Academia Sinica IBC summer internship oral presentation in 2019/08/30

1. Gene cloning for finding the
neurodegeneration-associated protein
interactors via APEX2
Speaker: Tzu-Hao Liu
Advisor: Dr. Chi-Kuang Yao
2019/08/30
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2. Some dysfunction of proteins cause
neurodegenerative disease
Parkinson’s disease(PD) ALS/FTD
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C9orf72
TDP-43
SOD1
FUS
associated protein
LRRK2
α-synuclein
synphilin-1
Parkin
UCH-L1
associated protein

3. LRRK2 is a large, widely expressed, multi-
domain and multifunctional protein
• LRRK2 is associated with Parkinson's disease.
• The most common mutation is LRRK2 G2019S.
• G2019S mutation cause LRRK2 kinase hyperactivity on
level of LRRK2 instead kinase domain per se.
• LRRK2 gene length is up to 10kb, it’s make me difficult to
transform.
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LRRK2 protein
J Neurosci Skibinski G et al.(2014), 34:418–433.
Li et al. Molecular Neurodegeneration 2014, 9:47

4. Codons repeats in C9orf72 cause neuron
toxic
 Expression of pure codons repeats in Drosophila caused neurodegeneration
 C9orf72 mutant in human was reported associate with ALS/FTD.
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https://ghr.nlm.nih.gov/gene/C9orf72#location
Gene Express in flies
Poly-GA100 Low toxic (Control)
Poly-GR100 Pathogenic
Poly-PR100 Pathogenic
Science. Sarah Mizielinska et al. (2014 September 5)
DPR

5. Drosophila eyes pattern and life time
difference between Poly-(PA/GA/GR/PR)
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Science. Sarah Mizielinska et al. (2014 September 5) Figure 3-A,B,D
IBC summer internship 2019

6. Find the possible interacting
protein to understand disease
developed mechanism
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Aim

7. We use APEX2 method to screen protein-
protein interaction
Braden T. Lobingier et al.(2017)
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We can know “when” and “where protein-
protein interaction happened in live cell by
APEX2 method.

8. Construct plasmid by gene cloning
technology
 Gene cloning is a method make us can recombine gene we
interesting to another gene fragments.
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Gibson reaction Ligation

9. Isolate genomic DNA from fruit fly
Use squishing buffer
Squish fruit fly
Heat in 37C
30min
98C for 3min
Sup. High
speed
Centrifugation
Gnomic DNA
involve in sup.
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squishing buffer
• 10mM Tris
• 1mM EDTA
• 25mM NaCl
• 200ug/ml proteinase K
Destroy proteinase K

10. Main flow of gene cloning
Draw gene
map
Primer
design
PCR
fragments
Digestion
Ligation
Transform
into E.coli
Colony PCR
test
Plasmid
purification
Restrictive
Enzyme test
DNA
sequencing
Finish
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LRRK2-wt
Poly-GR/PRPoly-GA

11. Problems and resolutions
 Because of GGGGCC repeats, primer will bind to multiple sites, I can’t
have sequence data of Poly-GA.
 Poly-PR and Poly-GR also have DPR codons repeats, thought I have DNA
sequence data, I can’t success PCR fragments because primer mis-
binding.
 pUAST-LRRK2-APEX-HA is a too large plasmid, I can’t success
transform to DH5-alpha
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12. Redesign a primer for specific binding
PCR condition
 98 °C for 3 min
 98 °C for 10 sec
 55 °C for 30 sec
 72 °C for 60 sec
 72 °C for 10 min
band at wrong site.
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100-200bp
1kb
35 cycles
pUASTattB-R
SV40term-R
IBC summer internship 2019So we redesign a new primer – pUASTattB-R

13. Problems and resolutions
 Because of GGGGCC repeats, primer will bind to multiple sites, I can’t
have sequence data of Poly-GA.
 Poly-PR and Poly-GR also have DPR codons repeats, thought I have DNA
sequence data, I can’t success PCR fragments because primer mis-
binding.
 pUAST-LRRK2-APEX-HA is a too large plasmid, I can’t success
transform to DH5-alpha
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14. 500 bp band gene in gel is a part of
pUASTattB
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https://blast.ncbi.nlm.nih.gov/Blast.cgi
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15. Change primer design strategy
Poly-(GR)excerpt Before Poly-(GR)excerpt After
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NotI
Change primers
IBC summer internship 2019

16. Problems and resolutions
 Because of GGGGCC repeats, primer will bind to multiple sites, I can’t
have sequence data of Poly-GA.
 Poly-PR and Poly-GR also have DPR codons repeats, thought I have DNA
sequence data, I can’t success PCR fragments because primer mis-
binding.
 pUAST-LRRK2-APEX-HA is a too large plasmid, the construct plasmid
have many mutant base pairs.
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17. We found 7 point mutations in LRRK2 DNA
sequencing data
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LRRK2 Restrictive enzyme test 7/19
LRRK2 DNA sequencing 7/21
1kb
10kb 6kb
Found 7 point mutations in plasmid
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18. I learned a lot of things in this summer.
 How to make agarose gel and run DNA electrophoresis.
 Knowledge and technology needed for gene cloning.
 How does DNA sequencing working.
 How to design primer for PCR and DNA sequencing.
 How to use restrictive enzyme and ligase.
 How to culture bacteria and simple sterile working.
 What is Gibson reaction and how to do it.
 How to transfer fruit fly and use CO2 to anesthetize fruit flies.
 How to pick larvae with phenotype we want.
 How to use fluorescence microscope.
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19. Thank you for your listening :)
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