Rapid-throughput teleost regeneration assay
View Patent ↗The present invention provides a method of simultaneously screening at least two candidate agents for an activity affecting regeneration of an embryonic teleost. According to this method, for each agent, a fin of the embryonic teleost is amputated. Next, the amputated teleost is incubated with the candidate agent for a specified period of time. After this period of time, the amputated teleost is imaged. This image is compared to an image of an amputated teleost that was incubated for the specified period of time in the absence of the candidate agent. When comparing the images, a change in morphology of the amputated fin of the embryonic teleost incubated in the presence of the candidate agent compared to the amputated fin of the embryonic teleost raised in the absence of the candidate agent indicates that the candidate agent affects regeneration.
1. A method of simultaneously screening at least two candidate agents for activity affecting regeneration of an embryonic teleost, comprising:
a) amputating a fin of each of at least two embryonic teleosts to create amputated teleosts;
b) incubating the amputated teleosts each with one of the at least two candidate agents for a specified period of time;
c) imaging each of the amputated teleosts after said specified period of time; and
d) comparing the images of each of the amputated teleosts to an image of an amputated teleost that was incubated for said specified period of time in the absence of the at least two candidate agents,
wherein a difference in the morphology between the fin of an embryonic teleost that was incubated in the presence of a candidate agent and the morphology of the fin of the embryonic teleost that was incubated in the absence of said candidate agent indicates said candidate agent affects regeneration.
2. The method as set forth in claim 1 , wherein said incubating is performed in a multi-well plate, and wherein each amputated teleost is incubated with a candidate agent in a different well.
3. The method as set forth in claim 1 , wherein said candidate agents are selected from a small molecule library of candidate agents.
4. The method as set forth in claim 1 , wherein said fin is a caudal fin.
5. The method as set forth in claim 1 , wherein said amputating occurs two or three days post fertilization.
6. The method as set forth in claim 1 , wherein said specified period of time is three days.
7. The method as set forth in claim 1 , wherein said telcosts are brafish.
8. The method as set forth in claim 1 , wherein said candidate agents are incubated at a concentration range between about 1 and about 25 kM.
9. The method as set forth in claim 1 , wherein said imaging is light microscopic imaging.
10. The method as set forth in claim 1 , wherein said amputating comprises using a sterile diamond blade mounted on a micromanipulator.
11. The method as set forth in claim 1 , further comprising determining the IC 50 of said candidate agent if said candidate agent is determined to affect regeneration.
12. The method as set forth in claim 1 , wherein said teleost are unstained prior to said imaging.
13. The method as set forth in claim 1 , wherein said teleost are not transgenic.