Living things now in existence have evolved over a long period of time through the mutatagenesis and the selection of mutants by the environment. The general evolution has very slow speed and passes through many generations to advance. On the other hand, in the case of antibody-producing cells of the immune system, the mutatagenesis and the selection by antigens are completed in one generation, and the next generation will not inherit the acquired function. Such a quick rate of evolution in the immune system is interpreted as being set against the mutation of an environment-dependent pathogenic microorganism.
The present inventor previously produced transgenic mice to which genes of bacteria-derived enzyme chloramphenicol acetyltransferase are introduced, and demonstrated that the mutation of an antibody gene is controlled by its promoter and enhancer and the mutation of any genes can be induced by controlling with a promoter and enhancer of an antibody gene (Azuma, T., et al., Int. Immunology, 5(2) 121-130 (1992)).
The currently used production process for a mutant is one of the mutant-producing methods in which deletion, insertion and/or addition mutation is suitably introduced into a desired DNA sequence, that is, site-specific mutagenesis to site-specifically replace a certain length of DNA sequences (Site-specific mutagenesis, Zoller, et al., Nucleic Acid Res., 10, 6487-6500 (1982); Zoller, et al., Methods in Enzymol., 100, 468-500 (1983))). In the general mismatch mutation using an artificially synthesized oligonucleotide as a primer, a complementary oligonucleotide consisting of around 20 bases is synthesized with an optional mutation in a base sequence nearby a site where mutation is to be induced, the oligonucleotide is hybridized to a target DNA, and then a DNA complementary to the remaining of target DNA can be produced using DNA polymerase. Thus it is possible to introduce a desired mutation into a desired site. However, this method cannot induce many mutants at once.
In the other mutagenesis systems, some examines the disappearance or the manifestation of a specific function in the presence of a compound that damages a gene (Myers, et al., Science, 232, 613-618 (1986)), and other uses bacteria etc. However, these methods are fundamentally different from the above one to induce mutation by the present inventor.
Variation (diversity) in the Ig V region of mice and humans is generated by the combinatorial joining of V, D, and J gene segments existing separately in germline, the deletion and addition of nucleotides at the junction of these segments during joining, and somatic hypermutation of joined V-(D)-J genes. The somatic hypermutation is related to the affinity maturation of antibody and has been frequently observed after stimulation of T cell-dependent antigen (TD) (Bothwell, A. L. M., et al., Cell, 24, 625 (1981): Gearhart, P. J., et al., Nature, 291, 29 (1981): Griffiths, G. M., et al., Nature, 312, 271 (1984): Maizels, N., et al., Cell, 43, 715 (1985): Wysocki, L. T., et al., Proc. Natl. Acad. Sci. USA, 83, 1847 (1986): Cumano, A., et al., EMBO. J., 5, 2459 (1986): Berek, C., et al., Cell, 67, 1121 (1991): Taketani, M., et al., Mol. Immunol., 32, 983 (1995): Furukawa, k., et al., Immunity, 11, 329 (1999): 2-10). The cis-acting elements responsible for the induction of somatic hypermutation have been identified using κ-chain (O'Brien, R. L. et al., Nature, 326, 405 (1987): Sharpe, M. J., et al., Eur. J. Immunol., 20, 1379 (1990): Sharpe, M. J., et al., EMBO J., 10, 2139 (1991): Betz, A. G., et al., Cell, 77, 239 (1994): Yelamos, J., et al., Nature, 376, 225 (1995): Peters, A., et al., Immunity, 4, 57 (1996): 11-16), λ-chain (Klotz, E., et al., J. Immunol., 157, 4458 (1996):17) and H-chain transgenic mice (Durdik, J., et al., Proc. Natl. Acad. Sci. USA, 86, 2346 (1989): Sohn, J., et al., J. Exp. Med., 177, 493 (1993): Tumas-Brundage, K. M. and Manser, T., J. Exp. Med., 185, 239 (1997):18-20).
As stated above, the present inventor prepared transgenic mice carrying chloramphenicol acetyltransferase (CAT) gene that is driven by VH17.2.25 (Loh, D. Y., et al., Cell, 33, 85 (1983): Grosschedl, R. and Baltimore, D., Cell, 41, 885 (1985): 22, 23) and J-C intron enhancer (hereafter abbreviated to Eμ) (Gillies, S. D., et al., Cell, 33, 715 (1983): Banerji, J., et al., Cell, 33, 729 (1983):24, 25)/matrix attachment region (hereafter abbreviated to MAR) (Forrester, W. C., et al., Science, 265, 1221 (1994):26). As a result, somatic hypermutation was detected in CAT but not in VH promoter or Eμ/MAR flanking. However, the frequency of mutation was approximately 1/10 that observed in endogenous VH-D-JH, suggesting that these cis-acting elements are critical or important for the induction of hypermutation and that other components such as Cγ, Cα of 3′ or the enhancer flanking of Cα (3′ enhancer) (Pettersson, S., et al., Nature, 344, 165 (1990): Dariavach, P., et al., Eur. J. Immunnol., 21, 1499 (1991): Lieberson, R., et al., EMBO. J., 14, 6229 (1995):27-29) might be responsible for high frequent somatic hypermutation (Sohn, J., et al., J. Exp. Med., 177, 493 (1993): Tumas-Brundage, K. M. and Manser, T., J. Exp. Med., 185, 239 (1997): Giusti, A. M. and Manser, T., J. Exp. Med., 177, 793 (1997):19, 20, 30).