Ctcfl and Ctcf expression and targeting. A B RNAse protection analysis of Ctcfl and Ctcf. For Ctcfl (A) RNase protection analysis (RPA) was performed on polyA purified mRNA with probes covering parts of Ctcfl exon 8 and 9 (left, small fragment) or a 5’end RACE product (right, large fragment). For Ctcf (B) the RPA was performed on total RNA with probes protecting Ctcf exon 2. The positions of the respective protected fragments are indicated with arrows. Ctcfl mRNA mRNA can only be detected in polyA purified mRNA from testis (t), whereas Ctcf is identified in total RNA from all three tissues tested. M, marker; p, input probe; c, tRNA control; h, heart; t, testis; b, brain. Aprt exon 3 is used as loading control and marked by an asterisk. This analysis identifies the first exon containing the ATG translation initiation codon in Ctcfl and shows that Ctcfl is expressed in testis. C Schematic overview of the modified Ctcfl alleles and targeting constructs. The Ctcfl locus is shown on scale, with the constructs (not on scale) used for homologous recombination in ES cells underneath. Targeting at the 5’end of Ctcfl yielded the Ctcflgpf- neo allele. Cre-mediated excision of the LoxP-embedded neomcyin resistance gene yielded the Ctcflgfp allele (not shown). The 3’end targeting was performed on the Ctcflgpf- neo allele, and yielded the Ctcflgfp-neo-puro allele. Cre-mediated excision of the sequence in between the outermost LoxP sites yielded the Ctcfldel allele, in which exons 1–8 of the Ctcfl gene are deleted (not shown). A major difference between the Ctcfldel allele described here and the Ctcfl knockout published earlier  is that in the Ctcfldel allele the GFP coding sequence is fused in frame with the CTCFL coding sequence. Black boxes represent exons, GFP tag, neomycin and puromycin cassettes. Probes a, b, c, d and e are indicated by lines. Oligos 1, 2, 3 and 4 are represented by arrowheads. White triangles are LoxP sites. B = BglII; N = NcoI; S = SpeI; A = AvrII. D DNA blot showing Ctcfl targeting. Probes a and b were used on DNA blots from ES cells for identification of the Ctcflgfp-neo allele and probes c and d for the Ctcflpuro allele. Probe e identifies the Ctcfldel allele from Ctcflgfp-neo-puro mice that were crossed to a chicken Actin-Cre transgene. Probe a, HindIII digest (wt 8.9 kb, ko 5.7 kb); probe b, EcoRI digest (wt 14 kb; ko 11 kb); probe c, BamHI digest (wt 16.1 kb; ko 6.8 kb); probe d, BamHI digest (wt 16.1 kb; ko 11.1 kb). E Absence of Ctcfl DNA in the Ctcfldel allele. PCR on tail DNA indicates that Ctcfldel/del mice are deleted for exons 1–8 (top three panels) and are positive for GFP (oligos 2 and 4). F Absence of Ctcfl RNA in Ctcfl mutant mice. PCR on cDNA derived from testis mRNA shows that Ctcfl is absent from Ctcfldel/del mice. Acrosin and Gapd function as positive controls. G Schematic overview of the Ctcf allele and targeting strategy for the Ctcfgfp-neo allele. The Ctcf locus is shown on scale, with the construct (not on scale) used for homologous recombination in ES cells underneath. Cre-mediated excision of the LoxP-embedded neomcyin resistance gene yielded the Ctcfgfp (or Ctcfki) allele (not shown). Black boxes represent exons, GFP tag and neomycin cassette. Oligos 5, 6, 7 and 8 are represented by arrowheads. White triangles are LoxP sites. E = EcoRI. H PCR confirming Ctcfgpf-neo allele. Identification of the CTCFgfp-neo (or Ctcfki) allele by PCR with oligos 7 and 8 or oligos 5, 6 and 8 (see panel G). I Western blot confirming GFP-CTCF expression from the Ctcfgfp allele. We isolated MEFS from E13.5 day wild-type (+/+), heterozygous Ctcfgfp/+ (or Ctcfki/+) or homozygous Ctcfgfp/gfp (or Ctcfki/ki) embryos, and identified the GFP-CTCF fusion protein by Western blot of MEF extracts using anti-CTCF or anti-GFP antibodies. Note the increased size of the GFP-CTCF protein compared to the CTCF protein due to the GFP tag.