CTCFL is functionally different from CTCF A Strategy for the rescue of CTCF-depleted ES cells. Ctcflox/lox ES cells were infected with lentivirus containing the Cre recombinase and/or fluorescently tagged CTCF(L) proteins. After infection neomycin-resistant colonies were picked and analyzed. m = mouse, g = chicken. B Analysis of Ctcflox/lox deletion. After infection with CRE-containing constructs, Ctcflox/lox ES cells were scored for the status of the conditional Ctcf alleles by DNA blot. The position of wild-type (wt), deleted (Ctcfdel, or del) and conditional (Ctcflox, or lox) loci in control ES cells (C), non-treated Ctcflox/lox ES cells (1) and lentivirally transduced Ctcflox/lox ES cells (2–5, see panel A for numbering of constructs) is indicated. Cells are considered rescued when both conditional CTCF alleles have been deleted. C Analysis of CTCF protein expression. Neomycin-resistant colonies were grown and analyzed by Western blot for CTCF (upper panel) or GFP (lower panel) expression. Note that rescued cells are negative for endogenous CTCF. D, E GFP-CTCFL is a functional protein. ES cells were transiently transfected and harvested after 1 day. ChIP (DNA, D) and RT-PCR (mRNA, E) analyses revealed that GFP-CTCFL binds Vps18, Stra8 and Prss50 promoters (D) and is able to induce expression of Gal3st1, Stra8 and Prss50 (E).