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• What did you learn from surfing FlyBase?
• Why do the inversions in Balancer chromosomes greatly reduce the frequency of crossing over in meiosis?
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• Autonomous vs non autonomous
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Nonautonomous
• Domineering - mutant cells disrupt the development of neighboring wild type cells.
• Submissive - wild type neighbors rescue mutant cells.
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• FLP/FRT can also be used to remodel chromosomes.
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RNAi in flies
ds RNA in cultured cells.
http://flyrnai.org/RNAi_goals.html
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RNAi in flies
1. Express a fold back RNA from a transgene.
2. This results in tissue/temporal specific gene knockdowns.
3. Collections of transgenic flies are available that contain transgenes to knockdown the expression of most fly genes.
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Gene targeting
• Until recently not available in flies.
• One difficulty is that one cannot select for a targeted cell.
• A technique has now been developed that has been shown to work on a number of genes.
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• Technique requires first getting a germ line transformant for the knock out construct.
• The DNA for targeting is excised by the activity of the FLP/FRT site specific recombination system.
• The excised circle is then cut by the induced expression of a very rare cutting RE.
• High rate of homologous targeting.
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In Vivo Imaging
• A number of developmental stages/tissues provide opportunities for in vivo imaging.
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Genetic Screens
• Recessive lethal mutations
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The iso la tion of recessive v isib le m uta tions
m utagenize X th st fz in ri/TM 3
screen for flies that are phenotypica lly or fz in .
these flies would be or fz*th st/fz in ri th st in*/fz in ri
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X
The iso la tion of recessive le tha l m utations
cn bw treat w ith m utagen (e.g. em s)
cn bw X G la/CyO
ind ividual cn bw /C yO X G la/CyO
* *
***
*
*
cross s ib lings X cn bw /C yO cn bw /CyO
cn bw / cn bwcn bw /C yO2
CyO /CyO
Look for v ia ls w ith no stra ight w inged flies
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Flp/FRT
• High efficiency of FLP/FRT clone induction has allowed new types of mutant screens.
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