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Petri dishes/100mm tissue culture dishes
Very fine tweezers
Plastic transfer pipettes
Frogs are killed by destruction of the head, ovaries are then dissected and put into MBS solution.
Preparation of the oocytes
- Wash the ovary as soon as possible with MBS solution to remove all traces of blood and debris.
- Gently pull the ovary apart with the tweezers.
- Select a small section of ovary, hold it with the tweezers in a dish of MBS, carefully dislodge the oocytes from the surrounding tissue with a wire loop. (more oocytes remain intact using this method rather than the razor blade/spatula method).
- Transfer intact oocytes into clean MBS.
- leave overnight in MBS at 20° C (oocytes are easier to inject after overnight incubation because the outer membrane becomes firmer).
Selection of oocytes for microinjection (injections should be completed within 48 hours of removal of the ovary from the frog)
- Examine oocytes under the microscope and transfer healthy looking ones to a new dish containing MBS.
- Select oocytes at developmental stage IV. Successful microinjection/expression depends upon selecting the correct oocytes at this stage. Oocytes at stage IV are the larger ones in the sample, they have good contrast between the black nuclear pole and the creamy coloured cytosolic pole. Oocytes which are jelly like or which have indistinct poles should be discarded. Healthy oocytes should be smooth, quite firm and should not burst too easily when gently manipulated.
3 Transfer these oocytes into clean MBS in the 20° C incubator.
- Pull a glass capillary, examine under the microscope and using the fine tweezers gently break off the end of the needle. (for cytoplasmic injections the needle width is not so important, but for nuclear injections it is easier if the needle has a very sharp thin point. Try to break of the needle as close to the end as possible but choose a point where the needle is fairly rigid otherwise it will bend when you try to inject.
- Backfill the capillary with mineral oil and assemble onto the injector*. (avoid air bubbles by slowly withdrawing the needle whilst continuing to inject oil, you may find it easier to leave the end of the needle intact before backfilling with oil.)
- Press the empty button on the injector for a few seconds to dispel any air bubbles (break the end of the needle first if you have not already done so.)
- Take a small petri dish and colour the surface over with marker pen. Cover with parafilm.
- Indent the surface of the parafilm with a pipette tip and pipette a small drop of sample onto it.
- Focus on the drop under the microscope and position the needle over it by eye, look under the microscope and make sure you can see the needle enter into the drop. Remember to hold the needle in place (as it will slowly move downwards) and press the fill button on the microinjector. Fill half way up the needle with sample. (make sure your sample is in a substance that is not miscible with the mineral oil, you should clearly see the difference between the two phases.) Check for air bubbles.
- Withdraw the needle from the droplet and focus on the needle point. Press the empty button for a few seconds until the sample flows from the point and any air bubbles have been expelled.
Microinjection of the oocytes
- After the sample has been loaded, take two microscope slides and place one overlapping the other. Use a transfer pipette to take about 10 oocytes from the pre selected batch. Line them up against the edge of the overlapping microscope slide.
- Put the slide under the microscope and examine the oocytes, manipulate carefully with the tweezers so that the pole to be injected is facing upwards.
Injections into the cytoplasm
- Change the switches on the microinjector so that the correct volume is injected. For cytoplasmic injections the switches should be up, up, up, down.
- Line the oocytes up with the cream coloured pole facing upwards.
- Put an upturned petri dish under the microscope and put the slide with the oocytes on top. (This makes moving the slide easier) Position the needle by eye over the oocytes to be injected. Focus under the microscope and aim the needle roughly over the centre of the cytoplasmic area, using a gentle stabbing action insert the needle into the cytoplasm. Remembering to hold the needle in place press the inject button (foot pedal is easier.)
- Hold the needle in the cytoplasm for a few seconds after injection, withdrawing too quickly will result in the sample oozing out of the oocyte.
- The oocyte should be smooth and moist if they have started to dry then, inject some of the sample over the injection site before attempting to insert the needle. Discard the oocytes if they have dried out too much i.e. the surface is cracked.
- Take the needle out of the oocyte and move the petri dish along to the next oocyte.
When all the oocytes have been injected wash them into a well of a six well T.C plate containing MBS and return them to the incubator.
- Change the position of the switch to Down, Up, Down, Down.
- Line the eggs up along the microscope slide so that the black pole is facing upwards.
- Position the needle by eye above the oocyte to be injected, focus under the microscope and insert the needle into the egg using a gentle stabbing motion. Make sure that the needle enters the oocyte exactly in the centre of the black pole. Withdraw the needle from the oocyte so that it is positioned just under the surface of the black pole.
- Hold the needle in position and press the inject button.
- Hold the needle in the oocyte from a few seconds and then withdraw it slowly.
- Inject the rest of the oocytes and then wash them into a well of a six well T.C plate containing MBS.
After injection leave the oocytes at 20° C for 48 hours, change the MBS solution each day and discard any unhealthy eggs.
Assaying for gene expression
- Detection of gene expression depends upon which reporter gene was in the DNA that was injected.
- Fill three petri dishes with the appropriate lysis solution.
- Snip the end off a blue pipette tip.
- Examine the injected oocytes under the microscope and only select healthy ones for assay i.e. the healthy ones have good contrast between the black and cream halves. Discard any oocytes, which are discoloured or damaged.
- Using a 1ml gilson and the blue tip that has been cut, select 5 oocytes and put them into the first petri dish. Transfer as little of the MBS over as possible. Transfer the oocytes from the first dish to the second dish, transferring as little of the first lysis solution over as possible. Finally transfer the oocytes from the second dish to the third dish, transferring as little of the second lysis solution over as possible.
- Pipette the 5 oocytes up with 500m l of the third lysis solution and transfer them to a 1.5ml eppindorf.
- Repeat this procedure for all of the oocytes.
- Using a glass pasteur pipette, break open the oocytes in sample by pipetting up and down.
- Centrifuge the samples at 15000rpm for 10 minutes.
- Carefully take out the eppindorfs from the centrifuge if the layers are disrupted then re centrifuge.
- Examine the samples there are 3 layers; the pellet with the remaining membranes etc. Above this is a clear layer, and on top is a cloudy layer containing protein etc.
- Take a 20m l sample of the clear layer above the pellet take care to wipe the outside of the pipette tip with a tissue before transferring it to a luminometer tube to prevent contamination with protein etc..
- Assay for Luciferase, b galactosidase or GFP activity (depending on which plasmid was used for injection) in the usual way.
* Modified Barths Saline (MBS)
15mM Hepes NaOH pH7.6
0.3mM Ca(No3) 2
50m g/ml Gentamycin
Since the MBS contains many chemicals it is easier to make 2 stock solutions which can be stored frozen and diluted in water before use
High salt stock
pH the solution to 7.6 and make upto 1000mls with water
Divalent Cation stock
1.9g Ca(No3)2 4H2O
2.25g CaCl2 6H20
5.0g MgSO4 7H2O
Store 40ml aliquots of the solutions at -20°C.
To use dissolve 1x 40ml aliquot of high salt stock and 1x 40ml aliquot of divalent cation stock into 1000mls of water and add gentamycin before use.