Transgenic Fly Lab

• Understand how construct and transpose DNA are used in producing a transgenic organism.
• Practice (through virtual procedures using a computer mouse) the steps for selecting and inserting DNA into fly embyos.
• Identify the steps used in producing a transgenic organism.
• Utilize transgenic organisms to understand gene expression, specifically through experimentation with fly circadian rhythm.

This virtual lab is an animated tutorial that is used to help participants become familiar with sophisticated lab procedures and learn how to make a transgenic organism. By following steps in the virtual lab, participants learn about construct and transpose DNA, how these are used to make a transgenic organism, and experiments that can be performed on transgenic organisms to understand gene expression. The virtual lab takes about an hour to complete if participants move through the proper steps, and subsequent experiments require more time.

This lab helps users learn techniques to make transgenic flies and then use these organisms to study circadian rhythms.

1. Go to the website entitled "The Virtual Transgenic Fly Lab." The MERLOT description and link can be found at http://www.merlot.org/merlot/viewMaterial.htm?id=83538 and the direct link can be found at http://www.hhmi.org/biointeractive/vlabs/transgenic_fly/index.html.
2. Read the Introduction to The Virtual Transgenic Fly Lab to learn the steps involved. These steps include: 1) Prepare DNA that will be incorporated into the fly genome, 2) Prepare fly embryos, 3) Inject fly embryos with DNA, 4) Breed flies, 5) Select transgenic offspring, and 6) Examine light output from transgenic adults.
3. Proceed to the lab, which requires the full version of Adobe Shockwave Player, and can be downloaded at http://get.adobe.com/shockwave/otherversions/.
4. The site is equipped with sections entitled "notebook," "quiz," "resources," and "help." Participants are led to these sections as necessary, or they can be accessed by clicking on the tabs as the lab progresses.
5. Once transgenic flies are made, participants may proceed to one of three experiments: 1) Measure per-luc gene expression while keeping lights off, 2) Measure per-luc gene expression while changing light on-off, and 3) Examine different body parts of the fly for per-luc expression.
6. The notebook and quizzes guide participants through the lab and provide question-and-answer to ensure the individual steps are understood.

While some laboratories may be equipped to make transgenic organisms, this virtual lab can be implemented for those who do not have resources available, or used as a pre-lab assignment to familiarize participants with procedures before doing the actual lab. The virtual lab helps guide students through proper steps, but there are places (such as injecting DNA into fly embryos) where participants must pay attention to proper protocol in order to achieve anticipated results. If mistakes or poor procedures are attempted, this virtual lab guides users through a troubleshooting protocol to learn where refined procedures can be implemented. This can be frustrating to participants who might be more accustomed to just moving the mouse and clicking at random, because improper positioning or selection of reagents can lead to error. The virtual lab helps re-focus participants on being more careful during each step when mistakes are made.

The Virtual Fly Lab is equipped with quizzes that participants use throughout the experiment to keep them focused on what they are doing. The quizzes are taken on-line, graded, and corrected throughout the individual steps of the lab.

MERLOT description and link to '"The Virtual Transgenic Fly Lab," which provides a very-well animated tutorial for producing a transgenic organism and using that organism to study gene expression. See http://www.merlot.org/merlot/viewMaterial.htm?id=83538

The following resources are also listed as follows at this site:

Allada, R., Emery, P., Takahashi, J.S., and Rosbash, M. 2001. Stopping time: The genetics of fly and mouse circadian clocks. Annual Review of Neuroscience. 24: 1091-119.

Bargiello , T.A., Jackson, F.R., and Young, M.W. 1984. Restoration of circadian behavioural rhythms by gene transfer in Drosophila. Nature 312 (5996): 752-5-4.

Brandes, C., Plautz, J.D., Stanewsky, R., Jamison, C.F., Straume, M., Wood, K.V., Kay, S.A., and Hall, J.C. 1996. Novel features of Drosophila period transcription revealed by real-time luciferase reporting. Neuron 16: 687-92.

Citri,Y., Colot, H.V., Jacquier, A.C., Yu, Q., Hall, J.C., Baltimore, D. and Rosbash, M. 1987.A family of unusually spliced biologically active transcripts encoded by a Drosophila clock gene. Nature 326 (6108): 42-47.

Darlington, T.K., Wager-Smith, K., Ceriani, M.F., Staknis, D., Gekakis, N., Steeves, T.D., Weitz, C.J., Takahashi, J.S., and Kay, S.A. 1998. Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim. Science 280 (5369): 1599-1-603.

Fujioka, M., Jaynes, J.B., Bejsovec, A. and Weir, M. 2000. Production of transgenic Drosophila. In Developmental Biology Protocols, vol. 2, ed. R.S. Tuan and C.W. Lo, 353-63. Methods in Molecular Biology, Vol. 136. Totowa, NJ: Humana Press.

Konopka. R.J., and Benzer, S. 1971. Clock mutants of Drosophila melanogaster. Proceedings of the National Academy of Sciences USA. 68(9): 2112-6.

Plautz, J.D., Kaneko, M., Hall, J.C., and Kay, S.A. 1997. Independent photoreceptive circadian clocks throughout Drosophila. Science 278: 1632-35.

Spradling, A.C., and Rubin, G.M. 1982. Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218: 341-47.

Stanewsky, R., Kaneko, M., Emery, P., Beretta, B., Wager-Smith, K., Kay, S.A., Rosbash, M., and Hall, J.C. 1998. The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell 95: 681-92.

Wager-Smith, K., and Kay, S.A. 2000. Circadian rhythm genetics: From flies to mice to humans. Nature Genetics. 26 (1): 23-2-7.

Wilsbacher, L.D., Yamazaki, S., Herzog, E.D., Song, E.J., Radcliffe, L.A., Abe, M., Block, G., Spitznagel, E., Menaker, M., and Takahashi, J.S. 2002. Photic and circadian expression of luciferase in mPeriod1-luc transgenic mice in vivo. Proceedings of the National Academy of Sciences USA. 99 (1): 489-94.

Young, M.W. 2000. The tick-tock of the biological clock. Scientific American 282(3): 64-71