1. Using reporter genes to monitor onset of circadian rhythm in embryonic hearts of Xenopus laevis
Lindsey Kuhagen and Kris Curran University of Wisconsin – Whitewater, Department of Biological Sciences
Introduction/Background
Many organisms exhibit circadian rhythms including cyanobacteria, plants, insects, and vertebrates.
Circadian rhythm can effect behavior, changes in physiology, and gene expression based on the time of day (Dunlap, 1999).
The clock works as a transcriptional negative feedback loop.
Transcription of clock controlled genes (CCG) leads to rhythmic expression of output genes (reviewed in Takahashi, 2015).
We use the promoter of a CCG (Nocturnin-LUC2) as a readout for Nocturnin gene expression.
Rev-erba is part of the central oscillator and it becomes rhythmic early in organ
development
(Sladek et al., 2007).
Results I cont.: xNoc-LUC2
Methods II cont.: Primer design
Figure 3: This figure shows before and after pictures of heart transplanted embryos and the 3-day luciferase assay of that embryo. A. and C. are pictures of the embryo that it looks like with the naked eye. B. and D. are a fluorescent pictures of that same embryo with an ultra-violet light on it to show where the transplant is in the embryo. E. is showing the luciferase assay. It did not display circadian expression of Nocturnin, which is not unexpected.
Stage 39/40 transplant
Stage 44 transplant A. B. C. D. E.
Figure 5: This figure shows the primers used to isolate and amplify the Rev-erbα promoter gene from the Xenopus leavis genome. The parts highlighted in yellow are extra bases added to the primers to account for any degradation. The parts highlighted in green are restriction enzyme sites. The left primers (L4 and L5) have BglII sites and the right primers (R5 and R6) have HindIII sites. The rest of the sequence was taken from the Rev-erbα promoter gene sequence that has already been sequenced.
Nocturnin
Partch et al
Results II: Putative Rev-erba amplicon
LAD
Per1 L2+R3
Per1 L2+R4
Per1 L1+R3
Rev L4+R5
Rev L5+R5
Rev L4+R6
Rev L5+R6
+ control
Per1 L1+R4
Figure 6: This shows a gel ran using the electrophoresis method. The first row on the left is the DNA 1KB ladder. The four lanes to the right of that are all results for a different gene’s PCR product. The four to the right of that (Rev) are the results from the PCR done with the 4 different primers that were designed. Four different combinations of the primers were used to see which one would amplify the Rev-erbα gene. The farthest right lane is a positive control to ensure that the PCR is working. A 3Kb PCR product was obtained from L5+R5 primer combination.
Methods I: xNoc-LUC2
Using the Noc-Luc2 reporter we can monitor the light expression of embryonic hearts to tell us when the developing heart attains a mature circadian rhythm.
1. Inject embryo with Noc-Luc2 and age to early to mid-gastrula stages (use embryos that show high light expression (>500 light counts))
2. Remove the dorsal marginal zone (DMZ)
3. Treat with suramin for 4 hours, culture overnight at 16℃
4. Transplant into stage matched uninjected host
5. Monitor light expression using Glomax Multimode reader, do 3 day luciferase assays once a week to see if heart is showing a rhythm
Methods II: Rev-erba-Luc2 Reporter
Like the Noc-LUC2 construct used in the heart experiment, this construct can be used to monitor the expression of Rev-erbα. Rev-erba is the first oscillator component to display a significant rhythm.
1) Design primers using sequenced Xenopus Genome (Xenbase) to isolate Rev-erbα promoter (3kb upstream of exon 1).
2) PCR to amplify promoter from isolated Xenopus genomic DNA.
3) Cleanup amplified product and clone into pGEMT bacterial vector.
4) Amplified product will contain engineered restriction enzyme sites. Use sites to clone into PGL4.1 vector containing the Luciferase coding sequence.
5) Remove Rev-erbα-LUC2 from pGL4.1 and clone into transgenesis vector containing SCE-1 meganuclease sites.
6) Digest Rev-erbα-LUC2 with SCE-1 and inject into one cell Xenopus laevis embryos.
Conclusion
We have found no significant change in Nocturnin expression in induced hearts transplanted into non-transgenic hosts.
This may be because of the embryos are losing the transgene.
Also could be because of the age of the embryos.
We have a putative 3Kb Rev-erbα promoter amplicon that we will ligate into pGEM-T vector.
Future experiments:
Once cloned into pGEM-T vector, we will sequence to determine if Rev-erbα promoter was amplified.
Work on increasing the number of heart transplants we can perform. This will increase that chances of doing one where the transgene has incorporated into the genome.
Results I: xNoc-LUC2 Reporter
Figure 2: This is a luciferase assay of early stage DMZ treated with suramin. These results are not from transplanted DMZ. This was done to see if we could see a rhythm before we tried more transplants. These were all taken from high light expressing embryos (>500 light counts).
Acknowledgements
Summer Undergraduate Research Fellowship