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[American3pt14]

I have an MCF7 breast cancer cell line stably infected with an AKT1-mCherry construct (via retroviral pMSCV vector) as well as an mCerulean-NLS construct (to image live nuclei). I picked out clones via single-cell dilution for the highest expression mCherry/mCerulean.

My problem: When I FACS sort the clones, while the mCerulean-NLS appears to be a single, tight peak, the AKT1-mCherry appears to be a combination of 2 peaks (sometimes looks like 1 bright peak and a long left shoulder, sometimes looks like almost 2 distinct but overlapping peaks).

I've been trying to find a good explanation for this and was wondering if it was possible that the gene insert is somehow "silence" (explaining 2 peaks) as cells do not like too high levels of exogenous AKT1. Perhaps the mCerulean-NLS is one peak because there is no "selection pressure" for overexpression?

Thanks you!
-Albert

Edited by American3pt14, 15 December 2010 - 10:50 AM.