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. 2008 May 9;4(5):e1000069. doi: 10.1371/journal.pgen.1000069

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This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

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(A) Screening for optimal integration sites in ES cells and classification of ES clones by X-gal staining. A representative ES clone is shown for each class with and without phase contrast for better imaging of ES cell morphology and X-gal staining, respectively. (B) Screening of class I clones for high inducibility. Forty three class I ES clones were transfected with a tTA expression vector and β-gal activity was quantified 48 hours post-transfection. Bars: open, without tTA; filled, with tTA. A luciferase expression vector was cotransfected to normalize β-gal activities. Three ES clones were designated as T1, T2 and T3 as shown in the figure, and were used to generate mice. (C) Gene regulation in mice generated from tightly regulated ES clones. Three mouse strains were established from ES clones T1, T2 and T3, and crossed to MMTV-tTA mouse. Top three panels: β-gal activity was measured in the following three genotypes: nontransgenic mice (lacZ(−)tTA(−), open bars); mice with lacZ gene but without tTA (lacZ(+)tTA(−), lightly shaded bars); mice with both lacZ gene and tTA gene (lacZ(+)tTA(+), filled bars). Values are shown as means with error bars of standard deviations from five male and five female animals. Values of nontransgenic mice (open bars) are common in all three panels. Bottom panel: tTA mRNA expression level quantified by real-time PCR from one male and one female mouse. Mean values are presented by filled bars. Note that values are shown in logarithmic scale.