Transcript Document

Characterization of Circadian Gene Expression in Xenopus laevis
Jessica Solis, Aaron Trow
Mentor: Dr. Kristen Curran
What is Circadian Rhythm:
In situ Hybridization:
The daily change in gene
expression, behavior,and physiology
that is subsequently maintained in
constant conditions. Light input has
the ability to reset this rhythm
(entrain).
Quantitate the level of gene expression
using REAL TIME RT-PCR:
Materials and Methods:
In situ hybridization uses a probe
specific to NAT tagged with
digoxigenin. An anti-digoxigeninalkaline phosphatase antibody binds to
the probe. An alkaline phosphatase
enzymatic color reaction shows where
in the embryo the gene is expressed.
Background:
When is circadian gene expression first
observed in developing Xenopus laevis
eyes?
Extract RNA from each sample
Copy total RNA into cDNA (reverse
transcription)
Amplify the gene of interest (Xbmal) using
Real time PCR
NAT expression in stage 46 eyes
Real Time PCR was used to measure
expression levels of circadian oscillator and
output genes in different stages of
development and different circadian times.
In situ hybridization was used to analyze
where each gene is expressed in these
embryos.
3.5
Relative quantitation
3
2.5
2
1.5
1
0.5
0
0
6
12
18
ZT
One central oscillator gene, Xbmal1 and one
output gene, NAT were analyzed.
NAT
Circadian Genes of Interest:
Xbmal and Clock
ZT 0
ZT 6
ZT 12
ZT 18
Per1 and Per2
Future Directions
Embryonic stage
Stage 26
Nocturnin and AANAT
Xbmal:
-oscillator gene
-transcription factor
-expected ZT18
Negative Control
No rhythmic expression
Conclusions:
Stage 32
Looking for expression
In Situ: NAT is present in the eye, pineal, and SCN. No
difference of level expression is seen at stage 46.
RT-PCR: Xbmal is rhythmically expressed in stage 46
eyes.
NAT:
Stage 46
Positive Control
We have rhythmic expression
Acknowledgements:
-output gene
-enzyme rate limiting step in Melatonin
synthesis
- expected at ZT 12
Outcome
Gastrula
Cleavage
Blastula
St 1
St 6
St 12
Neural Tube
St 20
Tadpole
St 40
McNair scholars Program
WISCAMP
Chancellor Telfer
Dean Stone
Dean Barnett
Dean Pinkerton
NASCO
NSF
UWW Undergraduate Research
UWW Faculty Development Grant