Inactivation of the mouse HPRT locus by a 203-bp retroposon insertion and a 55-kb gene-targeted deletion: establishment of new HPRT-deficient mouse embryonic stem cell lines.

To obtain useful hypoxanthine phosphoribosyl-transferase (HPRT)-deficient mouse ES cell lines, two different methods were employed: (i) selection of spontaneous 6-TG-resistant mutants and (ii) gene targeting of the HPRT locus. The first approach resulted in the establishment of E14.1TG3B1, a spontaneous HPRT-deficient cell line with an insertional mutation of 203 bp in the third exon of the HPRT gene. The insert is highly homologous to the B2 mouse repetitive element and has all the expected retroposon characteristics, thus providing an example of gene inactivation by retroposon insertion. This clone exhibited stable 6-TG resistance and high germ-line transmission frequency. Thus E14.1TG3B1 is a useful ES cell line for modifying the mouse genome using the HPRT gene as a selection marker and for transmission at a high frequency into the mouse germ line. The second approach resulted in a 55-kb deletion of the mouse HPRT locus, demonstrating the feasibility of replacement-targeting vectors to generate large genomic DNA deletions.

Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice.

We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

Production of antigen-specific human antibodies from mice engineered with human heavy and light chain YACs.

Our paper describes the introduction of large fragments of both the human heavy and light chain Ig genes into the mouse germline to create a mouse strain capable of producing a broad repertoire of antigen-specific, fully human antibodies. The human immunoglobulin gene sequences were functional in the context of the mouse machinery for antibody recombination and expression, either in the presence or absence of functional endogenous genes. This was demonstrated by their ability to undergo diverse rearrangement, to be expressed at significant levels, and to exclude expression of mouse immunoglobulins irrespective of their copy number or site of integration. The decrease in susceptibility to influence by adjacent genomic sequences may reflect the greater size, variable gene content, or structural integrity of the human Ig YACs and/or the presence of unidentified but important regulatory elements needed for optimal expression of the human immunoglobulin genes and their correct regulation. Our results show that mouse B cells coexpressing human heavy and kappa chains, upon immunization, can produce antigen-specific, fully human antibodies. Furthermore, the human heavy and kappa chain YACs induced differentiation and maturation of the growth-arrested B-cell lineage in mice with inactivated endogenous Ig genes, leading to the production of a diverse repertoire of fully human antibodies at levels approaching those in normal serum. These results suggest the potential value of these mice as a source of fully human antibodies for human therapy. Furthermore, it is expected that such mice would lack immunological tolerance to and thus readily yield antibodies to human proteins, which may constitute an important class of targets for monoclonal antibody therapy. Our findings suggest that the introduction of even larger portions of the human heavy and light chain loci, which should be achievable with the ES cell-yeast spheroplast fusion technology described, will result in strains of mice ultimately capable of recapitulating the full antibody repertoire characteristic of the human humoral response to infection and immunization. The present and future mouse strains may prove to be valuable tools for studying the molecular mechanisms and regulatory sequences influencing the programmed assembly and expression of human antibodies in the normal immune response, as well as the abnormal response characteristic of autoimmune disease and other disorders. The strategy we have described for the introduction of large segments of the human genome into mice in conjunction with the inactivation of the corresponding mouse loci may also have broad applicability to the investigation of other complex or uncharacterized loci.

Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells.

With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (VH) genes, the major diversity (D) gene cluster, the joining (JH) genes, the intronic enhancer (EH), and the constant region genes, mu (C mu) and delta (C delta). Two IgK locus-derived YACs each contain three variable (V kappa) genes, the joining (J kappa) region, the intronic enhancer (E kappa), the constant gene (C kappa), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form.

Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs.

We describe a strategy for producing human monoclonal antibodies in mice by introducing large segments of the human heavy and kappa light chain loci contained on yeast artificial chromosomes into the mouse germline. Such mice produce a diverse repertoire of human heavy and light chains, and upon immunization with tetanus toxin have been used to derive antigen-specific, fully human monoclonal antibodies. Breeding such animals with mice engineered by gene targeting to be deficient in mouse immunoglobulin (Ig) production has led to a mouse strain in which high levels of antibodies are produced, mostly comprised of both human heavy and light chains. These strains should provide insight into the adoptive human antibody response and permit the development of fully human monoclonal antibodies with therapeutic potential.

Germ-line transmission and expression of a human-derived yeast artificial chromosome.

Introduction of DNA fragments, hundreds of kilobases in size, into mouse embryonic stem (ES) cells would greatly advance the ability to manipulate the mouse genome. Mice generated from such modified cells would permit investigation of the function and expression of very large or crudely mapped genes. Large DNA molecules cloned into yeast artificial chromosomes (YACs) are stable and genetically manipulable within yeast, suggesting yeast-cell fusion as an ideal method for transferring large DNA segments into mammalian cells. Introduction of YACs into different cell types by this technique has been reported; however, the incorporation of yeast DNA along with the YAC has raised doubts as to whether ES cells, modified in this way, would be able to recolonize the mouse germ line. Here we provide, to our knowledge, the first demonstration of germ-line transmission and expression of a large human DNA fragment, introduced into ES cells by fusion with yeast spheroplasts. Proper development was not impaired by the cointegration of a large portion of the yeast genome with the YAC.