CMB Final Presentation

APPLYING MOLECULAR TECHNOLOGY TO MAP

THE CONNECTOME

HOW CAN WE USE NEW VISUALIZATION

TECHNOLOGY TO BETTER OUR UNDERSTANDING OF

NEURAL CONNECTIONS AND THE CONNECTOME?

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

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

● 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

SPINAL CORD DEVELOPMENT

Growth cones respond to navigational

cues

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

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:

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

● 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

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

● 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”

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

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

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

GFP reporter geneminimalpromoterECRtransgen

e

Plasmid Configuration

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

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

2.0 kb1.5 kb

1 kb DNA Ladder

ECR

1% Agarose/TAE Gel

1.8 kb

Confirming the ECR sequence length

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

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

CCGCGCGGATTGCTTAGGCTACCAATTAATTPacIAscI

CGCGCCTAACGAATCCGATGGTTAAT

GGATTGCTTAGGCTACCAAT

Plasmid

ECR

2.0 kb1.5 kb

1 kb DNA Ladder

ECR Fragment

1% Agarose/TAE Gel

1.8 kb

Purifying the ECR after making “sticky ends”

INSERTING THE ECR INTO THE PLASMID

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

AMPLIFYING THE PLASMID

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

2.0 kb1.5 kb

1 kb DNA Ladder

Plasmid

1% Agarose/TAE Gel

ECR: 1.8 kb

1 2

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

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

INJECTION OF THE TRANSGENETwo students injecting chick

embryos

A student injection of the

transgene into a chick embryo

membrane video and injection video

GETTING DNA INTO CELLS

In order to properly transform

the transgene into the chick

embryos, a process called in

ovo electroporation was used

Students removing the spinal cord from the chick embryo to

analyze select neurons

VISUALIZING THE NEURAL TISSUE

A dissected neural tube that will soon be cut and prepared for

microscopic visualization

COMPARISON

vs.comparison picture

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

CONNECTOME

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

Thank you Ralph and

Linda for a once in a

lifetime experience!