Quantitative analysis of gene expression in living adult neural stem cells by gene trapping John R Scheel 1 , Jasodhara Ray 1 , Fred H Gage 1 & Carrolee Barlow 1,2 The potential of neural stem cells (NSCs) for the treatment of neurodegenerative diseases makes the identification and characterization of genes involved in neural stem cell responses therapeutically important. Although technologies exist for measuring gene expression in cells, they often provide only a representative expression profile specific to a stimulus and time. We developed a complementary technology based on a retroviral- vector gene-trap approach that uses b-lactamase–induced disruption of fluorescence resonance energy transfer in the fluorophore CCF-2/AM. A library of ‘tagged’ adult rat NSCs was generated by transduction with gene-trap virus produced from a single-integrant packaging cell line that allowed us to quantitatively analyze dynamic gene expression changes in real time in living NSCs. Using this library we identified previously unknown genes regulated by oxidative stress, indomethacin and factors that induce differentiation, and show that one of the trapped genes, Sox6, is sufficient to induce astrocytic differentiation when overexpressed. NSCs are cells that maintain the capacity for self-renewal and multilineage differentiation, with the potential to replace or repair damaged tissue 1–3 . During neurodegeneration and ischemia, NSCs can migrate to areas of damage and actively participate in repair 4,5 . But in ischemia, for example, only 0.2% of newly generated neurons survive longer than five weeks 6,7 . A better understanding of the molecular mechanisms regulating the generation and main- tenance of NSCs, their choice of different cell fates, and their migration and survival properties is the foundation on which therapeutic strategies will be built. Cell culture of adult rodent NSCs provides a model of neurogen- esis in the adult brain because NSCs are primary nontransformed cells that can be propagated in vitro without losing their stem-cell properties 8,9 . Transplanted rat NSCs can partially repair brain da- mage resulting from ischemia by preferential migration and integra- tion of newly differentiated neurons into ischemic lesions, indicating that they maintain the capacity to respond to appropriate cues 10,11 . Therefore, cultured rat NSCs provide a platform for the study of important biological processes applicable to their behavior in vivo. Several technologies had been used to identify genes involved in the migration, proliferation and differentiation of NSCs under normal circumstances and in response to disease 12,13 . These stra- tegies, however, measure gene expression in heterogeneous popula- tions because it is difficult to obtain quantitative data from single NSCs. Studying the effects of a stimulus on NSC populations may give an inaccurate impression of the temporal pattern of gene expression because each cell in the population may have a different susceptibility to the same stimulus, based on its functional stage, that will determine when or if the cell will respond to the stimulus. Additionally, processes such as differentiation are controlled by tightly regulated genes that are only expressed for a short time, such that a readout from a population of NSCs might lead to dilution of the signal of important genes of low abundance. A recently described gene-trap system (p237, Aurora Biosciences) uses a b-lactamase reporter enzyme to cleave a nontoxic fluorogenic substrate, CCF-2/AM, which results in a shift in the emission wave- length from 530 nm (green) to 460 nm (blue), as expression of the trapped gene increases. This enzyme and fluorophore–based com- bination allows measurement of small changes in transcriptional activity through enzymatic amplification of the transcriptional signal and quantitative ratiometric analysis of gene expression, making the system less affected by background fluorescence and cell density 14,15 . Applying this strategy to living NSCs is difficult for several reasons. First, the gene-trap virus was produced by simultaneous transfection of several viral components, which allows the viral genome to re- arrange. A rearranged-genome gene-trap virus does not allow for quantitative analysis of gene expression. Second, the method relied on fluorescence-activated cell sorting (FACS) to isolate single cells. FACS and subsequent growth of single cells into a clonal population is difficult for NSCs because of the cellular stress caused by FACS, and the cells’ requirement for paratrophic growth factors. To resolve these problems, we developed a single-integrant packaging cell line that produces a uniform virus that can accurately reproduce the expression of the trapped gene. Additionally, we developed culturing and FACS conditions that allow the survival and expansion of single- tagged rat NSCs isolated by FACS, and here we describe the metho- dology necessary to develop this system in stem cells from other organisms (see Supplementary Protocol online). We also demon- strate how this high-throughput strategy can be applied to study changes in gene expression in real time in response to modulators of oxidative stress and factors that induce differentiation. p u o r G g n i h s i l b u P e r u t a N 5 0 0 2 © e r u t a n / m o c . e r u t a n . w w w / / : p t t h s d o h t e m PUBLISHED ONLINE 21 APRIL 2005; DOI:10.1038/NMETH755 1 Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA. 2 Present address: BrainCells Inc., 10835 Road to the Cure, San Diego, California 92121, USA. Correspondence should be addressed to C.B. (cbarlow@braincellsinc.com). NATURE METHODS | VOL.2 NO.5 | MAY 2005 | 363 ARTICLES