1. APPLYING MOLECULAR
TECHNOLOGY TO MAP
THE CONNECTOME

2. HOW CAN WE USE NEW
VISUALIZATION
TECHNOLOGY TO BETTER
OUR UNDERSTANDING OF
NEURAL CONNECTIONS
AND THE CONNECTOME?

3. THE BRAIN INITIATIVE
National Institutes of Health (NIH)
Food and Drug Administration (FDA)
National Science Foundation (NSF)
Defense Advanced Research Projects
Agency (DARPA)
● Set to begin in 2016
● $4.5 Billion over 10 years

4. CIRCUITRY MAPPING IMPLICATIONS
Nearly 1 in 6 of the world’s population
suffer from neurological disorders
“As humans, we can identify galaxies light years away and
we can study particles smaller than an atom, but we still
haven’t unlocked the mystery of the 3 lbs of matter that sits
between our ears”
- President Barack Obama on the BRAIN Initiative

5. ● Neurons: cells in the nervous system
● Action Potential: an electrical impulse sent
through neurons to transmit information
● Synapse: the connection between neurons
● Axon: the projection that carries the electrical
impulse
NEURON STRUCTURE AND FUNCTION

6. SPINAL CORD DEVELOPMENT
Growth cones respond to navigational
cues

7. WHAT CAUSES CELL DIFFERENTIATION
All organisms contain the same
genes or “building blocks”
Cell differentiation happens as a
product of the regulation of genes
The regulation of genes is controlled
by non-coding regions
These non-coding regions switch on
genes that give cells their unique
identity

8. EVOLUTIONARILY CONSERVED REGULATORY ELEMENTS
Only 1-3% of the
DNA sequence
is coding
regions
A non-coding DNA
region that can
act as a “gene
switch” for a
coding region
of DNA
ECR:

9. LOOKING AT TISSUES
Tested ECRs using a blue
reporter gene
Screening to identify the purpose
of ECRs in the gene
These ECRs were recorded in a
database
Mouse embryo

10. ● A database that contains
information on noncoding
fragments of DNA
● This provides a database for
scientists to identify tissue
specific sequences
● Aided in our selection of ECR
● Our experiment is a secondary
screening on the selected ECR

11. BIOLUMINESCENCE VS. FLUORESCENCE
Bioluminescence - light produced
chemically by an organism
Different organisms use different
biochemical strategies to emit light
Fluorescence - organisms take in light and
reemit it at a lower wavelength
The emitted light is only visible when the
stimulating source is present

12. ● GFP is a small protein and
biological marker that is
visible in living tissues
● GFP takes in blue light and
emits green light
GFP:GREEN FLUORESCENT PROTEIN
GFP was
derived from
the “Crystal
Jellyfish”

13. WHY GFP?
● GFP does not need another
cofactor to fluoresce
● Relatively small
● If we can trick the neurons
into producing these
proteins all we would need is
a stimulating light
● Easy for in lab use

14. ECR SPECIFICS
The ECR selected is active in
this region
ECR was tied to reporter
gene, expressing blue
Using our transgene, we can
see the entire neuron in
more detail

15. EXPERIMENTAL OVERVIEW
Transgene that includes ECR
and the GFP
A plasmid was used to transfer
the gene into chicken
embryos
The transgene will illuminate
specific neurons in the
neural tube using the GFP

16. GFP reporter gene
minimal
promoterECRtransgen
e
Plasmid
Configuration

17. PCR is the method of
amplifying our ECR
sequence through
the variation of
temperatures to
achieve...
1. Denaturation
2. Annealing
3. Elongation
REPLICATING THE ECR

18. ECR VERIFICATION
Gel electrophoresis was
used to confirm the
length of the ECR
A DNA Ladder was used
to show known
fragments so we can
identify the length of
ours

19. 2.0 kb
1.5 kb
1 kb DNA
Ladder
ECR
1% Agarose/TAE Gel
1.8 kb
Confirming the ECR sequence length

20. SEPARATING AND PURIFYING
ECR
We cut the ECR band out of the gel
and began the ECR purification
process
We purified our ECR using a silica
matrix to bind the DNA to it and
remove the unwanted waste

21. MAKING DNA PUZZLE PIECES
We used restriction enzymes to create “sticky ends” at the
ends of the ECR
This allows for the insertion of the ECR into the plasmid
which has the complementary “sticky ends”
GGCGCGCCTAACGAATCCGATGGTTAATTAA
CCGCGCGGATTGCTTAGGCTACCAATTAATT
PacIAscI
CGCGCCTAACGAATCCGATGGTTAAT
GGATTGCTTAGGCTACCAAT

22. Plasmid
ECR

23. 2.0 kb
1.5 kb
1 kb DNA
Ladder
ECR
Fragment
1% Agarose/TAE Gel
1.8 kb
Purifying the ECR after making “sticky ends”

24. INSERTING THE ECR INTO THE PLASMID
Added a binding agent to the ECR and plasmid so we
could seal the “sticky ends” through incubation.

25. AMPLIFYING THE PLASMID

26. PLASMID GROWTH & PURIFICATION
Selected colonies were incubated to
replicate the plasmid
Bacteria cells were burst open using
special buffer
Purified and isolated the plasmid
DNA
Added restriction enzymes to some of
the purified DNA
E. coli colonies containing the
transgene

27. 2.0 kb
1.5 kb
1 kb DNA
Ladder
Plasmid
1% Agarose/TAE Gel
ECR: 1.8 kb
1 2

28. SCIENTIFIC MODELS
Our model system in this experiment was the nervous system or more
specifically the developing spinal cord
Our model organism was the chicken (Gallus gallus)
Fruit flyRoundwormChickenZebrafish

29. WHAT DID WE DO?
Insert transgene containing a
specific ECR into the chick
embryo’s developing spinal
cord using electroporation
Examine the embryonic spinal
cord and visualize the
neurons that expressed the
GFP reporter

30. INJECTION OF THE TRANSGENE
Two students injecting chick
embryos
A student injection of the
transgene into a chick embryo

31. membrane video and injection video

32. GETTING DNA INTO CELLS
In order to properly transform
the transgene into the chick
embryos, a process called in
ovo electroporation was used

33. Students removing the spinal cord from the chick embryo to
analyze select neurons

34. VISUALIZING THE NEURAL TISSUE
A dissected neural tube that will soon be cut and prepared for
microscopic visualization

35. COMPARISON
vs.
comparison
picture

36. ECRS IN GENE MANIPULATION
An ECR, or a gene switch, will be
active within a specific population
of neurons
We can target specific neurons and
“knock out” receptors in them
Through mutated receptors we can
see the responses to specific
cues

37. CONNECTOME

38. IMPORTANCE OF DATABASES
In order for scientists to
learn from each
other, a collection of
work needs to be
established
Bioinformatics is the field that creates applications
and softwares for the safekeeping of biological
data

39. Thank you Ralph and
Linda for a once in a
lifetime experience!