American Society of Hematology

Figure 5.

$Figure 5. Determination of GFPcre-mediated recombination efficiency. (A-B) GFPcre-mediated recombination efficiency in adult mice. Bone marrow and spleen cells from 3 mice positive for the R26R reporter allele and hemizygous GFPcre positive (GFPcre/+) for the EpoR locus were sorted by MACS for Ter119-positive (+) and -negative (–) fractions. The EcoRV-digested DNA was analyzed by quantitative Southern blot analysis (A). The quantitation was performed by phospho-imager analysis and the values for the recombinant (del) as well as the nonrecombinant (flox) allele were calculated after background subtraction per lane in percent (table). The genomic organization of the reporter allele is depicted. The letters indicate the restriction site EcoRV (E) and the probe (P) used for hybridization. LoxP sites are shown as ◃. The size of the deleted (del) as well as the loxP-flanked (flox) reporter allele is indicated. Each cell fraction was analyzed by flow cytometry for Ter119 content (B). Data were plotted as relative fluorescence intensity versus the cell number. The Ter119-positive (Ter119+, gray line) and -negative sorted fractions (Ter119–, thick black line) of bone marrow (left) and spleen (right) were compared with the corresponding pool of unsorted cells (thin black line). The number in each panel represents the relative amount of Terr119-positive cells within the Ter119-positive sorted fraction as defined by the indicated gate. (C-D) GFPcre-mediated recombination efficiency in embryonic mice. FLCs derived from E14.5 embryos hemizygous for the R26R reporter allele and wild-type for the EpoR locus (+/+) or hemizygous positive for GFPcre (GFPcre/+) were sorted for Ter119-positive and -negative fractions. EcoRV-digested DNA derived from sorted FLC fractions were subjected to quantitative Southern blot analysis (C). The left lane displays the results obtained from Ter119-positive control FLCs, negative for GFPcre (+/+) and positive for the R26R reporter allele directly after sorting. The middle left, middle right, and right lanes in panel C show the results obtained from either Ter119-negative (–) or Ter119-positive (+) sorted FLCs hemizygous for GFPcre (GFPcre/+) and R26R directly after sorting (t = 0) or after 24-hour cultivation in Epo (t = 24). Sorted fractions were analyzed for Ter119 content by flow cytometry (D) either directly after sorting (0 h) or after 24-h cultivation in Epo (24 h).$

Determination of GFPcre-mediated recombination efficiency. (A-B) GFPcre-mediated recombination efficiency in adult mice. Bone marrow and spleen cells from 3 mice positive for the R26R reporter allele and hemizygous GFPcre positive (GFPcre/+) for the EpoR locus were sorted by MACS for Ter119-positive (+) and -negative (–) fractions. The EcoRV-digested DNA was analyzed by quantitative Southern blot analysis (A). The quantitation was performed by phospho-imager analysis and the values for the recombinant (del) as well as the nonrecombinant (flox) allele were calculated after background subtraction per lane in percent (table). The genomic organization of the reporter allele is depicted. The letters indicate the restriction site EcoRV (E) and the probe (P) used for hybridization. LoxP sites are shown as ◃. The size of the deleted (del) as well as the loxP-flanked (flox) reporter allele is indicated. Each cell fraction was analyzed by flow cytometry for Ter119 content (B). Data were plotted as relative fluorescence intensity versus the cell number. The Ter119-positive (Ter119⁺, gray line) and -negative sorted fractions (Ter119^–, thick black line) of bone marrow (left) and spleen (right) were compared with the corresponding pool of unsorted cells (thin black line). The number in each panel represents the relative amount of Terr119-positive cells within the Ter119-positive sorted fraction as defined by the indicated gate. (C-D) GFPcre-mediated recombination efficiency in embryonic mice. FLCs derived from E14.5 embryos hemizygous for the R26R reporter allele and wild-type for the EpoR locus (+/+) or hemizygous positive for GFPcre (GFPcre/+) were sorted for Ter119-positive and -negative fractions. EcoRV-digested DNA derived from sorted FLC fractions were subjected to quantitative Southern blot analysis (C). The left lane displays the results obtained from Ter119-positive control FLCs, negative for GFPcre (+/+) and positive for the R26R reporter allele directly after sorting. The middle left, middle right, and right lanes in panel C show the results obtained from either Ter119-negative (–) or Ter119-positive (+) sorted FLCs hemizygous for GFPcre (GFPcre/+) and R26R directly after sorting (t = 0) or after 24-hour cultivation in Epo (t = 24). Sorted fractions were analyzed for Ter119 content by flow cytometry (D) either directly after sorting (0 h) or after 24-h cultivation in Epo (24 h).

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