LGS's+Milestone+Map+and+Report

Zebrafish Milestone Map and Report

Lesley Sullivan Advanced Research December 21, 2007 Final Report

Lesley Sullivan Advanced Research P.10 December 21, 2007 Final Milestone Map and Report


 * Milestone Map**


 * 1) Learn about zebrafish (//Danio rerio//)
 * 2) Get license to work with animals in a lab
 * 3) Understand a lab set up and responsibilities
 * 4) Recreate own lab
 * 5) Undertake research project


 * Milestone Report**

//Notre Dame// //Katie and I were very lucky to have worked at Notre Dame’s QuarkNet Center last summer. Because of that wonderful opportunity, we were teamed up with James Whitcom, a biology doctoral student at Notre Dame, and also allowed to work in the same lab as esteemed biologists such as Dr. David R. Hyde. The Center for Zebrafish Research at Notre Dame allowed Katie and I to brag about having the best summer job! The opportunities we had last summer do not compare to many others. Thanks to QuarkNet and NDerC, our project took off.//

Our first task was to research zebrafish.
 * __I. LEARNING THE WAYS OF THE FISH__**


 * //__Danio rerio__//**


 * Zebrafish, //Danio rerio//, are freshwater fish that were originally found in slow streams and rice paddies and in the Ganges River in East India and Burma. Zebrafish embryos have become very popular worldwide as a means of understanding how not only fish, but all vertebrates, including people, develop from the moment that sperm fertilizes an egg.


 * Zebrafish Used in Research**


 * In the early 1970's, a scientist at the University of Oregon by the name of Dr. George Streisinger determined that the zebrafish is a wonderful model for studying vertebrate development and genetics. Since he began using them in his research, zebrafish embryos have become very popular worldwide as a means of understanding how not only fish, but all vertebrates including people, develop from the moment that sperm fertilizes an egg. The eggs are clear and develop outside of the mother's body, allowing scientists to watch a zebrafish egg grow into a newly formed fish under a microscope. Scientists will occasionally move a cell to another spot to see if it will still go to form the same part of the body as it is known to do in other embryos or if it will do something different. Occasionally a cell is removed or destroyed to see what the result is to the fish once it has developed. This is how scientists are discovering the causes of birth defects in human children and it's how they are trying to find a way to prevent these birth defects by understanding why they happen and what original cells are involved. So, this little obscure fish is helping us to learn about how all vertebrates develop and why sometimes things go wrong in that development to cause birth defects and other health problems. It is serving a very important role in our understanding and some day it may play a huge role in overcoming these things.


 * Zebrafish are ideal models for studying human genetics**


 * Zebrafish are vertebrates. Like humans, they have a backbone. This means that they are more closely related to humans than commonly used invertebrate models such as insects and worms (//Drosophilia// - fruit flies and //Caenorhabditis elegans -// nematodes) which do not have backbones. Because zebrafish are more closely related to humans, they are more likely to be similar to them in many biological traits than a more distantly related organism. These biological traits would include genes, developmental processes, anatomy, physiology, and behaviors. This is an advantage that invertebrate lab animals do not share with humans. The invertebrates are more appropriately used in comparisons at the cellular or biochemical level of organization where they share many features with humans.


 * No single model is perfect, but zebrafish have features that make them easy to maintain, manipulate, and observe in the lab. They do well in many environments, and their small size, their ability to be kept together in large numbers, and the ease with which they can be bred makes them a favored model. Breeding and getting eggs from the zebrafish is relatively easy. Their eggs are externally fertilized, produced regularly in large numbers, and are non-adhesive. Their embryos develop rapidly, and are clear throughout their development. Their embryos are also smaller than many vertebrate embryos and contain smaller numbers of cells. It is easier to trace the development of individual cells.


 * Females lay large quantities of eggs. For many types of genetic analysis you need to look at many different embryos at many different stages to understand what the problem is with a given mutation.
 * The embryos develop outside the mother's body, so you can have easy access to them. In contrast, mouse embryos develop inside the mother, and you have to kill the mother to get at them. This would have to be done at each of the stages of development you want to look at. Once you do this, of course, the embryos die as well as the mother, so you are very limited in the types of experiments you can do.


 * Zebrafish embryos are transparent. This means you can watch development as it happens in living embryos. You can see internal organs, such as the brain, heart, blood, muscles, etc. In addition, you can monitor the behavior of single cells in live embryos and watch the cells divide and through dyes, trace where each cell’s "daughters" go in making up the complete organism. It is not possible to achieve this resolution with other systems.


 * The embryos develop quickly. They go from a single cell to something that is recognizable as a tiny fish within 24 hours. Mice take 21 days.


 * You can physically manipulate the embryos. By this I mean you can transplant single cells or groups of cells into host embryos. This kind of experiment is performed frequently to analyze the behavior of cells at different stages, or to ask how mutant cells behave in wildtype embryos. This can give us a lot of information about how certain gene products act. In addition, fertilization of the egg can be manipulated so that the embryo contains only it's mother's genes. This is done by exposing the sperm to ultraviolet light which destroys the genes it contains from the male. This allows scientists to study recessive mutations since the characteristics and defects are inherited from only one parent.


 * There is a large community of researchers willing to share their knowledge of the more specific areas of zebrafish research.


 * Research using zebrafish**

· Features of the zebrafish strongly favor studying embryology and collecting and analyzing mutations and comparing them to other vertebrates. There are invertebrates that are equally or better suited for these types of studies, but they have very different anatomy patterns of development. These and other approaches allow a problem to be studied from many different experimental points of view, resulting in a better understanding of it.

· There is now a large base of established knowledge on the developmental biology and genetics of the zebrafish. Many useful genetic techniques and staining reagents have been developed that allow mutations to be more easily found, studied, and mapped.

· You can do genetic analysis. This means you can generate mutations, and identify genes required for a wide variety of biological processes. Genes direct synthesis of proteins, which do all the work in a cell. A mutation is a change of DNA that disrupts the resulting protein. Often, a defective protein will disrupt an essential biological process--and we can learn a lot about that process by analyzing how it can be screwed up. We can also learn much about the role of the normal protein by understanding what goes wrong when that protein is defective.

· There is a detailed genetic map of the zebrafish genome. This facilitates the identification of proteins disrupted by mutations. The genome is currently being sequenced and is due to be completed by the end of next year. This will make the process much easier. It will also facilitate evolutionary comparisons of the zebrafish genome with the mouse and human genomes.

· You can make transgenic fish--that means introducing foreign DNA in a heritable manner. This is crucial for analysis of gene regulation, and also for gene function.

Training is crucial to the proper care and use of animals in research and teaching. Personnel must be instructed in proper methods of handling, husbandry, animal and occupational health, and experimental methods.
 * __II. LICENSE TO WORK WITH ANIMALS__**


 * Policy on Training Investigators Using Animals**

· In order to work in the Freimann Lab, we had to pass an exam to demonstrate that we knew how to deal with animals in a lab. Responsible animal care and use is not only a regulatory requirement, it is an ethical imperitive. The policies and guidelines outlined in the manual we had to study are meant to serve as a general information source for investigators, students, and others using vertebrate animals in teaching and research. It was our responsibility to ensure that we were adequately trained to promote safe and humane animal use. · The PI resource manual can be found through Notre Dame’s website. In order to access the manual, however, you need an ND i.d.. It was a very exciting moment when Katie and I got our login names!
 * The federal Animal Welfare Act (administered by the USDA) and regulations of the Department of Health and Human Services require training and continuing education for all scientists, research technicians, and animal care technicians and others involved with animal care and use. These laws and regulations require that the Institutional Animal Care and Use Committee (IACUC) as an agent of the University of Notre Dame, determine that personnel wishing to conduct procedures on animals or animal tissues are qualified and trained to do so on the animal species proposed. To fulfill this responsibility, the following mandatory training program has been developed.


 * Laboratory Animal Training Association Video Module**
 * “Human Care and Use of Laboratory Fish” (2003)
 * Level 5: Test Questions 1-10 for “Human Care and Use of Laboratory Fish”


 * IACUC**
 * The IACUC is responsible for the overall assurance that individuals involved in handling animals or animal-derived tissues or fluids are properly trained. The IACUC must assure that mechanisms exist that will effectively communicate needed information to personnel.


 * PI Manual**
 * Administrative Policies
 * Training
 * Security
 * Emergency Procedures
 * Laws and Regulations
 * Federal Regulations
 * Institutional Regulations
 * Ethics and Scientific Issues
 * Basic Principles
 * Alternatives to Animal Use
 * Humane Endpoints in Animal Experimentation
 * Occupational Health and Safety
 * Laws and regulations
 * Confidential Medical Form
 * Education: Health Issue Summaryà Animal Allergies
 * Training
 * Equiptment
 * Technical Procedures
 * Anesthetics, Analgesics, Tranquilizers and Neuromuscular Blocking Agents
 * [|http://www.nd.edu/~ndflsc/piresourcemanual.html]

//We had the wonderful opportunity to learn from the best over the summer: before attempting to set up our own lab we got to observe the goings-on at the Freimann Zebrafish Lab.//
 * __III. UNDERSTANDING LAB SETUP__**


 * Freimann**
 * Katie and I got to learn about maintaining a laboratory first hand. The women who work breeding the fish and maintaining the colony work all year round, even over holidays. This is not a normal job. They do not simply feed the fish once a day as most people with fish tanks do. These women are in charge of tens of thousands of tanks of fish. They are never off their feet on the job. From spending a few days in the lab, we noticed that there were 3 different rooms with tanks lining every wall. The picture below is what just part of one room would look like. Besides taking care of the adult fish, they were also in charge of breeding the fish and taking care of the fish eggs and fry (baby fish). Katie and I learned not only daily procedures, but how much care is needed to keep the colonies alive and healthy. (We also learned the importance of closed toed shoes!)

//This picture shows a zebrafish lab with a flow-through system for water maintenance.// //Over the summer, we set up our own lab in the basement of Freimann. When the school year started, we recreated the lab at St. Joe and made minor adjustments to overcome slight obstacles.//
 * __IV. RECREATE LAB__**


 * Space for lab**
 * Requirements
 * Ability to control light
 * Access to power source
 * Access/ means of getting clean water for tanks
 * Summer 2007
 * Basement of Freimann (also had air source)
 * St. Joe
 * Supply closet off of 3rd floor lab
 * Access to power source
 * Plenty of space
 * Sink


 * Equiptment**
 * Tanks
 * Enough tanks to separate males and females, and preferably enough to separate wild type from albino.
 * 5 gallon tanks with cover
 * Rack
 * We have a 5-shelf rack
 * Filters
 * Air source
 * Thermometers
 * One for each tank
 * Very important because fish’s water has to stay at a constant 82° F
 * Heaters
 * One for each tank
 * To maintain the 82° F
 * Tubing
 * Gravel/marbles
 * We preferred marbles for the breeding tanks when we bred in the 5 gallon tanks so that the fish would not eat their eggs
 * Gravel is the normal choice for the regular holding tanks
 * Breeding tanks
 * When we were at Freimann we used the 5 gallon tanks with marble
 * At St. Joe we use smaller plastic tanks with slits in the bottom to let the eggs fall through. It’s a funky apparatus: the small plastic tank is submerged in a larger plastic container in a canopy-style so the plastic tank is slighty off the bottom of the larger container, allowing the eggs to fall safely away from the adult fish.
 * Food source
 * Brine shrimp
 * We hatch brine shrimp daily to feed to the fish
 * We hatch the eggs in salt water in a 2 Liter soda bottle; 8 grams of eggs every 2-3 days
 * The fish are fed three times daily with one dropper full of shrimp
 * Brine shrimp
 * We hatch brine shrimp daily to feed to the fish
 * We hatch the eggs in salt water in a 2 Liter soda bottle; 8 grams of eggs every 2-3 days
 * The fish are fed three times daily with one dropper full of shrimp

//The set up is similar to the one below. Our soda bottle, however, is right side up and contains a heater as well as an air tube.//

//Just over a centimeter in size, the adult brine shrimp is an extremely well known animal because of its importance as a food source for fish raised in home aquariums, aquaculture systems, and **laboratories**. You can buy brine shrimp at practically any pet store. It looks like a powdery brown substance but in reality it is thousands of eggs surrounded by protective cases (cysts). When added to water, these cysts will hatch into shrimp within a few hours.// //It is this ability of the brine shrimp cysts to remain dormant for long periods of time and then easily hatched that has made them a valuable organism for research. These cysts can withstand changes in temperature. (This is because they can lose and regain all their intracellular water.) Brine shrimp have been useful to researchers in genetics, histology, toxicology, radio biology, biochemistry, molecular biology, and ecology.//

//Close up magnification of single brine shrimp.//
 * Bleach
 * Tanks must be cleaned about once a month with bleach
 * **Before returning fish to newly cleaned tanks, it is very important to make certain that there is no more bleach left; check for bleach scent**
 * Gravels and marbles can also be cleaned with bleach (same precaution applies)


 * Clean water
 * We use approximately 2-3 gallons of water daily
 * The water has to be sterile; can NOT use regular tap water
 * At Freimann we used the same reservoir that Freimann used
 * At St. Joe we had to install a reverse-osmosis system; the system filters approximately 3 gallons of water a day (perfect!)


 * Petri dishes
 * Besides keeping tetramin (flake fish food) in the Petri dishes, we also store our collected eggs in these dishes because they don’t take up much space and can be easily stored in the incubator


 * Egg water
 * Special (blue) solution that eggs are placed in in incubator stage
 * Water contains nutrients for embryos
 * We get our supply from Notre Dame


 * Incubator
 * We used Freimann’s over the summer
 * We have our own incubator at St. Joe which we set at 28° C when there are eggs in it


 * Fish nets
 * For transfer of fish from tank to tank


 * Brine shrimp net
 * We don’t use this at St. Joe but over the summer we used it daily
 * Over the summer we would collect all the shrimp that had hatched overnight with the net and concentrate them in a smaller amount of water. We found that we got a lot more shrimp when we gave them more than one day to hatch and so we no longer use the net.


 * 2 Liter soda bottle
 * For our brine shrimp set up as explained above


 * Salt water (or salt to mix with water)
 * We mix one gallon of water with a cup of sea salt about every 2-3 days to reset our brine shrimp aquarium


 * Tape
 * We use color coded tape to keep track of which heater goes to what tank and which air tube is connected to what tank. We use them as a means of organization.
 * Used also to label. In a lab, recording is very important. We keep track of dates of breeding and last feeding and last time the tank was cleaned and so on.


 * Fish
 * At St. Joe we currently have 10 wild-type zebrafish (5 males. 5 females) and 12 albino zebrafish (6 males, 6 females)
 * We are very proud to say that none have died (we had about 7 deaths when at Freimann which is not very uncommon)


 * Keeping record**
 * Computer installed
 * Thanks to a number of people, we now have a computer in our lab with internet access. We can easily update our logbooks daily online and record our observations.
 * Feeding schedule on door of lab
 * We keep a sheet of paper on the door of our lab. Every morning, Katie marks down what time she fed the fish. Every lunch period, I also feed the fish and record it. This way we can make sure that the fish are being fed 3 times a day.


 * //Clay//** **//High School//** **//also set up their own zebrafish lab under Mark Balentine.//**


 * __V. UNDERTAKE RESEARCH PROJECT__**


 * Look up past projects**
 * PubMed: A service of the U.S. National Library of Medicine and the National Institutes of Health
 * Using our Notre Dame i.d.’s, we were able to use this search engine to look up past research and current research being done using zebrafish


 * Decide on research project of our own**
 * Dr. David R. Hyde
 * After spending a summer in the Hyde Lab we were able to meet with Dr. Hyde on a few occasions
 * Now that we are comfortable with our set up, we are ready to start using our fish for research
 * We have e-mailed Dr. Hyde asking if he might have any research topics we can expand on. We are still waiting for a reply.


 * In the mean time…**
 * Hopefully while we are waiting for a response, we can start conducting minor experiments using the fish that have been done before so we get a feel for using the fish for actual research. A few ideas are as follows:
 * The effect of temperature on heartbeat
 * Harvesting eyes (a common practice done on zebrafish)
 * Once we are comfortable with the stability of our breeding, we would still like to show the biology and genetics classes at St. Joe the development of the zebrafish because it is so much like our own (and it’s cool!). Seeing the development of the zebrafish embryo under a microscope proved to me just how amazing science can be and I know it will have the same effect on other students.