The A.CA strain (H-2f) lacks expression of the class II E molecule because of a stop codon in the leader sequence of the Ea gene and defective transcription of the Eb gene (Vu et al. 1989;Tacchini-Cottier et al. 1988). We previously reported that A.CA mice possessing an Ea d transgene express the Eo~ d polypeptide chain on the surface of lymphoid cells, presumably in association with AI3 chains. (Ishikawa et al. 1991). We have established transgenic mice expressing both the o~ and ~3 chain of the E d molecule to investigate the role in thymic selection of Eo~E~3 isotype-matched heterodimers in comparison with Ec~A[3 mixed-isotype heterodimers.
A 17 kilobase (kb) Kpn I fragment (Fig. 1 A) containing the Eb gene of the macrophage cell line, BAC1.2 (H-2d#) was isolated from a genomic library generated in Charon 40. The gene spanning from -5.7 kb to 11 kb was microinjected directly into A.CA fertilized eggs. The founder shows a characteristic 2 kb band present in Eco Rl-digested genomic DNA (Fig. 1 B). Northern blot analysis shows that transcripts of the transgene were detected in spleen, thymus, lung, and kidney, but not in heart or liver (Fig. 1 C). The Tg(o,Eb) line of the Eb transgenic mice was established and crossed to two A.CA strains transgenic for the Ec~a, Tg(a, Ea), and Tg(b, Ea) to obtain Tg(a/o, Ea/Eb) and Tg(b/o, Ea/Eb) double transgenic mice, respectively.
Fluorescence-activated cell sorter (FACS) analysis of lymph node cells stained with the 14.4.4S anti-E~ antibody showed that Tg(b/o, Ea/Eb) double transgenic mice express a higher density of E d molecules than Tg(a/o, Ea/Eb) double transgenic mice, even higher than the density of E d seen in BALB/c mice (Fig. 2A). In this mouse, the expression of E d is elevated on B220 + cells (data not shown). Single transgenic mice with the Correspondence to." B. Diamond.