1. Microinjection into Drosophila Embryos: A Guide for the Beginner (AKA The Crazy Person in the Backroom Screaming at the Weird Looking Thingy) Chantel T. Barrett, B.Sc., soon-to-be M.D. Department of Biology Memorial University of Newfoundland A presentation for partial fulfillment of my lab duties July 27, 2005

2. “Microinjection of Drosophila embryos is relatively easy to perform and has several important applications.” Ingham and Forbes, 1993  Uses:  Injection of DNA to make transgenics (what I’m doing)  Injection of RNA, oligonucleotides, metabolites, lineage tracers, cytoplasm, and antibodies  Transplantation of nuclei or cells between embryos

3. “Microinjection of Drosophila embryos is relatively easy to perform and has several important applications. Ingham and Forbes, 1993

4. ARGGHHHH!!!!

5. How exactly does microinjection work?  Transgenics created by insertion of known DNA sequence into chromosomal DNA, using transposon (P element)  P elements have transposase:  Allows insertion at any chromosomal site  Hopping from site-to-site within germ cells  Carrier P elements do not have transposase  Instead provided by helper P element (cannot insert into host chromosome)

6.  Both elements injected together into posterior end of egg where germ cells are made  Marker gene (white + ) added to P element  Red is dominant over white:  Red eyes in flies that have integrated and express P element  1 st generation: all white  2 nd generation: several red

7. So now that I know what’s going on, what exactly do I need?  Flies!  Mating cages (fancy word for cups ya pee in!)  Consist of inverted, perforated cup and apple juice plate smeared with yeast paste  To collect embryos to inject  Dechorionation materials  Water bottle, paintbrush, embryo basket (made of a cut Fisher tube and fine netting), dechorionation chamber, 50% bleach solution

8. And what else?  Stereoscope, forceps, 3% agarose green pad, slides prepared with double-sided tape, dessicating chamber (with calcium carbonate), timer  Injection materials  Injection mix (consisting of carrier and helper plasmids), micropipette, microloaders, femtotips (AKA “needles”), plumber’s tape, light and heavy oils, Vaseline, injection apparatus (inverted microscope, micromanipulator, plastic tubing attached to needle holder and syringe), humidor containing damp chromatography paper

9. AND??? PATIENCE!

10. Now that I have all my materials, what do I do?  About 3 days prior to injection, prepare new cups. Each cup should contain ~200 w 1118 flies. Change the apple juice plates daily.  On the day of injection change the plates every 60-70 minutes. The first two egg lays should be discarded as they will include eggs that have been withheld by the female, and have thus developed too much for your purposes.

11. Translation: There will be maggots crawling all over the green agarose pad when you are trying to line up the embryos…come on…who needs that! EWWWWWW!!!!

12. Next?  Once the plates are changed, take note of the time (embryos older than 70 minutes are no good!).  Flood the plates with water and loosen the yeast paste and embryos with a paintbrush (not too hard, as this will cause agar to loosen, and will be difficult to work around later). Pour contents into embryo basket and rinse with water.

13. Go on…  Immerse the little ones in the dechorionation chamber with 50% bleach solution. Swirl for ~60 seconds (until the respiratory filaments disappear).  Rinse the embryos with water, and blot the screen dry.

14. WARNING!!! Make sure that you have sufficiently swirled the embryos! Any amount of remaining chorion will cause you grief when you are trying to inject them later (i.e. the embryos will slide around when the needle hits them).

15. And then?  Transfer the (well-rinsed) embryos from the screen to the agarose pad by placing the screen embryo-side down and pressing gently (don’t squat them!). The pad should be tacky (i.e. not too dry, not too wet).  I find it helpful to place one drop of water from the bottle onto the pad and to smear it around with my finger.

16. Anything else?  Line up the embryos on the agarose pad with the forceps, facing their anterior ends towards the center of the pad.  The anterior end has the micropyle!  Transfer to double-sided tape and dessicate for ~3- 10 minutes.

17. Dessication Time  Depends on:  Weather  Humidity  Length of time aligning embryos  Calcium carbonate

18. In the meantime…  Make sure that the plungers containing the light and heavy oils, and the Vaseline, are full.  Set up the injection needle with ~2  L of appropriate injection mix. Seal the needle with plumber’s tape and attach to apparatus.  If you’ve thought ahead, these things should already be done! But hey, if you wanna run around like a maniac while you’re waiting, it’s your business!

19. Are we there yet?  Once embryos have dessicated, cover with heavy oil, and place slide on stage of injection apparatus.  Orient the embryos so that their butts are facing towards the needle!

20. Almost!  Bevel the needle using the side of the double- sided tape, ensure that DNA flows out when pressure is applied to the syringe.  Line the needle up with the posterior of the embryo, try to inject the area between the cytoplasm and embryo wall (where the pole cells are located).

21. After all flies on slide have been injected, remove slide from apparatus, surround embryos with Vaseline moat and cover with light oil. Place in moist humidor overnight.

22. OH YEAH! Make sure that the light oil does not flow over the Vaseline moat, as any surviving larvae could ESCAPE!!! OH NO!!!! This means that all your hard work will have literally left the premises!

23. Epilogue  Now that that part’s over, what now?  Pick out hatched embryos ~24 hours after injection, and place them into vials of food.  Hatched embryos will no longer be one of the “little soldiers” so nicely lined up on the double- sided tape! They will be “swimming in the moat”!  Hopefully the larvae will pupate and survive to adulthood. Cross survivors to w 1118 flies, and look for red-eyed offspring.

24. + TRANSGENIC FLY!!!

25. Record:  Number of:  Embryos injected  Embryos hatched  Pupae survived to adulthood  Fertile adults

26. So what’s so hard about that?  Locating the needle under the microscope can be impossible!  Once found, it must be in the same plane as the embryo for injection to be successful.  Needles are very fragile, and can break seemingly by breathing on them!  Even if injection is successful, there is no guarantee that the embryos will live through the night!

27. Questions or Comments?

28. This presentation brought to you by…  The letter “I” – for INSANE!!!! …or injection if you’re going to get smart about it!  And the number 33 …the number of times you will try to do this before going the “I” word!  And me…the “Crazy Person in the Backroom Screaming at the Weird Looking Thingy”!