleus in a better position, release the zygote from the holding pipette, use the injection pipette and/or the holding pipette to rotate it slightly, and then suck the zygote back onto the holding pipette.
  5. When satisfied with the position of the zygote, give the syringe controlling the holding pipette an extra twist to be sure the zygote is held firmly. The zona pellucida should be seen being pulled slightly into the opening of the pipette, but the zygote itself should not be deformed. Either of the two pronuclei may be injected.
  6. Refocus on the pronucleus to be injected, making sure that its borders can be seen sharply (the focus is set to the midplane of the pronucleus).
    1. Bring the tip of the injection pipette into the same focal plane as the mid-plane of the pronucleus.
    2. Move the injection pipette to the same y-axis position as the targeted pronucleus (either 6 o’clock or 12 o’clock of the embryo) and adjust the height of the pipette so that the tip of the pipette appears completely sharp (without changing the focus!). This is an important step that allows the pipette to target the pronucleus exactly.
  7. Move the injection pipette to a 3 o’clock position without changing its vertical level. Push the injection pipette through the zona pellucida, into the cytoplasm, and toward the pronucleus. Make sure that both the tip of the pipette and the outline of the pronucleus remain in focus; if the zygote moves and the pronucleus goes out of focus, the pipette will not hit the pronucleus.
    1. Continue pushing the pipette forward, entering into the pronucleus. Avoid touching the nucleoli as they are very sticky and will adhere to the pipette.
    2. When the tip of the pipette appears to be inside the pronucleus, apply injection pressure through the injector.
  8. If the pronucleus swells visibly, it has been successfully injected! Quickly pull the pipette out of the zygote.
    A pipette pulled out slowly frequently will remain attached to nuclear components (perhaps the nuclear membrane or chromosomes). Also, flow of the solution may disturb the plasma membrane if the withdrawl is slow.
  9. If the pronucleus does not swell, the pipette has become clogged or has not punctured the oocyte plasma membrane.
    1. If a small round “bubble” forms around the tip of the pipette, then the pipette has not punctured the plasma membrane. The plasma membrane is very elastic and can be pushed far back into the zygote, even into the pronucleus, without being pierced. In this case, try pushing the pipette right through the pronucleus and out the other side; then pull back on the pipette slightly so that the tip is again inside the pronucleus. The maneuver frequently moves the pipette through the plasma membrane and into the pronucleus.
    2. Another sign that the pipette has actually pierced the membrane is that at the point of entry the membrane will be roughly perpendicular to the wall of the pipette, whereas if the membrane has not been pierced, it will appear to be indented.
  10. It is difficult, if not impossible, to accurately control the volume of DNA solution introduced into the pronucleus. Most investigators estimate that 1-2 picoliters (pl) is injected, but the fraction of DNA remaining in the nucleus is unknown. The size of the pronucleus varies from zygote to zygote, and the injected volume has to be adjusted accordingly. As a guideline, injection should be continued until a clear increase in pronucleus size has been achieved.
  11. Cytoplasmic granules flowing out of the oocyte after removal of the injection pipette are a clear sign that the zygote will soon lyse. In this case, or if nuclear components are sticking to the tip of the injection pipette after injection, the oocyte should be discarded. If the zygote appears to be intact and successfully injected, it should be sorted into the group of “good zygotes”, and another zygote should be picked up for injection.
  12. The same injection pipette can be used as long as it continues to inject successfully. Switch to a new injection pipette if (a) you are unable to get into the pronuclei of several zygotes, even though the pronuclei can be seen clearly; (b) two oocytes in succession lyse immediately after injection; (c) the tip of the pipette becomes visibly “dirty”, or nuclear contents stick to the pipette; (d) the tip of the pipette breaks and appears to be more than 1 ųm in diameter; or (e) the pipette clogs and cannot be cleaned by flushing through with high pressure (“Clean” function on the FemtoJet).
  13. When all the zygotes in the chamber have been injected, they should immediately be moved back into either M16 or  KSOM medium and incubated at 37ºC. A new group of zygotes can be transferred into the injection chamber, and the injection procedure continued until all zygotes are injected.
  14. Some injected zygotes will inevitably lyse due to the mechanical damage caused by the injection procedure.  Lysing will take place 5-30 minutes after completed injection. These lysed zygotes can be easily distinguished from healthy ones. Lysed zygotes appear translucent, fill out the whole zona pellucida, and give a lightweight impression (they will swim up more easily if blown upon with media). Healthy zygotes have a distinct space between the plasma membrane and zona pellucida, and the cytoplasm appears compact and evenly shaped.  Typically, about 75% of the zygotes survive the injection.

Comments

  • It is of utmost importance to carefully select only good-quality zygotes for injection. There should be no signs of sperm under the zona pellucida. The oocyte should not fill out the whole zona pellucida (there should be a marked previtelline space), but it should also not appear “shrunken.” Two pronuclei should be clearly visible. No frangmentation should be apparent.
  • If only one pronucleus can be detected, the oocyte is most likely unfertilized. If more then two pronuclei can be detected, the oocyte is polyspermic (fertilized by multiple sperm). The absence of a second polar body is not always a reliable sign of failed fertilization, because occasionally polar bodies are fragmented, making them difficult to count accurately. However, the oocyte itself should not show any signs of fragmentation.
  • The quality assessment can be performed while injecting, but care should be taken to sort the normal and successfully injected oocytes from abnormal or unsuccessfully injected ones immediately, since this discrimination will be impossible to do at a later stage. Electronically controlled micromanipulators can aid the sorting of good- and low- quality embryos by making use of programmed positions of the holding pipette. In this case, set the position 1 far to one side of the microinjection drop, and the position 2 to the other. Each injected zygote can then be automatically moved to the corresponding group.
  • To minimize the risk of the zygote turning in the holding pipette’s grip during injection, it is important to make sure that the targeted pronucleus lies approximately in the mid-plane of the cytoplasm. If this is the case, the outline of the pronucleus will be sharp at the same time as the zona pellucida appears as the most “crisp” with most details visible in it.
  • Most modern electronic injectors (such as the FemtoJet) have an “Automatic” and a “Manual” injection mode. The difference here is simply the mode of injection in relation to the release button or foot pedal action. In case of the “Automatic” mode, the length of time during which injection is triggered is preset and equal each time. In the “Manual” mode, however, the duration of the injection is determined individually; as long as the button or foot pedal is held, injection proceeds. Because pronuclei can vary significantly in size, the “Manual” mode should be used, so that the appropriate pronuclear swelling can be achieved for each individual oocyte.
  • The settings of injection pressure and constant flow pressure have to be determined empirically and are dependent on the inner diameter of the injection pipette. As basic setting for the FemtoJet, Pc can be set to 10-15, and Pi set to 40-50. If during injection the pronucleus swells very fast, resulting in nucleoli pressed out into the cytoplasm, the pressure should be reduced. If, on the other hand, the pronucleus swells very slowly, the pressure should be increased.
  • The microinjection setup is virtually the same whether normal-sized or large constructs are injected.  However, it is important to tkeep in mind the higher viscosity of high-molecular-weight DNA solutions, and the danger of shearing the DNA when pushing it through a very small pipette tip. The injection pressure should be kept as low as possible, and if the injection pipette is clogged, it should immediately be changed. The inner diameter of the injection pipette should be slightly increased to allow an easy flow of DNA without increasing the injection pressure. This can most easily be achieved by “ticking” the tip of the injection pipette onto the holding capillary until it breaks at a larger dimension. If the new diameter is broken too widely, the injection pipette should be changed. The lysing rate will grow proportionally with the tip diameter, but this is a drawback one should accept when working with large constructs, rather than risking the shearing of the DNA by pushing it through a very small pipette tip.
  • It is easiest to target the larger of the two pronuclei and/or the one that is closest to the injection pipette. If the embryos are well timed, the pronuclei should both appear in the center of the embryo, both be large and clearly visible. The advice to inject the male pronucleus is outdated. At the optimal time for injection, it is often difficult to tell which pronucleus is the male and which is the female. The only criterion for the choice should be the ease of injection: The pronucleus that is largest and/or nearest to the injection pipette should be targeted.
  • If the pronuclei are small, the embryo has not yet reached the optimal time point for injection. It may help to bring the embryos back to the incubator for an hour and start the injection process later.  In case the embryos are left too long at 37ºC before the injection is started, the two pronuclei will have fused and injection will be impossible. The developmental stage of the oocytes highly correlates with the timing of the hCG injection and the light cycle in the animal room, so that the injection time point easily can be influenced by the superovulation protocol.
  • The microinjection chamber should be removed from the microscope stage immediately after each injection session to prevent any accidental spillage of oil or medium into the optics.
  • Microinjection is not a trivial process. Allow a considerable learning time before high efficiencies can be achieved.  Embryos are living material, which should be handled with care. It is not sufficient for them barely to survive the manipulation and in vitro culture; they should be treated in such a way that after replacement in vivo they can develop into healthy mice. This is an important picture to keep in mind during all embryo micromanipulations, culturing, and handling.

Troubleshooting Guide

The process of creating genetically altered mice through pronuclear injection is very sensitive. Numerous potential problems may arise, eventually leading to either a low number of pups born or no founders among the offspring. This troubleshooting guide gives an overview of the most common difficulties, their possible underlying causes, and suggestions for solutions.