Optimization of Gene Excision in Mouse Blood Brain Barrier Endothelial Cells
Additional Funding Sources
The Bridges to Baccalaureate program is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award No. R25GM123927.
Abstract
The blood-brain barrier (BBB) is formed by endothelial cells that line the interior of the blood microvessels. BBB dysfunction is implicated in neurological disorders such as Multiple Sclerosis, stroke, and Alzheimer’s disease. To better understand the mechanisms that regulate BBB function, cell culture models are needed. Unfortunately, common strategies for gene manipulation are not ideal as they can impair BBB function in isolated cells. Although the Cre-loxP system of DNA recombination is often used for gene manipulation in animals, its value in cell culture is limited as it can be challenging to achieve high recombination.
In this study, we administered varying concentrations of a Cre enzyme fused with a cell-penetrating peptide to endothelial cells isolated from the brain tissue of a red fluorescent Cre reporter mouse line.
Based on preliminary findings using live-cell fluorescent microscopy and PCR, we have identified likely suitable concentrations of Cre for efficient DNA recombination.
Future studies will include characterizing barrier function in Cre-treated cells to ensure the BBB function of the cells is not impaired and validating the effectiveness of the identified concentrations in endothelial cells isolated from the brain tissue of other mice carrying loxP-flanked DNA sequences.
Optimization of Gene Excision in Mouse Blood Brain Barrier Endothelial Cells
The blood-brain barrier (BBB) is formed by endothelial cells that line the interior of the blood microvessels. BBB dysfunction is implicated in neurological disorders such as Multiple Sclerosis, stroke, and Alzheimer’s disease. To better understand the mechanisms that regulate BBB function, cell culture models are needed. Unfortunately, common strategies for gene manipulation are not ideal as they can impair BBB function in isolated cells. Although the Cre-loxP system of DNA recombination is often used for gene manipulation in animals, its value in cell culture is limited as it can be challenging to achieve high recombination.
In this study, we administered varying concentrations of a Cre enzyme fused with a cell-penetrating peptide to endothelial cells isolated from the brain tissue of a red fluorescent Cre reporter mouse line.
Based on preliminary findings using live-cell fluorescent microscopy and PCR, we have identified likely suitable concentrations of Cre for efficient DNA recombination.
Future studies will include characterizing barrier function in Cre-treated cells to ensure the BBB function of the cells is not impaired and validating the effectiveness of the identified concentrations in endothelial cells isolated from the brain tissue of other mice carrying loxP-flanked DNA sequences.