And Mutant Mouse Strains

Transgenic mice constructed on an inbred background provide a novel approach to the study of class I gene expression and antigen function. In the transgenic system, a single well-defined gene can be placed on an inbred background in one generation. If the transgene is properly expressed and the locus carrying the transgene can be bred to homozygosity, the resulting mouse strains would be, in principle, analogous to congenic lines. These transgenic mice would be identical to the parental inbred mouse strain, except for the addition, at one histocompatibility locus, of a foreign allele introduced by microinjection. Yoshioka et al. introduced the H-2Dd class I gene into the C57BL/6 mice by microinjection (7). The H-2Dd transgene was found to be expressed in a tissue-specific manner that paralleled that of the endogenous H-2Kb gene. The H-2Dd transgene was inducible in response to interferon and suppressible by transformation with the human adenovirus 12. In addition, all the transgenic H-2Dd mice were found to be tolerant of the H-2Dd antigen. Therefore, spleen cells from the transgenic H-2Dd mice failed to generate an anti-H-2Dd CTL response in vitro, suggesting that the H-2Dd antigen is recognized as a self-molecule in these animals. Similar observations were made when transgenic mice were generated by microinjecting the human HLA genes into mouse embryos. The human HLA-B27 molecules could be used by the mouse T-cell as a restriction element against viral antigens in HLA-B27/human [}-2M double-transgenic mice (8). Moreover, the introduction of HLA-B27 into C57BL/6 or (C57BL/6 x SJL/J)F1 mice in the absence of human p-2M gene could also result in the expression of the HLA-B27 molecules at a level comparable to the endogenous H-2b and H-2S class I molecules. Transgenic mice carrying the HLA-A2.1 gene were also generated (9). Spleen cells from these transgenic mice expressed the HLA-A2.1 molecules on their cell surface in association with mouse f}-2M. The cells, however, contain more HLA mRNA than endogenous H-2 class I mRNA. In contrast, the amount of HLA:p-2M is low. There is also a large pool of non-p-2M-associated HLA heavy chain inside the cell. These results indicated that in these transgenic mice, HLA-A2.1 seems to compete poorly with the H-2 heavy chains for mouse (3-2M.

In most of the transgenic mice produced, the introduced MHC molecules behave as self-antigens, and could function as transplantation antigens in skin graft reactions and as restriction elements in antiviral or antibacterial T-cell response. The introduction of MHC genes by microinjection of mouse embryos would therefore circumvent the painstaking task of developing congenic mouse strains by classical breeding method and through the isolation of spontaneous class I mutant mice.

2.3. Identification of Tissue-Specific Transcription Regulatory DNA Sequences

Many studies involving generation of transgenic mice are aimed at understanding normal developmental processes. By introducing genes that are normally expressed in a tissue-specific manner, one can assess whether cis-acting DNA elements involved in developmental programming of gene expression are present. Then, by producing transgenic mice with a series of mutant genes, one can define the precise sequence required for tissue-specific expression. In this respect, the class II MHC molecules provide a good model system for the identification of multiple-enhancer elements for expression and compart-mentalization. In the thymus, the class II molecules occur on epithelial cells of the cortex, and more abundantly on mterdigitating reticular cells and epithelial cells of the medulla. It is in these well-defined areas of the thymus that "education" of T-lymphocytes takes place, that is, the shaping of the T-cell receptor repertoire toward recognition of foreign antigens in association with self-MHC and away from reactivity against self-MHC alone. Widera et al. (10) microinjected two fragments containing 1.4kb of 5'-flanking and 0.5 kb of 3'-flank-ing region of the Ea gene into (H-2bxs) mice that do not express their endogenous Ea gene. The transgene was expressed in thymic tissue and in adherent spleen cells, and was induced in peritoneal exudate cells by y-interferon. However, in contrast to normal mice, there was no expression of Ea in B-lymphocytes of the transgenics. Since transgenic mice made with constructs containing 3.2 and 2 kb of 5'-flank-ing sequences show the normal expression pattern of the Ea gene, it appears that deletion of 5'-flanking sequences between -1.4 and -2 kb inactivated or eliminated regulatory sequences required for expression of Ea specifically in B-cells. The presence of this B-cell control region at -2 to -1.4 kb was also confirmed by van Ewijk et al. (11), who analyzed the Ea gene expression in a more detailed fashion by the creation of a set of transgenic mouse lines after introduction of Ea genes carrying deletions in its promoter/enhancer elements. By this approach, the authors further demonstrated a dissociation of expression in thymic cortical vs medullary cells, in B-cells vs non-B cells, and in germinal center vs follicular B-cells. Besides allowing conclusions about the role of individual promoter elements in controlling gene expression in different tissues, the set of transgenic lines described by these authors offered the potential for additional studies on the role of class II MHC molecules in various compartments of the immune system. The transgenic mouse system also allows the detection of crucial transcriptional regulatory elements. In vitro studies demonstrated the importance of the conserved regulatory sequence motifs, the X and Y boxes, in the 5'-flanking region of the MHC class II genes. Dorn et al. (12) confirmed the importance of these sequences by investigating the effect of their deletion on Ea RNA synthesis in transgenic mice. Although mutant genes that had either the X or Y box deleted could still be transcribed, the efficiency of transcription was drastically reduced, and furthermore, the RNA initiation was no longer accurate.

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