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X. Laevis Microinjection Protocol

Before starting: Training, Notes and Logbook

___When you use laevis for the first time, be sure to have someone from lab come along and show you exactly where things are and help you prime and boost. Laevis are big and wrangling them can take some practice.

___Print out the abbreviated protocol as a guide.

___After you are confident, you can use the abbreviated protocol and go on your own.

___Be sure you complete the steps in and fill out the laevis logbook (white binder), located on the laevis work bench upstairs each day. If you are unsure how to fill it out or how to do anything required in the book, ask Maura or Emily Mis.

___Make sure there is 3% Ficoll made up for the embryos. If not, ask whoever is responsible for it to make some because it needs to be autoclaved before use.

___The laevis incubator needs to be locked any time there are frogs in it. The key is in an empty tip box on the first shelf above the laevis bench, right hand side.

3 days before: Get HCG from Sarah

___Three days before you plan to inject, contact Sarah and let her know you will need a syringe of HCG to prime 3 female Laevis (approx 0.2 ml; this is enough to give each female 50 units (0.05ml) plus a bit extra for loss). Tell her you will also need 3 full syringes for boosting, 0.75 ml each. She will make these up and leave the syringes in the small refrigerator in the trop kitchen. Have someone show you where this is. She will label the syrings with tape that says "laevis".

___ Collect 3 females from "For Use" tank into transfer tank using fresh net kept near the sink. It is best to get three frogs that look different, so you can keep track of which ones you injected. Prime the 3 females with 50 microliters of hCG.

Prime females 2 nights before (around 5pm):

___Bring large transportation tank and lid from shelf over sink in laevis area and the 1 ml syringe with hCG for priming + a few extra needles to laevis room on a cart. Make sure you go to trop room to get HCG before going to laevis room. Don't go back to the trop room after you go to the laevis room to avoid transferring pathogens from the laevis to trops. Fill the tank with about 6 liters of system water from the hose in the corner. Make sure to turn off all the red water valves you turned on. It is best to have someone from lab who knows how to use the water show you first to avoid a flood.

___ Collect 3 females from "For Use" tank into transfer tank using fresh net from container kept near the sink. It is best to get three frogs that look different, so you can keep track of which ones you injected. Prime the 3 females with 50 microliters of hCG. Discard the needle in the sharps box.

___After priming, place the females in one of the individual holding tanks above the large tanks in the laevis room. Use one of the tanks on the left hand side of the room at the end. Make sure the tank is empty first.

___Cage card: Get a cage card from the grey file box on the shelf by the sink, filed under the Khokha lab tab. Put cage card on the tank with frog information completed. The small extra barcode at top should be torn off and placed in plastic folder on laevis room door.

___ Put the used net in the"Dirty nets" container on the lower shelf on the cart by the sink.

___ Fill in any relevant information in the log book upstairs.

1 night before (around 5pm):

___Bring down and fill 3 plastic tanks with about 6 L water from laevis room

___ Put the 3 primed females in one tank. Collect a male from the Khokha tank labeled "Males" into a second tank. Keep the third tank empty to use as a water change the next day.

___ Bring the 3 primed females, the male and the empty tank up to lab. Boost females with 0.5 - 0.6ml hCG. The higher dose might be needed if females have been laying late or if they haven't been used in 6 mos. - 1 year.

___ Place frogs in the incubator you have previously set to 16 - 18 deg C. Cooler they will take longer to lay, warmer they will lay sooner. Be sure incubator is locked. They should be starting to lay by the time you get to lab in the morning. Leave the third empty tank of water out on the counter overnight. If the frogs are not laying when you get in, you can put them in this warmer water to speed up laying.

___ Fill in any relevant information in the log book.

Injection Day

___Euthanize male by anesthetizing in benzocaine and decapitating with shears.

___ Dissect testes from male and place in a small petri dish of ice cold 1XMR. Testes can be stored at 4 degrees for up to a week or so.

___ Cut off 1/4 to 1/3 of a testicle and place in an epindorftube filled with 0.5ml of water.

___ Squeeze females for eggs into separate dishes so you can assess quality and only use the good quality eggs.

___Release sperm from testes with pestle.

___ Pour sperm on eggs, mix well with pestle and wait 3 minutes.

___ Start fertilization by flooding with water. You can put the eggs into the incubator to slow down development.

___ Wait 10 minutes or until you are ready to start.

___ During this time, make 3% cysteine in 1/3 X MR (pH 7.8), 2.5% ficoll in 1/3 X MR (should be pre-made) and 0.1X MBS ph 7.8 -8.0.

___ During this time, pull needles and rinse dishes.

___ Once at least 10 minutes have passed, cysteine embryos for 5 minutes or until de-jellied.

___ Rinse 3 times with 0.1X MBS, then 2 times with 2.5% ficoll (NOT 3% Ficoll- this is for trops). Leave embryos in 2.5% ficoll. They can then be kept at room temperature or in 16-18 degree incubator, depending on how much time you will need for injections.

___ Fill needle with RNA or other injection solution using pipette.

___ Break needle using tweezers, test with oil (10 nl = 13 hash marks). For injections, remove media above embryos so that surface tension keeps embryos in place.

___ Label dishes, after injections are done, add ficoll back to dish. Rest 1 embryos for 1 hour at RT.

___ Change solution to 1/3 x MR +/- GENT, put embryos at 18°C or desired temperature. See temperature/development chart for laevis under Developmental Timetables Link.

___ Put the females in fresh water in the afternoon of injection day. You can use the bucket of water you brought up the previous day if it is unused. Otherwise prepare fresh water by filling a tank with ~6L DI water(NOT TAP WATER_THIS WILL KILL THE FROGS) from the white faucet at the lavis sink. Add NaCl from the container on the shelf above the sink. The amount of salt to add is marked on the 50 ml Eppendorf tube in the NaCl container.

____Check the females to be sure they look happy and healthy. Check for any red marks on the skin or lethargy/bloating. If you are unsure, ask Maura to take a look. Never return a sick frog to the laevis room.

____Put the females back in the incubator for another night so they finish laying in the bucket and don't dirty the main tank downstairs. Make sure the incubator door is locked.

___ Fill in any relevant information in the log book.

Day After Injections

___ Check the females to be sure they still look good, and bring them back down to the laevis room.

____Return them to the "Return" tank. Try to avoid carrying eggs from the bucket into the tank.

____Bring the empty buckets back upstairs, spray or rinse the tanks/lids with a light solution of bleach, rinse our bleach and any eggs, and leave to dry on the shelf.

___ Fill in any relevant information in the log book, clean up the area.