Download Figure 1 - York College of Pennsylvania

Zebrafish (Danio rerio) as a Proposed Model Organism to Study The Expression of
Ankyrin-3 and its Link to Bipolar Disorder
http://zdmsociety.org/zebrafish-and-human-disease/
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Introduction
Bipolar disorder is a debilitating brain disorder that causes
extreme mood swings and abnormal shifts in energy and activity
levels. It affects 2.6% of the adult American population (Luessis,
et al. 2013).
There is a strong association between bipolar disorder and
mutations in a gene called Ankyrin 3, also known as ANK 3 in
humans (Luessis, et al. 2013).
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Ankyrin 3 is a scaffold protein located in the brain and is
responsible for the formation and maintenance of the axon
hillock and at the nodes of Ranvier (Roberto, et al. 2011).
•
Ankyrin 3 links voltage-gated sodium and potassium channels to
the cytoskeleton and is required for the production and
propagation of action potentials mediated by these channels
(Luessis, et al. 2013).
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Alicia Dillman
York College of Pennsylvania, Department of Biological Sciences
Table 1. Primer sets for Ankyrin 3A and B
Ankyrin 3A
Primer 1
Forward:
ATGCAGCAACCAAGAAAGGAA
Reverse:
AAGAGTGTCGCTACGTTGATG
Size: 484 base pairs
Ankyrin 3B
Primer 1
Forward:
GACTGGTGTGGACATCAACAT
Reverse:
CGTTGTGGTCGTTCTGCAGGA
Size: 497 base pairs
Primer 2
Forward:
ATGTCAGCATTCAAGTAGGTA
Reverse: AACTCGATGTAAGCTATACGT
Size: 480 base pairs
Primer 2
Forward:
ATCAAGAGATTCTTAAGGATG
Reverse:
CACCAAGATCATTCATTAAGT
Size: 417 base pairs
Primer 3
Forward:
ATAGAAGAGCATAAGAGTCCT
Reverse: TGCATTGGCATTGGATGTCTC
Size: 480 base pairs
Primer 3
Forward:
CCTTGGAATCCTCCAGTACAT
Reverse:
CTTCCAGCAATGTCACAATAT
Size: 419 base pairs
Zebrafish are small, freshwater, tropical fish that are
advantageous animal models compared to the traditional lab rat
due to the low cost, fully sequence genome, transparency during
development and are amenable to genetic, behavioral,
Ankyrin 3A
physiological, pharmaceutical, and developmental studies
(Nguyen, et al. 2014).
Zebrafish contain two Ankyrin 3 genes, Ankyrin 3A and B. It is
believed that the zebrafish genome went through a duplication
during evolution and the functions of the genes were split
between the genes (Postlethwait, et al. 2000).
318
13,766
STOP
571
9,061
1,055
1
12,801
2
9,541
13,181
3
Ankyrin 3B
1
Objective
Clone and investigate the expression of
Ankyrin 3A and B genes in adult zebrafish
tissue.
STOP
425
EA2 EB3 GA2 GB3 HA2
HB3
Conclusions
Figure 4. RT-PCR analysis of Ankyrin 3A and B in eye,
gut, and heart tissue . The Ankyrin 3A and B genes were
amplified using adult zebrafish eye (E), gut (G), and heart
(H) cDNA. The 500 base pair ladder is represented with
BP and the Ankyrin 3A and B primers are represented as
A or B, respectively.
BP
A1
A2
A3
B1
B3
B2
Figure 5. RT-PCR analysis of Ankyrin 3A and B in muscle
tissue. The Ankyrin 3A and B genes were amplified using
adult zebrafish eye (E), gut (G), and heart (H) cDNA. The
500 base pair ladder is represented with BP and the
Ankyrin 3A and B primers are represented as A or B,
respectively.
3
2
ATG
285
13,355
BP
5,881
11,581
Table 2. Amino acid alignments between human Ankyrin
3 and zebrafish Ankyrin 3A and B sequences.
Amino Acid Sequences
921
1
6,297
2
12,000
3
Figure 1. ANK 3A and B genes and primer sets. Ankyrin 3A and B
genes from start to stop codons. Primer sets are labeled 1-3 with
forward and reverse primers indicated by the arrows.
Zebrafish ANK 3A Amino Acid
Sequence
Zebrafish ANK 3B Amino Acid
Sequence
Human ANK 3 Amino Acid
Sequence
59% identical
Leussis, M.P., Berry-Scott, E.M., Saito, M., Jhuang, H., Hann,
G., Alkan, O., Luce, C.J., Madison, J.M., Sklar, P., Serre, T.,
Root, D.E., and Petryshen, T.L. 2013. The ANK3 Bipolar
Disorder Gene Regulates Psyciatric-Related Behaviors That
Are Modulated by Lithium and Stress. Biological Psychiatry.
73: 683-690.
3.85 kb
Postlethwait, J.H., Woods, I.G., Ngo-Hazelett, P., Yan, Y., Kelly,
P.D., Chu, F., Huang, H., Hill-Force, A., and Talbot, W.S.
Zebrafish Comparative Genomics and the Origins of
Vertebrate Chromosomes. Genome Research. 10:18901902.
Primers were designed to target a 500 base pair sequence of each
gene (Table 1)
Nguyen, M., Stewart, A.M., Kalueff, A.V. 2014. Aquatic blues:
Modeling depression and antidepressant action in zebrafish.
Progress in Neuro-Psychopharmacology and Biological
Psychiatry. 55: 26-39.
Figure 2. pDrive vector. Plasmid used with space for the 500 base
pair insert.
Results
RNA Extraction
BP
A1
A2
A3
B1
B3
Ruberto, G., Vassos, E., Lewis, C.M., Tatarelli, R., Girardi, P.,
Collier, D., and Frangou, S. 2011. The Cognitive Impact of the
ANK3 Risk Variant for Bipolar Disorder: Initial Evidence of
Selectivity to Signal Detection during Sustained Attention.
PLoS ONE. 6:1-7.
B2
Woods, I.G., Wilson, C., Friedlander, B., Chang, P., Reyes,
D.K., Nix, R., Kelly, P.D., Postlethwait, J.H., and Talbot, W.S.
2005. The zebrafish gene map defines ancestral vertebrate
chromosomes. Genome Research.15:1307-1314.
PCR (Figure 1)
PCR product was ligated into a pDrive vector and transformed into
E. coli (Figure 2)
Plasmids from the bacteria containing the Ankryin 3A and B DNA
were isolated by mini prep and sequenced
• Isolate, clone, and investigate
the expression of Ankyrin 3A
and B in zebrafish embryo brain
tissue
• Complete an Ankyrin 3A and B
knockdown
References
Predicted mRNA sequence for ANK 3A and B were identified by
searching NCBI’s Gene Database
Reverse Transcription
Future Directions
58% identical
Methods
Adult Zebrafish Tissue Dissection
(muscle, gut, brain, heart, and eyes)
• Ankyrin 3A and B were
successfully extracted and
cloned from adult zebrafish
brain tissue
• Amino acid identities and
synteny support this Is the
homologous gene to human
Ankryin 3
• This suggests that zebrafish
may be a promising model
organism to study the
involvement of Ankyrin 3 in
the development of bipolar
disorder
3
2
1
ATG
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https://www.premedhq.com/nodes-of-ranvier-propagation-of-nerve-impulses-along-the-axon
Figure 3. RT-PCR analysis of Ankyrin 3A and B in brain tissue . The
Ankyrin 3A and B genes were amplified using adult zebrafish brain
cDNA. The 500 base pair ladder is represented with BP and the
Ankyrin 3A and B primers are represented with A and B,
respectively.
Figure 6. Synteny chart from Woods, and colleagues
showing synteny conservation between zebrafish and
human chromosomes (2005). Ankyrin 3A is present on
zebrafish chromosome 17 and shares 6 genes with
human chromosome 10. Ankyrin 3B is present on
zebrafish chromosome 12 and shares 27 genes with
human chromosome 10.
Acknowledgements
I would like to thank Dr. Wendy Boehmler for her
mentorship and patience throughout the duration of
this project.