Figure 1
Figure 1. Establishment and validation of knockout/knockin mice that lack the intracellular FasL domain. (A) Targeting strategy for the establishment of FasL ΔIntra mice. DNA sequences coding for the first 73 amino acids were deleted in exon 1, and a tk-neo selection cassette flanked by loxP sites was inserted into intron 1. Subsequent excision of this cassette by Cre recombinase left a single loxP site in the genome. (B) T and B lymphocytes from homozygous FasL ΔIntra mice express the truncated FasL molecule lacking the intracellular domain. Left panel, surface expression of full-length and mutant FasL molecules at the surface of activated lymphocytes isolated from wild-type and homozygous FasL ΔIntra mice. Freshly isolated lymphocyte populations from spleen were stimulated with plate-coated 5 μg/mL anti-CD3 antibody (T cells), or with 10 μg/mL LPS (B cells) for 24 hours. One representative experiment of 4 is shown. Wild-type cells are represented by the solid lines, FasL ΔIntra cells by the dotted lines, and the isotype control by the wide dashed lines. Right panel, RT-PCR analysis reveals FasL ΔIntra mRNA expression in T and B cells. Total RNA was isolated from purified splenic wild-type and FasL ΔIntra T and B cells, which had been stimulated for 24 hours with 1 μg/mL anti-CD3 (T cells) or for 4 hours with 1 μg/mL anti-IgM (B cells). The primer pair used for RT-PCR amplification of the FasL ICD allowed to distinguish cDNA from genomic DNA template (intron-spanning). (C) T cells isolated from homozygous FasL ΔIntra mice retain the ability to kill in a FasL-dependent manner. The killing capacity of restimulated T cell blasts (10 μg/mL anti-CD3 antibody, 24 hours) from wild-type, FasL ΔIntra and both homozygous and heterozygous gld mice was determined by coculture experiments. Fas-sensitive, CFSE-labeled A20 target cells were used at an effector:target ratio of 2.5:1 for 6 hours in the presence of 10 μg/mL anti-CD3 antibody. Killing capacity is expressed as the percentage of apoptotic subG1 A20 target cells minus the percentage of dead cells observed in the presence of 100 μg/mL Fas:Fc. Columns represent the mean and bars the SE of 4 individual experiments.

Establishment and validation of knockout/knockin mice that lack the intracellular FasL domain. (A) Targeting strategy for the establishment of FasL ΔIntra mice. DNA sequences coding for the first 73 amino acids were deleted in exon 1, and a tk-neo selection cassette flanked by loxP sites was inserted into intron 1. Subsequent excision of this cassette by Cre recombinase left a single loxP site in the genome. (B) T and B lymphocytes from homozygous FasL ΔIntra mice express the truncated FasL molecule lacking the intracellular domain. Left panel, surface expression of full-length and mutant FasL molecules at the surface of activated lymphocytes isolated from wild-type and homozygous FasL ΔIntra mice. Freshly isolated lymphocyte populations from spleen were stimulated with plate-coated 5 μg/mL anti-CD3 antibody (T cells), or with 10 μg/mL LPS (B cells) for 24 hours. One representative experiment of 4 is shown. Wild-type cells are represented by the solid lines, FasL ΔIntra cells by the dotted lines, and the isotype control by the wide dashed lines. Right panel, RT-PCR analysis reveals FasL ΔIntra mRNA expression in T and B cells. Total RNA was isolated from purified splenic wild-type and FasL ΔIntra T and B cells, which had been stimulated for 24 hours with 1 μg/mL anti-CD3 (T cells) or for 4 hours with 1 μg/mL anti-IgM (B cells). The primer pair used for RT-PCR amplification of the FasL ICD allowed to distinguish cDNA from genomic DNA template (intron-spanning). (C) T cells isolated from homozygous FasL ΔIntra mice retain the ability to kill in a FasL-dependent manner. The killing capacity of restimulated T cell blasts (10 μg/mL anti-CD3 antibody, 24 hours) from wild-type, FasL ΔIntra and both homozygous and heterozygous gld mice was determined by coculture experiments. Fas-sensitive, CFSE-labeled A20 target cells were used at an effector:target ratio of 2.5:1 for 6 hours in the presence of 10 μg/mL anti-CD3 antibody. Killing capacity is expressed as the percentage of apoptotic subG1 A20 target cells minus the percentage of dead cells observed in the presence of 100 μg/mL Fas:Fc. Columns represent the mean and bars the SE of 4 individual experiments.

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