8/4/2019 Nick Cho Poster

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Results (cont.)

Nicholas Cho, under the supervision of Prof. Lawrence Schook, in collaboration with Kyle Textor, Molly MelhemUniversity Laboratory High School and the Department of Animal Sciences, Division of Biomedical Science, University of Illinois at Urbana-Champaign

Ligation of dsRED vector with mouse VCAM-1

Acknowledgments

Conclusions and Future Work

Had the ligation been successful, the nextstep would be to insert the dsRED vector into

blank cells so that they glow red and indicatethat VCAM-1 is present. These VCAM-1expressing cells would then be tested to see

if they bind with neutrophils. Once theneutrophils are seen to stick to VCAM-1, a

layer of peptides will be placed over thesetwo proteins. Then, neutrophils (extractedfrom mouse blood) will run through and

observations will be made to see the efficacyof the peptides to inhibit neutrophil invasion.

AimThe goal of the project is to use peptides to

help inhibit neutrophil invasion into the hearttissue. To do so, these peptides bind toICAM-1 and VCAM-1. To help visualize this

interaction, the DNA of ICAM-1 and VCAM-1needs to be inserted into a vector called

dsRED so that these proteins show up as redwhen observed under a microscope and can

be used when testing the peptide binding.

Introduction

Heart attacks are caused by plaque thatobstructs blood flow to the heart. This results

in an environment in which the heart receivesno oxygen and nutrients. When blood flowreturns to tissue in such an environment, this

causes inflammation and ultimately scarringof the heart tissue. Because of this scarredtissue, the remaining living tissue must work

harder to perform its necessary tasks. Whatcauses this tissue scarring in the first place

are neutrophils, cells that immediatelyrespond whenever any inflammation is foundin the body. These neutrophils travel through

the body via blood vessels. When they reachthe heart, the neutrophils interact with theICAM-1 and VCAM-1 that line the tissue

(Figure 1). This neutrophil invasion ultimatelyreplaces working tissue with scar tissue and

results in added stress on the heart.

Method

1. Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) used toreplicate specific DNA of mouse VCAM-1multiple times

PCR is a cyclical process and undergoesthree steps (Figure 2):

Denaturation: The mouse VCAM DNA is

heated up to 95 degrees Celsius in order tosplit it into two separate strands.

Annealing: Primers, strands of nucleic acid,attach themselves to the strands of DNAand serve as the starting point for the DNA

synthesis.

Elongation: Enzyme known as KOD readsthe DNA strand and replicates thecorresponding strand.

Once expected product (specific piece ofVCAM) was produced, DNA was purified

via DNA purification kit, which containedseries of buffers to clean out the liquids and

salts surrounding DNA as well as DNAitself.

2. Enzyme cut

To insert the VCAM sequence into the vector,

enzymes Xho-I and Xma-I were used to cutboth the VCAM and dsRED vector (Figure 3).Through ligation, VCAM would line up and

insert itself into dsRED vector at the multiplecloning site (MCS).

3. Transformation

Once this process is completed, the product

was then placed in a E. coli cell culture inorder for it to grow. The newly formed vector

enters into E. coli so that it can grow.Antibiotics will kill off all other bacteriabesides those containing newly formed

vector. Observations will be made to see ifligation was successful and the vectorcontains sequences for both dsRed and

VCAM .

Results

Primers with enzymes Xho-I and Xma-I weremade for the PCR in order to form the

necessary enzyme sites in the dsRED andVCAM.

PCR successfully replicated wanted DNAsequence. Figure 4 shows a picture of theresult of the PCR.

The dsRED vector and VCAM were

successfully cut by the enzymes Xho-I andXma-I. Based on observations made from E.coli cell culture, dsRED vector did not grow

and ligation was unsuccessful.

Figure 4: Picture of DNA ladder of VCAM in EGFPvector. The bands of light seen in the picture indicatethat the DNA successfully binded with the primers at

2000 base pairs long and successfully replicated thecorrect sequence of DNA.

Figure 3: Diagramof dsRED vector. At

the location namedMCS is where the

strand of VCAM wasinserted.

Figure 2: Diagram of PCR

Figure 1: Diagram of neutrophils enteringthe heart from the blood vessels