LEARNING IN THE MANCHESTER LAB

An Internship Presentation of Learning by Jeanette Gillie

WHO AM I?

11th grade student at High Tech High North County in San Marcos, CA Participated in the COSMOS program at UCSC
Nanotechnology Cluster Instructed by Shaowei Chen, PhD and Roger Terrill, PhD Individual project studying applications of nanotechnology in biosensors, particularly those used to detect various cancers

Intern in the Manchester Lab at Skaggs School of Pharmacy and Pharmaceutical Sciences, UCSD

THE MANCHESTER LAB

Members
Primary Investigator: Dr. Marianne Manchester Lab Manager: Diane Thomas Gr vaduate Students: Marisa Hovlid, Jason Del Rio Undergraduate Students: Vicki Luo

Research Focus:
The use of Viral Nanoparticles as molecular imaging and therapeutic agents

WHAT ARE VIRAL NANOPARTICLES?

Nano-sized virus particles (1-100 nm) Platforms that can be functionalized for various purposes
Molecular imaging agents Therapeutic agents

Why nano?
Higher specificity Less invasive
Image Credit: Marianne Manchester

MY PROJECT: HOW DOES CPMV INTERACT WITH GL261 CELLS?

What is CPMV?
Cowpea mosaic virus ~30 nm diameter Mammals can be dosed with up to 10^16 particles per kg of body weight without any toxic side effects Cell Binds to the protein vimentin which is highly expressed in aggressive tumors Vimenti n What are Gl261 cells? CPMV Line of mice glioblastoma cells (brain tumor cells)

Why should we care?

Glioblastoma multiforme = most common and aggressive malignant brain tumor Low prognosis, no effective treatment

CPMV PROPAGATION

CPMV PURIFICATION

200 g infected leaves -> 1.44 x 10^17 particles

EVALUATING SURFACE VIMENTIN EXPRESSION

Immunofluorescence
Flurophore Secondary Antibody Primary Antibody Target Protein Cell

Flow Cytometry

EVALUATING SURFACE VIMENTIN EXPRESSION

Trial 1

EVALUATING SURFACE VIMENTIN EXPRESSION

EVALUATING SURFACE VIMENTIN EXPRESSION

EVALUATING SURFACE VIMENTIN EXPRESSION

Trial 2
Diluted blocking reagent Different primary antibody, Increased concentration

EVALUATING SURFACE VIMENTIN EXPRESSION

EVALUATING SURFACE VIMENTIN EXPRESSION

EVALUATING INTERNAL VIMENTIN EXPRESSION

EVALUATING INTERNAL VIMENTIN EXPRESSION

Coverslip 6 Gl261 Cells, Fixed in Ethanol, Primary & Secondary Reagents

Coverslip 13 HeLa Cells, Fixed in Ethanol, Primary & Secondary Reagents

INFECTING CELLS WITH CPMV

EVALUATING CPMV UPTAKE

H1 White Light Not Infected With CPMV, Secondary Reagent Only

H1 Fluorescence

H2 White Light Not Infected With CPMV, Primary and Secondary Reagents

H2 Fluorescence

H3 White Light Not Infected With CPMV, Primary and Secondary Reagents

H3 Fluorescence

H4 White Light Infected With CPMV, Primary and Secondary Reagents

H4 Fluorescence

H5 White Light Infected With CPMV, Primary and Secondary Reagents

H5 Fluorescence

G6 White Light Not Infected With CPMV, Secondary Reagent Only

G6 Fluorescence

G7 White Light Not Infected With CPMV, Primary and Secondary Reagents

G7 Fluorescence

G8 White Light Not Infected With CPMV, Primary and Secondary Reagents

G8 Fluorescence

G9 White Light Infected With CPMV, Primary and Secondary Reagents

G9 Fluorescence

G10 White Light Infected With CPMV, Primary and Secondary Reagents

G10 Fluorescence

POSSIBLE ISSUES
Mounted poorly Epifluorescent microscope not powerful enough need to use confocal Improperly stored

SUMMARY
Gl261 has higher expression of surface vimentin compared to HeLa
(~30% of Gl261 cells expressed surface vimentin compared to 10% of HeLa)

CPMV uptake study inconclusive If Gl261 cells uptake CPMV, it may be a very appropriate and effective VNP for imaging and therapeutic purposes, but more investigation must still be done.

REFLECTION
Communication & Advocacy Skill sets accrued:
Chimera Technical skills pipetting, using the epifluorescent and confocal microscopes, centrifuges, and incubators, preparing dilutions, Familiarization w/ different volumes, special vocabulary, working in a sterile environment, flow cytometry, biohazardous materials, general lab safety)

My future

ACKNOWLEDGEMENTS
Marianne Manchester, PhD Diane Thomas Marisa Hovlid

Jason Del Rio

UCSD Flow Cytometer Research Core Facility Jeff Speir

Jennifer Santini
Alicia Johal Parag Chowdhury