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.

1. Low plugging rate.

  • The stud males are too young or too old. Optimal age is 2-6 months.\
  • The stud males have been mated too often. They should not be mated with superovulated females more than twice per week, and they should be allowed at least 1 day rest between matings.
  • The batch of PMSG/hCG used is a low quality, or the hormones may have been prepared incorrectly.  Hormones should be kept in frozen stocks, and not allowed to be thawed longer than 1 hour prior to use.
  • The light-dark cycle is not constant from day to day. It is important that the cycle be kept exactly the same each day, with absolutely no variations. No entry into mouse rooms should be allowed after the start of the dark period.
  • Environmental problems in the animal facility. High noise levels-especially noise that is not of a constant nature-vibrations, temperature fluctuations, and suboptimal humidity should be avoided as much as possible.

2. Poor yield of zygotes.

  • Problems with the light cycle, hormones, or environment; see point 1.
  • Suboptimal age of donor females. Depending on the strain or hybrid cross used, it may be necessary to use older or younger females that respond better to the hormone regimen.
  • Incorrect hormone dosage. The dose of PMSG/hCG required for superovulation varies between strains and hybrid crosses, and also with the age of females. It is therefore important to determine the optimal dosage empirically.

3. Few or no zygotes display two pronuclei:

  • The females were not actually plugged.
  • The oocytes are not fertilized. This may be due to the use of too-old or overworked males, or too-young females.
  • The zygotes were fertilized very recently. In this case, the pronuclei will become visible after a few hours in in-vitro culture. Administer the hormones at an earlier time.
  • The dissecting or injection microscope is poorly adjusted, the optics are incorrectly aligned, and/or the objectives are not clean.
  • The injection microscope is not used with DIC or Hoffman optics, or plastic dishes are used in combination with DIC optics.
  • See also point 5.

4. The pronuclei are too small to inject easily.

  • Injection has been attempted too soon after fertilization. Administer the hormones earlier, or inject later in the day.
  • Some strains have small pronuclei that are difficult to inject. In case flexibility in the choice of genetic background is acceptable, try a different strain or hybrid combination.

5. The pronuclei cannot be seen properly.

  • See point 4.
  • Small oil bubbles may have collected under the surface of the medium drop in the microinjection chamber, which alters the image. This often happens when oocytes are moved in and out of the drop. The oil drops can be removed with the mouth pipette while focusing on them at low-power magnification, or a new chamber should be set up.
  • The medium drop in the injection chamber may not be completely covered by oil. This is most likely to happen if the medium drop is relatively high and the oil cover thin. Also in this case, the image will be distorted. Add more oil on top of the medium drop.
  • DIC or Hoffman optics are not properly aligned and adjusted. Ask the microscope manufacturer for help with accurate optimization.
  • The eyepiece or the objective is dirty (clean carefully using ethanol and water with dust-free soft paper lens tissue). Take good care never to let any oil drop down from the microinjection chamber onto the objectives! For this reason, it is important to remove the microinjection chamber from the microscope stage immediately after each injection session.

6. Strands of nuclear material stick to the injection pipette after withdrawl from the pronucleus.

  • The injection pipette has touched the nucleoli resemble small bright round spots within the pronucleus. They are extremely sticky and will immediately stick to the injection pipette if touched.  Try to avoid touching the nucleoli during injection.
  • The pipette is dirty and should be replaced. Extremely small particles, which cannot be seen even under high magnification, make the outside surface of the injection pipette sticky, which in turn results in an increased risk of the tip touching and attaching to nuclear material.
  • The injection pipette has been pulled out too slowly. Try to pull out faster after the pronuclear swelling.
  • One possible method for reducing this problem is to siliconize the pipettes before use with Sigmacoe (Sigma).

7. The injection pipette clogs repeatedly.

  • Flush with the “Clean” button if an electronic injector is used.
  • The opening in the pipette is too small. Alter the settings on the pipette puller, or enlarge the opening by chipping on the holding capillary.
  • The injection pressure is too low. Increase the constant flow pressure.
  • Impurities are present in the DNA solution. Alter the purification method.

8. The injection pipette or the holding pipette drifts when the joystick is not being touched (using Leitz micromanipulator).

  • Reduce the movement ratio by twisting the joystick collar in the appropriate direction.
  • The joystick is too far out of position. Reposition the pipette with the positioning screws on the manipulator, resetting the joystick to a middle position.

9. The tip of the injection pipette moves during injection.

  • The tip of the pipette is not symmetrical: Adjust the pipette puller so that the capillary is centered inside the heating filament.
  • The silicone rubber tubing inside the Leitz micromanipulator pipette holder is worn out, and should be replaced.

10. Too many zygotes lyse after injection.

  • The tip of the injection pipette is too wide. Replace the pipette. Adjust the settings on the puller.
  • The angle between the long axis of the pipette and the plane of the microscope stage is too great.  Try to enter the injection pipette in as low an angle as possible.
  • The injection pipette is dirty. Replace it.
  • The DNA is impure. Improve the purification protocol.
  • The DNA concentration is too high. Reduce the concentration to half.
  • The injection pipette is moved in the yaxis when inserted into the zygote. Take more care not to move the injection pipette other than straight into the embryo.
  • The injected volume is too high. Stop injection as soon as the pronucleus has expanded.
  • The injection pipette is pulled out too slowly. Try to pull out the pipette with a swift movement.
  • The injection and/or constant flow pressure is too high. Reduce the DNA flow in the pipette.
  • Microenvironmental problems such as extensive vibration on the microscope stage, too low a temperature in the microinjection room, or suboptimal medium composition may also contribute to reduced embryo survival.

11. The plasma membrane or pronucleus is difficult to penetrate.

  • The injection pipette is dull. Use a sharper pipette, or try tapping the pipette against the holding capillary to break off a small piece at the tip.
  • The injection pipette is dirty. Replace it.
  • Try to push the injection pipette through the pronucleus into the distal cytoplasm, then pull back so that the tip of the pipette is inside the pronucleus.
  • Reorient the zygote, and position a larger pronucleus in line with the center of the holding pipette.
  • When inserting the pipette, aim exactly at the middle of the pronucleus.
  • Make sure that the z axis of the injection pipette lies exactly in the mid-plane of the pronucleus (see point 6).

12. The zygotes do not divide (if cultured) after injection.

  • The zygotes were not fertilized. Make sure only to inject oocytes with two visible pronuclei.
  • The zygotes were fertilized by more then one spermatozoon each. Screen away any zygotes that display three or more pronuclei.
  • The culture medium is of poor quality, or the incubator is set to the wrong temperature/CO2 concentration, or the humidity in the incubator is not sufficient. Check the culture conditions by culturing uninjected zygotes. Make sure that the water reservoir in the incubator is filled.
  • The zygotes were maintained for too long at room temperature during injection. The temperature in the injection room may be too low, or the microscope stage suffers from vibrations.
  • The zygotes were mechanically damaged by the injection (see point 9 above).
  • The DNA contains toxic impurities. Compare injection of a different DNA or a buffer control.
  • The injected DNA concentration or volume was too high.
  • Transient expression of the injected gene is toxic to the zygote.

13. The pregnancy rate among pseudopregnant recipients is low, or the percentage development to term is low.

  • See point 12.
  • The females were not actually plugged, or the plug date was misrecorded. Foster mothers for oviduct transfer should be plugged on the same day as transfer is done, or on the following day if injected zygotes are cultured overnight.
  • The zygotes/embryos were not successfully implanted into the oviduct.
  • The females were overly traumatized by surgery or anesthetic.
  • The reproductive organs were damaged or traumatized during the transfer procedure. Try to avoid touching the ovary, and handle the oviduct with great care during transfer.
  • A large proportion of the zygotes were damaged by the injection procedure or DNA preparation (see point 11, above).
  • Noise, smell, vibration, etc. in the animal facility may affect pregnancy rates (see Chapter 3, p. 154).

14. The proportion of founders among the pups born is low.

  • The volume of injected DNA was too low; increase the degree of pronuclear swelling.
  • The DNA concentration was lower than estimated; remeasure or increase the concentration.
  • The DNA was diluted by backflow of culture medium into the injection pipette; make sure that the constant flow pressure is adequate. If injection is performed by use of a syringe, squeeze on the plunger once before entering the injection pipette into the zygote.
  • The DNA was not injected into the pronucleus because (a) the pronuclear membrane was not penetrated, resulting in DNA deposition in the cytoplasm, or (b) neither the pronuclear membrane nor the oolemma was penetrated, resulting in DNA deposition outside the zygote plasma membrane. Use sharper pipettes. Adjust the optics if the pronucleus is difficult to see.  Make sure only to transfer zygotes where a clear pronuclear swelling can be detected.
  • The vasectomized males were not correctly vasectomized, so that the foster mother carried her own fertilized embryos in addition to the injected zygotes. For example, if the CD1 outbred strain is used both as vasectomized males and foster females, any dark coat color can be used for the injected zygotes. If albino mice are detected in the litters, the vasectomized males must be suspected of not being properly vasectomized. Vasectomized males may be tested for sterility before being used (see Chapter 6).
  • The expression of the transgene is toxic to the developing fetus.

15. The transgenic founder mouse breeds but does not transmit the transgene to the progeny as expected.

  • The founder is not transgenic, but a false positive.
  • The founder mouse is mosaic, with a few or no transgenic cells contributing to the germ line.  Breed further litters, use other founders, or produce additional founders.
  • The expression of the transgene is toxic to developing F1 mice, but the founder survived because of mosaicism. If the founder is made, timed matings may be set up, and embryos analyzed at various developmental stages.

16. The F1 offspring from a founder show segretating integration and/or expression patterns.

  • The fonder was mosaic, with two or more separate integration patterns. As a general rule, all F1 offspring from a founder mouse should be carefully analyzed not only with PCR, but also with means to determine integration patterns.