Using RNA interference mosaics to map
Download
1 / 21

Background - PowerPoint PPT Presentation


  • 292 Views
  • Updated On :

Using RNA interference mosaics to map retained mutant phenotypes HHMI Internship Megan Kelly Mentor: Dr. Barbara Taylor Dept. of Zoology www.berkeley.edu/.../07/images/fruitflies.jpg Background How do genes work in creating body parts and controlling function? I focused on

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Background' - jaden


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Slide1 l.jpg

Using RNA interference mosaics to map retained mutant phenotypes

HHMI Internship

Megan Kelly

Mentor:

Dr. Barbara Taylor

Dept. of Zoology

www.berkeley.edu/.../07/images/fruitflies.jpg


Background l.jpg
Background

How do genes work in creating body parts and controlling function?

I focused on

answering this question

for the female fruitfly

reproductive tract

Ovaries

Oviducts

Uterus

http://flybase.net/static_pages/imagebrowser/imagebrowser10.html


One method for analysis l.jpg
One method for analysis

Using mutation in general to disrupt gene expression causing mutant phenotype

Changing gene expression in only specific cells to locate mutant phenotypes

I altered (retained) gene expression in cells of the female and found that in resulting phenotypes no eggs were laid - a result of retention.


Background4 l.jpg
Background

retained(retn) gene in fruit flies plays role in female reproductive tract development

Strong mutation, prevents the gene from transcription, therefore no expression. Results in embryonic lethal

Weak mutation, (missense). Adults viable, results in female sterility, and abnormalities in courtship behavior.


Hypothesis l.jpg
Hypothesis

Female sterility is due to

a retained mutant

phenotype in the

reproductive tract

If true, females should be

able to mate but not

release an egg from the ovaries

Wildtype reproductive tract

http://flybase.net/reports/FBim0000078.html


Slide6 l.jpg

Goal

Map the location of cells responsible for female sterility by comparing retn flies to retnRNA-interference mosaics

Mosaics:

Groups of cells in the body vary in gene expression

Ex: Calico cat has variation in pigment due to genotypic variation in skin cells

http://tigerpixie.com/tigerpixieart/HalloweenaSM.jpg


Slide7 l.jpg

Wildtype(CSA)

retained(retn)

Mosaic

= retn expression location

X


Slide8 l.jpg

UAS retn-RNAi

hthgal4

UAS

X

gal4 gene

homothorax gene

retn-RNAi gene

Gal4 protein

gal4 gene

homothorax gene

retn-RNAi gene

Creating mosaics

retn-RNAi I

retn-RNAi III

retn-RNAi ARID


Slide9 l.jpg

Normal homothorax expression

  • homothorax

  • hthgal4

  • UASretn-RNAi

Normal retn expression

  • retn expressing

  • retn expressing

  • homothorax

Loss of retn function

  • UASretn-RNAi

  • hthgal4


Comparing retn mutants to our rnai mosaics in three ways l.jpg
Comparing retn mutants to our RNAi mosaics in three ways:

  • Behavior before and during copulation

  • Fertility

http://www.pnas.org/misc/archive072803.shtml

http://www.csus.edu/indiv/h/hollandb/Pictures/fruit%20flies/fly%20laying%20egg.jpg

  • Physical makeup of the reproductive tract through dissections

http://flybase.net/reports/FBim0000078.html


Behavioral test l.jpg

Latency to courtship

Latency to copulation

Copulation duration

Behavioral test

Wildtype latency to courtship average: 1-2 minutes

Wildtype latency to copulation average:2-5 minutes

Wildtype copulation duration average 18-30 minutes


Slide12 l.jpg

Behavioral Results:

Wildtype

retn mutant

retn control

Mosaic controls

Mosaics

Analysis of variance concluded no behavioral difference due to genotype


Behavioral results l.jpg
Behavioral results

Wildtype

retn mutant

retn control

Mosaic controls

Mosaics

Analysis of variance concluded no behavioral difference due to genotype


Slide14 l.jpg

Behavioral test results

Wildtype

retn mutant

retn control

Mosaic controls

Mosaics

Analysis of variance concluded no behavioral difference due to genotype


Fertility results l.jpg

18

24

18

20

20

20

19

23

22

retn/cyo

retn/retn

23

hthgal4/RNAi I

hthgal4/RNAi III

Wildtype

RNAi ARID/+

RNAi I/+

hthgal4/+

hthgal4/RNAi ARID

RNAi III/+

retn mutant

retn control

Mosaic controls

Mosaics

Mosaics

Fertility results


Slide16 l.jpg

Reproductive tract

Wildtype

retn mutant


Slide17 l.jpg

retn mutant

Mosaic


Conclusion l.jpg
Conclusion

  • Results gear us toward understanding that sterility in mosaic is due to another phenotypic mutation resulting from retn degredation

  • retn mutants may be sterile due to another product of retn degredation and not fully because of the loss of the common oviduct


Future work l.jpg
Future Work

  • Continue comparative work in retn reproductive tract

  • Use a different driver than Gal4 to increase strength of sterility

  • Localize retn degradation to the nervous system and not the reproductive tract


One step closer to understanding the process of cell expression l.jpg
One step closer to understanding the process of cell expression

www.anatomy.unimelb.edu.au/.../life_cycle.jpg


Acknowledgments l.jpg
Acknowledgments expression

Howard Hughes Medical Institute

Dr. Barbara Taylor (Department of Zoology)

Dr. Kevin Ahern (Department of Biochemistry/Biophysics)

Taylor Lab


ad