Figure 4.
Figure 4. Expression of an Ly108-H1 BAC-based transgene in Sle1b mice. (A) The BACLy108-H1 transgene vector encoding Ly108-H1 was generated by deleting exons 7 and 8 of Ly108 and the SLAM and CD84 genes from the B6 BAC clone RP23-77A8. Alteration of the 200-kb B6 BAC clone by Red/ET recombineering is described in Materials and methods. The resulting DNA fragment BACLy108-H1 is ∼100 kb. S1, S2, S3, and S4 are homology arms for Red/ET recombination. KANA is a kanamycin resistance gene cassette. (B) RT-PCR prepared with thymic mRNA using Ly108-H1–specific primers. (C) Haplotype (Hap)-specific RFLP fragments were obtained by BsrI digestion of PCR-amplified thymic Ly108 cDNA. B6 and Sle1b mice exclusively express haplotype 1– and haplotype 2–specific fragments, respectively. Because Sle1b.BACLy108-H1 mice express both fragments, the transgene expresses the B6 based fragment. PCR primer pairs specific for Ly108 exons 2–4 (common for Ly108-1, Ly108-2, and Ly108-H1) were used, as described in Materials and methods. (D, left) RT-PCR primers specific for Ly108 exons 2–7 (common for Ly108-1 and Ly108-2 only) were used to isolate a cDNA from the thymus. Haplotype 1– and haplotype 2–specific RFLP used BsrI digestion, as in C. (middle) RT-PCR primers specific for SLAM exons 1–5 were used to isolate a cDNA from the thymus. Haplotype 1– and haplotype 2–specific RFLP fragments were obtained by TaqI digestion. (right) RT-PCR primers specific for CD84 exons 1–2 were used to isolate cDNA. Haplotype-specific RFLP fragments were obtained by MspI digestion. (E) Cell surface expression of Ly108 using T lineage cells isolated from Sle1b, Sle1b.BACLy108-H1, or B6 mice. Thymic and splenic T cells were incubated with the monoclonal α-Ly108 antibody (13G3) conjugated with DyLight 649 and then washed and analyzed by flow cytometry. The mean fluorescent intensity (MFI) of gated cell populations in the DyLight 649 channel is shown (three to four individual mice per group). PCR experiments were performed a minimum of two times. DN, double negative; DP, double positive.

Expression of an Ly108-H1 BAC-based transgene in Sle1b mice. (A) The BACLy108-H1 transgene vector encoding Ly108-H1 was generated by deleting exons 7 and 8 of Ly108 and the SLAM and CD84 genes from the B6 BAC clone RP23-77A8. Alteration of the 200-kb B6 BAC clone by Red/ET recombineering is described in Materials and methods. The resulting DNA fragment BACLy108-H1 is ∼100 kb. S1, S2, S3, and S4 are homology arms for Red/ET recombination. KANA is a kanamycin resistance gene cassette. (B) RT-PCR prepared with thymic mRNA using Ly108-H1–specific primers. (C) Haplotype (Hap)-specific RFLP fragments were obtained by BsrI digestion of PCR-amplified thymic Ly108 cDNA. B6 and Sle1b mice exclusively express haplotype 1– and haplotype 2–specific fragments, respectively. Because Sle1b.BACLy108-H1 mice express both fragments, the transgene expresses the B6 based fragment. PCR primer pairs specific for Ly108 exons 2–4 (common for Ly108-1, Ly108-2, and Ly108-H1) were used, as described in Materials and methods. (D, left) RT-PCR primers specific for Ly108 exons 2–7 (common for Ly108-1 and Ly108-2 only) were used to isolate a cDNA from the thymus. Haplotype 1– and haplotype 2–specific RFLP used BsrI digestion, as in C. (middle) RT-PCR primers specific for SLAM exons 1–5 were used to isolate a cDNA from the thymus. Haplotype 1– and haplotype 2–specific RFLP fragments were obtained by TaqI digestion. (right) RT-PCR primers specific for CD84 exons 1–2 were used to isolate cDNA. Haplotype-specific RFLP fragments were obtained by MspI digestion. (E) Cell surface expression of Ly108 using T lineage cells isolated from Sle1b, Sle1b.BACLy108-H1, or B6 mice. Thymic and splenic T cells were incubated with the monoclonal α-Ly108 antibody (13G3) conjugated with DyLight 649 and then washed and analyzed by flow cytometry. The mean fluorescent intensity (MFI) of gated cell populations in the DyLight 649 channel is shown (three to four individual mice per group). PCR experiments were performed a minimum of two times. DN, double negative; DP, double positive.

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