Fluorescent Labeling to Generate Green Fluorescent Protein (GFP) Positive Neural Stem Cells

School Name

Governor's School for Science & Mathematics

Grade Level

12th Grade

Presentation Topic

Physiology and Health

Presentation Type

Mentored

Mentor

Mentor: Ana Martin-Villalba, German Cancer Research Center

Written Paper Award

3rd Place

Abstract

Neural stem cells (NSC’s) often follow the same path of migration throughout the brain, traveling from the ventricles to the olfactory bulbs via the rostral migratory stream. However, they sometimes shift their migration to the striatum for an unknown reason during ischemia. The goal of this research is to develop a new method of labeling NSC’s with green fluorescent protein (GFP) in order to create a large source of accessible cells that could be used to visually record the patterns in the shift of migration. The episomal DNA injected into the mice created with EF1α, CMV, and UBC promotors, GFP, and a kanamycin resistance gene. This episomal DNA was then injected into mice at varying concentration of DNA and PEI (branched-polyethyleneimine). The mice rested for three days, and the 15 µm left-ventricular samples were stained using immunofluorescence. The NSC’s with the highest expression of GFP were found with the CMV promoter, but the quantity was not enough to create a stable cell line. The same experiment was run for a 7-day incubation period; however, resulted in no successfully transfected cells. It was hypothesized that this may be due to the PEI toxicity after extended periods. An in-vitro transfection with the EF1α, CMV, and UBC promoters and nucleofection was performed over three weeks. A FACS analysis showed that the EF1α promoter NSCs generated a large viable pool of cells and could potentially become a new method to create GFP-labeled cells with further trials and testing.

Location

Wall 318

Start Date

3-25-2017 12:00 PM

Presentation Format

Oral and Written

Group Project

No

COinS
 
Mar 25th, 12:00 PM

Fluorescent Labeling to Generate Green Fluorescent Protein (GFP) Positive Neural Stem Cells

Wall 318

Neural stem cells (NSC’s) often follow the same path of migration throughout the brain, traveling from the ventricles to the olfactory bulbs via the rostral migratory stream. However, they sometimes shift their migration to the striatum for an unknown reason during ischemia. The goal of this research is to develop a new method of labeling NSC’s with green fluorescent protein (GFP) in order to create a large source of accessible cells that could be used to visually record the patterns in the shift of migration. The episomal DNA injected into the mice created with EF1α, CMV, and UBC promotors, GFP, and a kanamycin resistance gene. This episomal DNA was then injected into mice at varying concentration of DNA and PEI (branched-polyethyleneimine). The mice rested for three days, and the 15 µm left-ventricular samples were stained using immunofluorescence. The NSC’s with the highest expression of GFP were found with the CMV promoter, but the quantity was not enough to create a stable cell line. The same experiment was run for a 7-day incubation period; however, resulted in no successfully transfected cells. It was hypothesized that this may be due to the PEI toxicity after extended periods. An in-vitro transfection with the EF1α, CMV, and UBC promoters and nucleofection was performed over three weeks. A FACS analysis showed that the EF1α promoter NSCs generated a large viable pool of cells and could potentially become a new method to create GFP-labeled cells with further trials and testing.