1. tpa-1 in C. elegans
How do tubes of cells coordinate?
How is the oocyte pushed in the correct
direction through the spermatheca?

2. Biological Tubes
• Examples;
– Bronchial tubes
– Arteries
– Reproductive tubing
• Coordinated cell
contractions via
signaling through
protein cascades

3. The spermatheca

4. Project Goal:
Determine role
of tpa-1 in
ovulation and/or
oocyte transit

5. Steps
• Clone tpa-1 construct
– Design and order primers for tpa-1 gene
– Amplify gene via PCR
– Ligate into T-vector and transform into bacteria
• RNAi and see what happens
– DNA prep and restriction digest
– Ligate insert into L4440, transform bacteria
– Test with T7 primer
– DNA prep and digest again, transform RNAi
HT115(DE3)
– Grow worms and observe

6. What is tpa-1
• Encodes 2 protein kinase C isoforms
• Role in gpa-12/G protein-mediated signaling
that modulates feeding and growth
• Nicotine induced adaptation
• Widely expressed- nervous system, pharynx,
musculature, reproductive system

7. How did we clone it?
• cDNA RT-PCR –
failure
– First try, gel shows
that amplification did
not work 9/29
– Second attempt 10/3
also produces no
bands for new
primers
~400 bp
Primer dimer
Expected length 830bp

8. Cloning the gene
• Genomic DNA PCR
– success (10/7)
– Ran gel with various
combinations of F1,
F2 and R1, R2 tpa-1
primers and cDNA
or genomic DNA
Approx 1885bp,
expected size

9. Clone into T-vector
• 10/23 gel of restriction
digest of T-vector with
insert, later
retransformed in T-
vector, new restriction
digest gel 10/30
• Validation by PCR
• Restriction digest

10. Then L4440
• Ligated into L4440, PCR gel with T7 primer
11/13 shows that some colonies (3, 5, 6, 8)
have the insert
1 2 3 4 5 6 7 8 9 +ladder
1885bp
Expected size

11. RNAi
• RNAi is often used for gene silencing
• The tpa-1 gene was isolated and transformed into
bacteria
• The worms consume the bacteria containing
dsRNA of the gene of interest
• RNAi response is activated, knocking out the gene
of interest
• Observe results of any abnormalities due to gene
silencing
• Determine possible functions/applications of the
gene

12. Experiment Set-up
• Egg prep 11/22 at 8 am
• 54 hours old, viewed 11/24 at 2:30 pm
• Imaging with UN1108
• Viewed with DIC and GCamp

13. Observations and Images
• Head vacuoles observed as a common
phenotype in several of the tpa-1 silenced
worms

14. Observations and Images
• Abnormal oocyte stacking observed in worms

15. Observations

16. Abnormal Phenotypes
• Vacuoles
– Tpa-1 is expressed in head neurons and the pharynx,
interference may cause the formation of vacuoles in the
heads of the worms
• Sensitivity to TT
– RNAi tpa-1 worms experience more extreme and faster
paralyzation with TT than control worms
– Tpa-1 is involved with nicotine induced adaptations,
possible that TT has similar chemical structures/functions
as nicotine, both act on acetylcholine receptors
• Weird oocyte stacking
– Tpa-1 also expressed in reproductive system, may cause
entry defect leading to oocyte stacking

17. Next Steps
• Follow up or move on?