Axolotl

Maintaining the axolotls

• Running tap Water… water change should be 10% of tank capacity everyday
• BIO and Mechanical Filters
• Measure Nitrite and Nitrate once a week
• If higher then 100 change water, or if required filter… first mechanical and last bio
• Room Temp 200C can be higher up to 240C for fast growth
• Lower temp is better as bacteria doesn’t grow well
• Also low temp eggs seems better

• Water temp 16-18 0C
• pH- 7 to 8
• Clean tanks 2-3 times a week

• Make a log book for Room temp, Tank water Temp., Water is a good habit.

• Heavy belly has a problem laying eggs.

Before Dissection

• O.03% Benzocaine to make axolotl sleep.

Mating

If eggs are required for injections, it is recommended to set up mating in the afternoon (3-4 pm),

1. Action in the mating database must be taken first: tanaka(\\storage.imp.ac.at\groups\tanaka) -> Organisms -> Axolotl -> Mating History Vienna full list
2. Preparation of the mating tank: Choose a clean tank and check the water temperature (15-16C) at the display of the water supply.
3. The stock tanks with the two mating partners are put on a trolley and moved to the mating tank area.
4. Each animal is taken out of the tank with a net.
5. The transponder should be checked with the handheld scanner while the animals are in the net. The reader just responds, when it is close to the animal.
6. The animals are put into the mating tank. (Start with the male)
7. The handwritten ID sticker should be moved from the stock tanks to the mating tanks.
8. The water supply needs to be stopped by pulling up on the inlet valve until you see a yellow strip.
9. 10-15 clean plastic leaves are put into the mating tank, swimming below the surface.
10. A lid is placed on top of the mating tank.
11. The empty stock tanks are labeled with purple tape and date.
12. The empty stock tanks go back to their stock position.
13. Poster walls are placed in front of the mating area, to protect the animals.
14. Axolotls don’t need to be fed during the mating.
15. Control after 24h, if spermatophores are visible, after 48h if eggs are visible.
16. Successful mating: use a bucket with tap water to put the plastic leaves in. If you want to grow the animals up, collect the eggs into a white square box and put them on the metal shelf. Not successful mating: maximal mating time should not be more than one week, otherwise animals need to be fed again.
17. Animals plus ID sticker are put back to the stock tanks. (Please make a check on the purple tape, if the mating was successful.)
18. Cleaning of mating tank and leaves: rinse the leaves carefully with tap water and put them back. Re-start circulation in the mating tank and label it with a sticker PLEASE CLEAN, then the animal caretakers take over.
19. Results must be reported in the database.

Feeding

Artemia (Brine shrimp eggs)

Brine shrimp eggs are metabolically inactive and can remain in total stasis for two years while in dry oxygen-free conditions, even at temperatures below freezing. This characteristic is called cryptobiosis meaning "hidden life" (also called diapause). While in cryptobiosis, brine shrimp eggs can survive temperatures of liquid air (−190 °C or −310.0 °F) and a small percentage can survive above boiling temperature (105 °C or 221 °F) for up to two hours.

Once placed in brine (salt) water, the cyst-like eggs hatch within a few hours. The nauplii, or larvae, are less than 0.5 mm in length when they first hatch. Brine shrimp have a biological life cycle of one year, during which they grow to a mature length of around one centimeter on average. This short life span, along with other characteristics such as their ability to remain dormant for long periods, has made them invaluable in scientific research, including space experiments. This ability has also enabled the use of a hybrid of brine shrimp, bred to grow larger and live longer, as Sea-Monkeys.

Artemia protocol (Vienna)

1. Prepare the artemia incubator funnel for hatching artemia cysts: 2 incubators per day (Saturday and Tuesday 3 incubators).
2. Insert heating element (adjust to 28°C).
3. Add 10L warm water from tap.
4. Wait until water is warm enough (lukewarm).
5. Put aerator into the funnel and start air bubbler and put lid on the top of the funnel.
6. Add Holtfreter´s solution: 50 ml.
7. Add 250g sea salt.
8. Wait 5 minutes so that the salt is dissolved completely.
9. Add 65g artemia cysts.
10. Hatch artemia for 48h (36-48h).
11. Remove lid and aerator.
12. Remove heating element.
13. There are 3 liquid phases in the funnel: 1. on top: the egg shells, 2. in the middle: nauplii (orange), 3. at the funnel outlet: unhatched cysts
14. Cover the upper half of the funnel with an opaque bag.
15. Attach a light source to the funnel outlet to attract nauplii.
16. Wait 5-10 minutes.
17. Prepare 1 x 5L-beaker, add 50ml Holtfreter´s solution and fill up with warm tap water .
18. Prepare 2 x empty 2L-beaker into which the nauplii have to be transferred.
19. Empty the funnel by opening the discharge screw on the bottom, discharge unhatched cysts (phase 3).
20. Collect nauplii (phase 2) in a strainer.
21. Discharge egg shells (phase 1).
22. Empty the strainer by pouring nauplii into the 2 empty beakers (equal parts).
23. Turn around the strainer and rinse the net with warm water from the 5L-beaker (A) so that the remaining nauplii attached to the net don´t get lost.
24. Fill up the 2L-beakers with warm tap water to 2L each from the 5L-beaker (A).
25. Wait 5-8 minutes so that the artemia phases have time to separate.
26. Empty the 2L-beakers into the 5L-beaker, and make sure that phases 1 (on Top) + 3 (on the bottom) are discharged (concentrate nauplii by doing so).
27. Add 50ml Holtfreter´s solution to both parts and fill up with warm water to 5L.
28. Empty the 5L-beaker with nauplii and pour nauplii into the 2 empty beakers (equal parts).
29. Repeat steps 24-28 as often as necessary.
30. Put an aerator into both beakers.

Cleaning of incubators: Rinsing the funnels with cold water (do not forget the outlet connection). Clean with a soft sponge, do not scratch the plastic surface. Rinsing the funnels with hot water.

Artemia Hatching Preparation: Protocol for 15 Ltr (Dresden).

Switch on the bubbler.
Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.
Switch on the heating. And make sure that the light on the top is on ☺ Add 50-60 ml full of dried artemia (Stored at 40C) to this.

Hatching preparation (Dresden)

• The following protocol is considered for 15 l solution.
• Switch on the bubbler.
• Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.
• Switch on the heating. And make sure that the light on the top is on.
• Add 50-60 ml full of dried artemia (Stored at 40C) to this.

Keep it like this for 2 days with aeration. And >200 Lux of light.
Usually they hatch within few hours but It is good to keep for 2 days to achieve a good hatching.
During these two days temperature can be 18°-20°C.
Make sure hatching container is not getting clogged during these two days.

In vitro fertilization

1. Inject HCG into the dorsal muscle of the hind limb thigh of the axolotl No need to put the animals asleep, just cover the head with the wet tissue and hold them tightly while injecting.

• HCG stock preparation: 10000/bottle, add 10 ml H2O to make 1000 U/ml . Freeze 1ml aliquots and keep at -20C
• Inject females approx 24-26h before intended egg collection time with 350 U HCG per 100g body weight
• Inject males with 200 U HCG per 100g body weight min 18-20 h before sperm collection time

1. Sperm and egg collection

• It is easier to squeeze the sperm/eggs when the animals are put to sleep first.
• For both, males or females apply the pressure on the lower abdomen starting from 2–3 cm cranial to cloaca and moving towards the cloaca. A bit like squeezing mayonnaise out of the tube.
• Collect the sperm first; it will remain motile and functional for about 24h when kept on ice. Eggs should preferably be used immediately after collection.
• Collect released semen into an Eppendorf tube and keep on ice. Check motility in collected sperm. The sperm in big clumps moves very vigorously. Single sperm much less.
• The semen will typically drain down the animal’s tail along with the droplets of benzocaine solution where the animal was kept to fall asleep. That is OK, doesn’t seem to affect the motility or fertilization.
• Collect the eggs onto a Petri dish. Avoid the water dripping from the animal to mix with the eggs.

1. 3. Fertilization

• Drop small droplets of semen onto eggs making sure to touch every egg individually. Leave like this for 5 minutes, then flood with Fertilization solution.

Fertilization solution (acc. to Mansour) 20 mmol/l NaCl,

 1 mmol/l  KCl, 
 1 mmol/l  Mg2SO4, 
 1 mmol/l  CaCl2, 
 3 mmol/l  NaHCO3, 

10 mmol/l TRIS, pH 8.5

(TRIS pH 10 may work even better)

Interesting observations

Below you can find the list of observations along with the name of the person who did the observation.

Person	Description	Date

Genotyping

• Navigate to galaxy.imp.ac.at and log in using your IMP credentials
• Upload the multiplexed BAM file by either clicking the arrow in the top right corner of the tools panel (left-hand side of the window) or clicking Local file(s) > Upload file

• A dialog window will pop up. You can select the local files or drag and drop them into the window. Once you selected all files, click Start to start the upload. After that you can close the dialog. The upload will continue in the background.
• Once the file upload finishes, the files will appear in the right-hand part of the window – the History section.

• Select the Genotyping tool in the tools section on the left-hand side by typing “genotyping” into the search field.

• Select the BAM file to analyze
• If you used the default Tanaka lab barcodes, set the Barcodes option to YES
• Otherwise, specify the JSON file containing the barcodes (contact Sergej for that)

• Specify the minimum overlap between the paired-end reads. When running the tool for the first time with a new BAM file, set this value to 4. This value only needs to be adjusted, if the tool fails.

• If your samples are in a few wells of the plate, it may be much faster to only process those wells, instead of the entire plate.
• It is highly recommended to pre-process the reads. To do that, you need to upload the file “Illumina.fa” into your history (see step 1). The file can be found on the fileserver under /groups/tanaka/Data/adapters.
If the length of your PCR product is shorter than the read length, e.g. 150bp, while the reads are 250bp, it may be better to trim the reads to the expected length. This can be achieved by specifying the cropped length (last field in the screenshot).

• Specify the amplicon name, the expected amplicon sequence (i.e. the sequence of the PCR product), and the gRNA sequence (18nt) in the 5’->3’ orientation. The gRNA sequence should not contain the PAM sequence (NGG for spCas9). 
Make sure the sequences contain no invalid nucleotide sequences, spaces or newlines since it may confuse the tool.

• The following parameters are optional and should only be changed in special cases:

1. If you used other CAS9 protein that has a different cleavage site. The default value of -3, which is suitable for the commonly used SpCas9.
2. You expect modifications at the positions that are offset with respect to the cleavage site (extremely unlikely)
3. You want to include more than 15nt on either side of the cleavage site to the indels analysis (rare)
4. Ignore certain types of modifications. This can be advantageous if you see many irrelevant polymorphic sites and want to exclude them from the analysis.