Presentation Title

Utilizing Fluorescent Ubiquitin Cell Cycle Indicators to Identify and Target Quiescent Cells for Novel Cancer and Cardiac Therapies

Faculty Mentor

Dr. Alexander Zambon

Start Date

18-11-2017 2:15 PM

End Date

18-11-2017 3:15 PM

Location

BSC-Ursa Minor 73

Session

Poster 3

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Cardiovascular disease and cancer are known to be two of the leading causes of death worldwide as stated by the World Health Organization. Currently, cancer therapies are not as effective as they can be due to the resistance of dormant cancer cells. Fetal cardiomyocytes lose the ability to re-enter the cell cycle as they become adult cardiomyocytes, therefore cardiovascular disease causes damage to the tissue that surrounding cells cannot repair. For this reason, identifying and understanding the quiescent phase of cells is necessary to be able to control how cells are affected by therapies or diseases. The transition between a proliferating and quiescent cell, or G1 and G0 phases, is difficult to measure without markers, so in this project, we integrated fluorescent indicators, or FUCCI systems, into the genomes of three different cell lines: an HT1080 line and 2 MCF7 lines. Ki67 mAG/mCherry 2K7BSD and Ki67 mAG Ki67mCherry 2K7BSD indicators were used to identify cells as red in the G1 phase and green in the S, G2, and M phases. Blue fluorescent protein was used to mark every single living cell blue. This method made it easy to identify cells that were only blue under a lumascope as potentially quiescent. To create the cell lines with indicators, a virus was created, which then inserted genes into another mammalian cell line. Using the lumascope after integrating the indicators, we were able to determine whether the virus was successfully incorporated into the genome of the mammalian cell line.

Summary of research results to be presented

In creating the cell lines with indicators, a gateway recombination reaction is necessary to create a plasmid with three vectors. A transformation of the plasmid into chemically competent E.coli was performed by plating on agar plates with ampicillin, but the first time transforming, there was no growth on the agar plate. The second time there was growth.

After picking the colonies and culturing them, a miniprep (or midiprep if more quantity is needed) was performed to lyse and elute the DNA from the E.coli. A nanoquant machine told us how much DNA was extracted from the sample. We used three samples of cells; the first two had very low amounts of DNA so a midi prep had to be run on those two samples to extract more DNA. From the midi prep, We acquired sufficient amounts of DNA.

Next, we confirmed that we had the correct DNA sequence using a restriction digest and gel electrophoresis. For each sample, the bands matched up to the 2-log ladder, therefore it was the correct sequence.

The last step of the process is to confirm that the indicators were incorporated into the genome of the cells by using a lumascope to see the fluorescence. The cells did, in fact, fluoresce red and green as well as blue, indicating that the BFP virus was successfully incorporated into the genome of the cell line.

This document is currently not available here.

Share

COinS
 
Nov 18th, 2:15 PM Nov 18th, 3:15 PM

Utilizing Fluorescent Ubiquitin Cell Cycle Indicators to Identify and Target Quiescent Cells for Novel Cancer and Cardiac Therapies

BSC-Ursa Minor 73

Cardiovascular disease and cancer are known to be two of the leading causes of death worldwide as stated by the World Health Organization. Currently, cancer therapies are not as effective as they can be due to the resistance of dormant cancer cells. Fetal cardiomyocytes lose the ability to re-enter the cell cycle as they become adult cardiomyocytes, therefore cardiovascular disease causes damage to the tissue that surrounding cells cannot repair. For this reason, identifying and understanding the quiescent phase of cells is necessary to be able to control how cells are affected by therapies or diseases. The transition between a proliferating and quiescent cell, or G1 and G0 phases, is difficult to measure without markers, so in this project, we integrated fluorescent indicators, or FUCCI systems, into the genomes of three different cell lines: an HT1080 line and 2 MCF7 lines. Ki67 mAG/mCherry 2K7BSD and Ki67 mAG Ki67mCherry 2K7BSD indicators were used to identify cells as red in the G1 phase and green in the S, G2, and M phases. Blue fluorescent protein was used to mark every single living cell blue. This method made it easy to identify cells that were only blue under a lumascope as potentially quiescent. To create the cell lines with indicators, a virus was created, which then inserted genes into another mammalian cell line. Using the lumascope after integrating the indicators, we were able to determine whether the virus was successfully incorporated into the genome of the mammalian cell line.