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Puzzling Low Surface Expression of H-DreI-B7 in IgL-targeted Clones

unstained. dsDre4. dsAni1. Puzzling Low Surface Expression of H-DreI-B7 in IgL-targeted Clones. DT40 TetR WT. DT40 Ψ -/- AID R. Dre #2. Dre #3. Dre #4. HA-APC. IgM-FITC. Dre #2. Dre #4. Dre #3. Cell count (%). dsOligo-Biotin/SAv-PE.

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Puzzling Low Surface Expression of H-DreI-B7 in IgL-targeted Clones

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  1. unstained dsDre4 dsAni1 Puzzling Low Surface Expression of H-DreI-B7 in IgL-targeted Clones DT40 TetR WT DT40 Ψ-/- AIDR Dre #2 Dre #3 Dre #4 HA-APC IgM-FITC Dre #2 Dre #4 Dre #3 Cell count (%) dsOligo-Biotin/SAv-PE

  2. Hypermutation optimized-DreI-B7/Hypermut2 clones 1 week in culture: I-AniI/Hypermut2 control 5B8 HDreI 3D5 3D5 5B12 HDreI 3 weeks in culture: 3F4 HDreI 5A8 HDreI 3F5 HDreI 5F8 HDreI HA-tag-APC IgM-FITC Better surface expression, but no integration into rearranged IgL allele

  3. Good recognition of H-DreI target sequence unstained dsDre4 Control cells DT40 H-DreI C4 HyperDreI 5A8 HyperDreI 5F8 HyperDreI 4E12 HyperDreI 5E6 Cell count (% of maximum) dsDre4-BT:SAv-PE dsDre4 staining corresponds well to HA-tag staining

  4. 1.0 1.0 1.0 1.0 1.0 1.0 2.6 6.3 14.0 2.4 1.3 7.8 Flow cytometry analysis of CTLMA2 target MFl B10 MFl DT40 MFl DT40 = 1.0 I-AniI WT: I-AniI CTLMA2: 5’ TGAGGAGGTTTCTCTGTAA 3’ 5’ AGAGGACCTTCATCTGTCA 3’

  5. Flow cytometry cleavage assay for CTLMA2 target dsAni1 wt dsAni1 +1C+2A DT40 B3 I-AniI B10 I-AniI Cell count (% of maximum) 3D5 I-AniI dsOligo-Alexa647

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