Fig. 3.
Fig. 3. Insertion of cassettes 2, 3, 234 and 0 at the RL1 locus and excision of MCneo. Panels (A) through (C) demonstrate insertion of cassettes 0, 2, 3, and 234 by RMCE at the RL1 locus using three different probes. In all three panels, lanes labeled RL1 contains DNA with the PGK-Hyg gene inserted at RL1 while lanes A and B contains DNA isolated from two independent clones. The blots in panel (A) were probed with a DNA fragment containing the entire coding sequence of the Neomycin resistance gene. The band present when the PGK-Hyg gene is at RL1, disappear after RMCE, and is replaced by a band of a size characteristic of each cassette. In the case of cassette 234, presence of an internal Xho I site (XhoI*) leads to a band smaller than the band observed when the PGK-Hyg gene is at RL1. The restriction site marked by a superscript “c” is a chromosomal site located close to the integration site. In panel (B) the blots were probed with a segment of the β-globin promoter (region −374 to +40 relative to the cap site). The size of the fragment varies as expected, according to the size of the cassettes. As expected, the PGK-Hyg gene at the RL1 locus did not hybridize to the β-globin promoter probe (data not shown). In panel (C), the blots were probed with a segment of the PGK-Hyg gene containing the coding sequence of the Hyg gene and the mouse PGK polyA signal. The 1.5-kb band present in all lanes represents the endogenous PGK polyA gene and serves as a useful loading control; the upper band is a PGK-Hyg specific band (lane RL1): as expected it disappears when one of the cassettes is at RL1 (all other lanes). This shows that the PGK-Hyg gene is exchanged during RMCE rather than simply inactivated by the insertion. Panel (D) shows excision of the reconstituted MCNeo gene. For each cassette, one subclone containing the MCneo gene (for instance Cas 2 A Neo) and two subclones having lost the MCNeo gene (for instance Cas 2 A Δ, and Cas 2 B Δ) are shown. The blots were probed with a β-globin promoter fragment (see panel B). With all cassettes, excision is demonstrated by an increase in size of the fragment due to the loss of an EcoRI site present in the MC Neo promoter. Neo cs, neomycin resistance coding sequence (also includes polyA sequence); β-G, β-globin promoter; L1 and L2, Lox sites differing by a mutation in their spacer region; F, FLP recognition target (FRT); MC, MC promoter.

Insertion of cassettes 2, 3, 234 and 0 at the RL1 locus and excision of MCneo. Panels (A) through (C) demonstrate insertion of cassettes 0, 2, 3, and 234 by RMCE at the RL1 locus using three different probes. In all three panels, lanes labeled RL1 contains DNA with the PGK-Hyg gene inserted at RL1 while lanes A and B contains DNA isolated from two independent clones. The blots in panel (A) were probed with a DNA fragment containing the entire coding sequence of the Neomycin resistance gene. The band present when the PGK-Hyg gene is at RL1, disappear after RMCE, and is replaced by a band of a size characteristic of each cassette. In the case of cassette 234, presence of an internal Xho I site (XhoI*) leads to a band smaller than the band observed when the PGK-Hyg gene is at RL1. The restriction site marked by a superscript “c” is a chromosomal site located close to the integration site. In panel (B) the blots were probed with a segment of the β-globin promoter (region −374 to +40 relative to the cap site). The size of the fragment varies as expected, according to the size of the cassettes. As expected, the PGK-Hyg gene at the RL1 locus did not hybridize to the β-globin promoter probe (data not shown). In panel (C), the blots were probed with a segment of the PGK-Hyg gene containing the coding sequence of the Hyg gene and the mouse PGK polyA signal. The 1.5-kb band present in all lanes represents the endogenous PGK polyA gene and serves as a useful loading control; the upper band is a PGK-Hyg specific band (lane RL1): as expected it disappears when one of the cassettes is at RL1 (all other lanes). This shows that the PGK-Hyg gene is exchanged during RMCE rather than simply inactivated by the insertion. Panel (D) shows excision of the reconstituted MCNeo gene. For each cassette, one subclone containing the MCneo gene (for instance Cas 2 A Neo) and two subclones having lost the MCNeo gene (for instance Cas 2 A Δ, and Cas 2 B Δ) are shown. The blots were probed with a β-globin promoter fragment (see panel B). With all cassettes, excision is demonstrated by an increase in size of the fragment due to the loss of an EcoRI site present in the MC Neo promoter. Neo cs, neomycin resistance coding sequence (also includes polyA sequence); β-G, β-globin promoter; L1 and L2, Lox sites differing by a mutation in their spacer region; F, FLP recognition target (FRT); MC, MC promoter.

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