An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit

Abstract

The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.

Figure 1

Development of the ROSA-TET system. (A) Experimental strategy to generate the ROSA-TET locus and the desired locus. The ROSA26 locus was targeted by the knock-in vector pMWROSATcH, and the region from loxP to loxPV of the resultant locus was replaced by the exchange vector. Puromycin selection and subsequent assay for hygromycin sensitivity was used to isolate desired clones. E, EcoRV; and X, XbaI. (B) Representative results of Southern analysis using a 5′ probe; results using an internal probe are not shown. (C) Success ratio of the knock-in step from Southern analysis, measured as the numbers of correctly targeted clones relative to total hygromycin-resistant clones. The total numbers of hygromycin-resistant clones analyzed are shown. (D) Efficiency of RMCE reaction on the ROSA-TET locus, measured as the ratio of hygromycin-sensitive clones (in Tc− medium) to total puromycin-resistant clones. Each bar represents the results using the exchange vectors carrying different cDNAs. The total numbers of puromycin-resistant clones analyzed are shown. (E) An example of Southern analysis of hygromycin-sensitive clones using a Puro probe. The 2.6 kb bands are indicated by an arrow. U, Untargeted clone; and T, Targeted clone.

Figure 2

Induction of expression in the ROSA-TET system. (A) Schematic representation of the induction of expression. The endogenous ROSA26 promoter was trapped by SA-tTA. In Tc− medium, tTA binds to the hCMV*-1 promoter, inducing expression of cDNA-IRES-Venus. (B) Induction of expression in EBRTcPGATA6 cells. mRNA and protein samples were prepared at 24 h after the induction. GAPDH and Oct-3/4 are used to normalize expression of Gata-6-specific mRNA and protein, respectively. It is known that endogenous Gata-6 protein is expressed at detectable level even in undifferentiated ES cells, and Oct-3/4 is expressed at the same level within 24 h after the induction of Gata-6 expression (25). (C) Morphological changes in EBRTcPGATA6 cells during induction of Gata-6 expression. Cells were observed 24 (panels a and b), 48 (panels c and d), and 72 (panels e and f) h after induction of Gata-6 expression through a bright field (panels a, c, e and g) and through a YFP filter (panels b, d, f and h). In the non-induced cell population, stem cell colonies without fluorescence were observed after 72 h (panels g and h). (D) Expression induction in the cellular population. Open and striped boxes indicate undifferentiated and differentiated colonies, respectively. A marked increase in the number of differentiated colonies was observed 6 days after induction, whereas undifferentiated colonies were almost eliminated. Error bars represent mean ± 2 × SEM (n = 3).

Figure 3

FACS analysis of the induction of the expression. EBRTcPGATA6 cells were cultivated for the indicated concentrations of Tc for 24 h and 1 × 10⁴ cells were analyzed for the intensity of GFP fluorescence. Note that in the absence of Tc 96.9% of cells are GFP positive.

Figure 4

Induction of expression in completely differentiated cells. EBRTcPOct3 cells, constructed by introducing Oct-3/4 cDNA into EBRTcH3 cells, were induced to differentiate into neuronal cells by the SDIA method with Tc for 8 days, and with (A and B) or without Tc (C and D) for an additional 6 days. The cells were fluorescently immunostained with anti-TuJ (A and C) and anti-GFP (B and D) antibodies, which showed that almost all the TuJ-positive colonies were GFP positives.