Live cell monitoring of gene modulation provides a better understanding of gene function (946.12)

Abstract

One of the most common methods for understanding gene function within a live cell is modulating the gene through knockdown or over‐expression systems. These experiments provide information regarding the role a particular gene plays within a given biological process. Unfortunately, determining the modulation in gene expression is currently performed at the population level as in the case of qPCR and or is ultimately an endpoint determination as in the case of RNA FISH.We propose a new method for determining the RNA expression levels of target genes using a nanoparticle technology which can be used in live cells without the need for transfection reagents. Live cell gene expression detection affords the ability to monitor changes in expression levels over time. More data can be collected over the course of a single experiment at the cellular level while monitoring gene expression in a dynamic manner as opposed to studying many samples at varied time points in the case of RT‐PCR or FISH.Here we present data illustrating the benefits of detecting gene expression in live cells while modulating both Survivin and ER alpha genes. U2OS cells which have an ER alpha knock‐in under the control of an inducible promoter were monitored using the nanoparticle detection method prior to and during the induction process enabling the monitoring of the gene modulation. Knockdown experiments were also performed using Survivin as a target gene to illustrate down regulation of genes and the ability to monitor the loss of function as well.Monitoring the modulation of genes within live cells can assist in determination of gene function by allowing for the collection of many more data points within a single experiment without the need for duplicate samples at various time points. Furthermore, as this method is non toxic the live cells can be further characterized or used in subsequent experiments providing even more data from the same cells.