inducible gene expression system is a versatile tool tostudy protein function. It provides an alternative to transientsystems wherein constitutive over-expression of ectopic pro-teins can introduce deleterious effects such as cell cycle arrest(1) or apoptosis (2). The ability to switch off/on the expres-sion of a specific protein is particularly useful for comparativestudies using the same cell system. To date, a range of induci-ble systems have been described such as those responsive tometal (3), glucocorticoid (4), or isopropyl-beta-D-thiogalac-toside (IPTG) (5). Since metal ions and glucocorticoids areknown to affect the expression of many endogenous proteins,IPTG remains a more popular choice of inducer. Nevertheless,these systems share a common drawback in that generation ofinducible cell lines can be labor-intensive, time-consuming(up to several months), and inefficient (1–2 cell lines perstudy). Here we use the LacSwitch II Inducible MammalianExpression System (Agilent Technologies, Santa Clara, CA) asa typical model to describe an improved approach to generateinducible cell lines.The LacSwitch system is designed for inducible geneexpression in mammalian cells. This system employs two vec-tors, the pCMVLacI vector which confers hygromycin resist-ance and encodes Lac repressor, and the lac-operator-contain-ing pOPRSVI/MCS or pOPI3CAT vector which confers G418resistance and encodes the gene of interest. When both vectorsare transfected into a cultured cell line, the Lac protein isexpressed and binds to the lac-operators to repress expressionof the gene of interest. Such repression is constitutive until theinducer IPTG is added to the cell culture to inactivate the Lacprotein. There are different approaches to generate induciblecell lines using this system. While the transfection conditionsvary among different studies, the screening procedures areapparently consistent in that individual transfected clones aremanually isolated and expanded for examination of geneexpression by Western blot analysis. Since transfection effi-ciency and expression levels of ectopic proteins can be highlyvariable, it is often necessary to screen dozens to hundreds ofindividual clones to select the specific ones with desired induc-ible phenotypic characteristics. Although multiple methodssuch as cloning rings (6), trypsin-soaked filter circles (7), andserial dilution (8) are available to facilitate the isolation ofindividual clones, the screening process remains extremelylaborious and poses additional risks for tissue culture contam-ination. To address these concerns, we propose a two-stepapproach using fluorescent protein as a selection marker andfluorescence-activated cell sorting (FACS) to automate thescreening procedures.This FACS-based approach starts with co-transfection ofboth the pCMVLacI vector encoding Lac repressor and thepOPRSVI/MCS or pOPI3CAT vector encoding fluorescentprotein. Since transfection efficiency of individual vectors var-ies among cells, four types of clone variants are anticipatedwith integrated gene expressing (i) Lac only; (ii) fluorescentprotein only; (iii) both Lac and fluorescent protein; or (iv) noectopic protein, due to undesired genomic recombination.The IPTG-dependent expression profiles of individual clonesare summarized (Fig. 1A). Given the unique property of thedesired inducible type III clone, specific isolation is possibleby applying different combinations of induction and cell sort-ing criteria (Fig. 1B). The strategy is to first keep the mixed