Transgenic Animals Derived by DNA Microinjection.

Human antibody expression in transgenic rodents has been significantly improved by the integration of large chimeric DNA constructs linking human VH, D, and JH segments to rat constant (C) regions and regulatory sequences. Light-chain constructs were fully human. The Ig loci were cloned and modified in artificial minichromosomes and added to the rat germline by DNA microinjection into fertilized oocytes. Silencing of all endogenous Ig loci was achieved using zinc finger nuclease constructs, which accomplished targeted gene disruption of sometimes several kilobases. DNA rearrangements provided extensive diversity, and near-normal or wild-type levels of transgenic IgM and IgG were produced. Hypermutation was readily found and many high affinity antibodies could be produced by cell fusion. For therapeutic applications, the rat C-region could be easily replaced by a desired human C without the loss of antigen binding.

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