Abelson lines infected with MCiGfp efficiently excise the Dnmt1 locus. (A) Schematic diagram of the genomic Dnmt1 locus. The genotype of the Abelson lines are 2lox/N before cre-mediated excision. WT, wild type. (B) Southern blot analysis of the Dnmt1 locus from purified populations of infected Dnmt^cond Abelson lines infected with either MiGfp or MCiGfp and sorted for Gfp⁺ viable cells (G⁺ or CiG⁺, respectively). The DNA was digested with SpeI and probed with a fragment from the Dnmt1 locus. The bands are labeled on the right.

Abelson lines are efficiently infected by retroviruses. (A) Schematic diagram of retroviral vectors used in this study. The MSCV LTR drives the expression of the enhanced Gfp downstream of an IRES in the control vector, MiGfp. The cre recombinase is cloned upstream of the IRES GFP cassette and also is driven by the MSCV LTR, MCiGfp. MCS, multiple cloning site. (B) Fluorescence-activated cell sorting analysis measuring Gfp expression of the Abelson line Dnmt^cond1 infected with either MiGfp or MCiGfp at 2 days postinfection gated on live cells by scatter properties. (C) Measurement of the percentage of live Gfp⁺ cells as a function of time postinfection either infected with MiGfp or MCiGfp as labeled. Each line represents an independent experiment.

Loss of methylation at the kappa locus activates transcription in Dnmt^cond2. (A) Schematic diagram of the murine kappa locus. The large arrow designates the promoter of the k_o transcript. The primers k_o and Ck used for PCR and the Jk2 primer used as a probe to detect the k_o product are shown schematically. (B) RT-PCR analysis of the expression level of k_o in the Dnmt^cond1 line under different conditions. Lanes 1–3 are unsorted cells either untreated (lane 1), or treated with LPS (lane 2) or HC toxin (lane 3) for 24 h. Lanes 4–7 are derived from cells 5 days postinfection. The cells were either infected with MiGfp (G) or MCiGfp (CG). Lanes 4 and 5 are unsorted infected cells, and lanes 6 and 7 are Gfp⁺ sorted cells. Lane 8 is the product of an RT-PCR with no RNA template. Lane 9 is the product of a PCR assay with no DNA template. (C) RT-PCR analysis of expression of k_o in the Dnmt^cond2 line under different conditions. Lanes 1–3 are unsorted cells either untreated (lane 1), or treated with LPS (lane 2) or HC toxin (lane 3) for 24 h. Lanes 4 and 5 are derived from cells sorted for Gfp expression 5 days postinfection either infected with MiGfp (G) or MCiGfp (CG). Lane 6 is the product of a PCR assay with no DNA template.

V(D)J recombination of the kappa locus is unaffected by loss of methylation. (A) Schematic diagram of the murine kappa locus. The degenerate primer Vk is homologous to many members of the Vk gene segment families and is used with the primer Jk2. These primers can only amplify a product once V(D)J recombination has brought the gene segments into close proximity. The product is detected by Southern blot analysis of the PCR products probed with the primer Jk1. (B) PCR-Southern analysis of genomic DNA from Dnmt^cond2 infected with either MiGfp or MCiGfp and sorted at the labeled time points postinfection. The cells were sorted for both Gfp⁺ and Gfp⁻ populations and assayed as described in A. (C) PCR-Southern blot of genomic DNA from unsorted Dnmt^cond1 or the Abelson line 230–238 previously demonstrated to respond to LPS treatment. The cells were either untreated (lanes 1 and 3) or treated with LPS for 24 h (lanes 2 and lane 4). Lane 5 is a 5-fold dilution of the genomic DNA in lane 4 and amplified in parallel.

Loss of Dnmt1 results in the loss of genomic methylation by day 4 postinfection. (A) Schematic diagram of the murine kappa locus. The map shows the V and J gene segments, constant region (Ck) and the intronic enhancer (Ek). The methylation-sensitive restrictions sites, HhaI (H), are shown as well as the probe used for analysis (Jk). The diagram is not to scale. (B) Southern blot analysis of the methylation status of the kappa locus in Abelson lines under different conditions. The DNA was digested with HhaI and probed with a Jk probe that spans the J region of the kappa locus. Lanes 1–8 are derived from Gfp⁺ sorted Abelson lines infected with the MiGfp (G) or MCiGfp (CG) at the time points indicated postinfection. Lanes 9–12 are generated from unsorted bulk populations of cells. Lane 9, untreated; lane 10, treated with LPS; lane 11, treated with butyrate; lane 12 treated with HC toxin. Lane 13 is derived from untreated Dnmt^−/− ES cells. (C) Southern blot analysis of the methylation status of IAPs in Abelson lines. The DNA was digested with HpaII and probed with a fragment of a known, endogenous IAP. Lanes 1 and 2 are controls, derived from ES cells that are wild type or mutant for the Dnmt1 locus, respectively. Lanes 3–7 are derived from Gfp⁺ or Gfp⁻ sorted Dnmt^cond1 Abelson line infected with either MiGfp (G) or MCiGfp (CG) at the time points indicated. The unmethylated fragments are shown on the right.