CLONE CHALLENGE RECIPIENT:
Gregory Poon, Ph.D., B.Sc.Phm., Professor of Chemistry, Georgia State University
The Challenge
Our work routinely involves knocking in two or more unrelated fluorescent proteins into a single cell line by lentiviral methods. A significant challenge has been the isolation of strongly expressing all-positive clones for use as reporters. A more facile approach to knocking in and isolating such clones would greatly accelerate our work over engineering alternative construct designs.
A Success Story
We supplied THP-1 cells (blue) with a lentiviral construct encoding two fluorescent proteins (red), and were then provided with a panel of twenty clones. The brightest clones (such as that shown in orange) were very rare outliers in the supplied population, which we verified by genomic PCR. The negligible fluorescence overlap of these clones with untransduced controls simplify our experiments and workflow.
Cell Microsystems was able to isolate clones of THP-1 monocytes that are highly fluorescent for our assays:
Figure: Flow cytometric analysis of THP-1 cells that have been transduced with a lentiviral construct expressing two fluorescent proteins (red) and selected (orange). Untransduced control is in blue. Contours are shown at 10% intervals with outliers as dots. All presented fluorescence has been compensated using single-color controls.