Presence of the neo-tk cassette results in a hypomorphic ( Cbx3hypo) allele. (a) Row 1, expression of HP1γ in MEFs. HP1γ expression was dramatically reduced to almost undetectable levels in MEFs derived from embryos that are homozygous for the targeted allele shown in row 3 of Figure 1 (Cbx3hypo/hypoMEFs). After expression of FlpE in the Cbx3hypo/hypoMEFs (FlpE-treated MEFs), the expression of HP1γ returned to wild-type levels. HP1γ protein expression in the FlpE-treated MEFs could be extinguished by expression of Cre recombinase, giving rise to Cbx3-/- MEFs. Hnrnpa2b1 protein expression was not affected by the presence or absence of the neo-tk cassette, as shown in row 2. Row 3, actin loading control. (b) Row 1, immunofluorescent HP1γ staining of MEFs. The typical punctate HP1γ pattern of expression in wild-type MEFs was reduced to very faint, almost background, levels in Cbx3hypo/hypoMEFs. Strikingly, HP1γ staining levels returned to wild-type levels after the neo-tk cassette was excised after Flpe treatment (FlpE-treated MEFs). HP1γ staining was lost after expression of Cre recombinase giving rise to Cbx3-/- MEFs. Row 2, DAPI staining; row 3, merged images. Bar = 20 μm.