Antibody Expression from the Core Region of the Human IgH Locus Reconstructed in Transgenic Mice Using Bacteriophage P1 Clones

Wagner, Simon D.; Gross, Gideon; Cook, Graham P.; Davies, Sarah L.; Neuberger, Michael S.

doi:10.1006/geno.1996.0379

Cited by 29 publications

(16 citation statements)

References 8 publications

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“…Chimeric IgM was present in M⌬G⌬ and chimeric IgG in both M⌬G⌬ and G⌬ serum. In nonimmunized adults, the chimeric IgM (Ϸ50 g͞ml) and IgG (200-1,000 g͞ml) are present at levels comparable with those seen in WT or mice with a normal human IgH locus (25). All six G⌬ mice had HCAb IgGs with a molecular mass of Ϸ80 kDa under nonreducing and Ϸ40 kDa under reducing conditions, consistent with HC dimers lacking a LC and each HC lacking CH1 (11 kDa shorter than the control human IgG; Fig.…”

Section: Mouse Light Chains Do Not Rearrange In M⌬g⌬ and G⌬ Micementioning

confidence: 52%

“…Antigen challenge results in antigen-specific chimeric HCAb of different classes (dependent on locus composition) expressed at levels comparable with WT or conventional human IgH transgenic mice (25). Only two VHHs were used, yet antibodies with diverse specificity were isolated successfully to almost all of the totally unrelated proteins we tested, demonstrating the efficiency and efficacy of diversity generated by CDR3 (27).…”

Section: Discussionmentioning

confidence: 96%

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Generation of heavy-chain-only antibodies in mice

Janssens¹,

Dekker²,

Hendriks

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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We have generated transgenic mice containing hybrid llama͞ human antibody loci that contain two llama variable regions and the human D, J, and C and͞or C␥ constant regions. Such loci rearrange productively and rescue B cell development efficiently without LC rearrangement. Heavy-chain-only antibodies (HCAb) are expressed at high levels, provided that the CH1 domain is deleted from the constant regions. HCAb production does not require an IgM stage for effective pre-B cell signaling. Antigenspecific heavy-chain-only IgM or IgGs are produced upon immunization. The IgG is dimeric, whereas IgM is multimeric. The chimeric HCAb loci are subject to allelic exclusion, but several copies of the transgenic locus can be rearranged and expressed successfully on the same allele in the same cell. Such cells are not subject to negative selection. The mice produce a full antibody repertoire and provide a previously undescribed avenue to produce specific human HCAb in the future.immunoglobulin rearrangement ͉ transgenic C onventional antibodies contain two heavy and light chains (LC) coded for by heavy and LC loci. B cell development and antibody production starts in the bone marrow (BM) by heavy chain (HC) VDJ recombination and expression of IgM associated with a surrogate LC on the cell surface. In a second round of recombination, one of the LC rearranges in pre-B cells. If successful, the B cells undergo selection, affinity maturation, and switching to different HC constant regions to result in B cells, which express tetrameric antibodies of different isotypes (IgA, IgG, and IgE). Normally absence of HC or LC expression leads to arrest of B cell development. However, some species produce HC-only antibodies (HCAb) as part of their normal B cell development and repertoire. The best-known HCAb (i.e., no LC) are IgG2 and IgG3 in camelids (1). They undergo antigen-mediated selection and affinity maturation, and their variable domains are subject to somatic hypermutation (2, 3). HCAb are thought to recognize unusual epitopes, such as clefts on the antigen surface (4). The first domain of the constant region, CH1, is spliced out because of the loss of a consensus splice signal (5, 6). CH1 exon loss also has been described in other mammals, albeit associated with disease, e.g., in mouse myelomas (7) and human HC disease (HCD) (8-10).Camelid HCAbs contain a complete VDJ region. Its size, stability, specificity, and solubility have generated considerable biotechnological interest. The antigen-binding site, a single-variable domain (VHH), resembles VH of conventional Abs. However, differences in FR2 and CDR3 prevent VHH to pair with a variable LC, whereas hydrophilic amino acids provide solubility (11). HCAb of the IgM class have not been found in camelids, suggesting that the IgM ϩ stage of HCAb formation is very transient and͞or circumvented.Murine NSO myeloma cells can express a rearranged camelid VHH-␥2a gene (12) and, recently, the same gene was expressed in transgenic mice (13). Here, we describe transgenic mice containing various...

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Section: Mouse Light Chains Do Not Rearrange In M⌬g⌬ and G⌬ Micementioning

confidence: 52%

Section: Discussionmentioning

confidence: 96%

Generation of heavy-chain-only antibodies in mice

Janssens¹,

Dekker²,

Hendriks

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…1. The integrated IgH and IgL transloci were then generated by coinjecting multiple BACs into fertilized rat oocytes, exploiting the previous finding that coinjection of overlapping DNA constructs often leads to cointegration into the genome (29). Thus, the IgH translocus was created by coinsertion of BAC6-VH3-11 (a 182-kb AsiSI-AscI fragment containing 13 V H s) with BAC3 (a 173-kb NotI fragment containing 10 V H s) and BAC3-1N12M5I8 (human/rat Annabel, a 193-kb NotI fragment containing human V H 6-1 and all Ds and J H s followed by the rat C region).…”

Section: The Human Igh and Igl Locimentioning

confidence: 99%

“…Construction of the human Ig loci employed established technologies to assemble large DNA segments using YACs and BACs (23,29,(38)(39)(40). As multiple sequential BAC modifications in E. coli frequently led to the deletion from the BAC of repetitive regions such as Ig switch sequences or of elements in the vicinity of the IgH 39 enhancers, a strategy was developed to assemble these large transloci by homologous recombination in S. cerevisiae as cYAC and, subsequently, converting such a cYAC into a BAC.…”

Section: The Human Igh and Igl Locimentioning

confidence: 99%

High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Igκ/Igλ Loci Bearing the Rat CH Region

Osborn

Avis

et al. 2013

The Journal of Immunology

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Mice transgenic for human Ig loci are an invaluable resource for the production of human Abs. However, such mice often do not yield human mAbs as effectively as conventional mice yield mouse mAbs. Suboptimal efficacy in delivery of human Abs might reflect imperfect interaction between the human membrane IgH chains and the mouse cellular signaling machinery. To obviate this problem, in this study we generated a humanized rat strain (OmniRat) carrying a chimeric human/rat IgH locus (comprising 22 human VHs, all human D and JH segments in natural configuration linked to the rat CH locus) together with fully human IgL loci (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ). The endogenous Ig loci were silenced using designer zinc finger nucleases. Breeding to homozygosity resulted in a novel transgenic rat line exclusively producing chimeric Abs with human idiotypes. B cell recovery was indistinguishable from wild-type animals, and human V(D)J transcripts were highly diverse. Following immunization, the OmniRat strain performed as efficiently as did normal rats in yielding high-affinity serum IgG. mAbs, comprising fully human variable regions with subnanomolar Ag affinity and carrying extensive somatic mutations, are readily obtainable, similarly to conventional mAbs from normal rats.

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“…They uniformly consisted of sIgM + B cells without exception. Interestingly, Burkitt's lymphoma cells are Buluwela and Rabbitts, 1988); Cm, a 1.2 kb EcoRl fragment from lC75 (Milstein et al, 1984); dM2, a 0.35 kb PCR fragment from the human Cdgene (Wagner et al, 1996); 3'IgH, a 0.4 kb EcoRI fragment from plasmid pM5-1-23, which contains 3.4 kb of DNA adjacent to the acentric arm of the HuIgH YAC and was obtained by plasmid rescue (C Mundt, unpublished), and c-myc and Em described in Figure 2). Separation was on conventional TAE gels except for D where pulsed-®eld gel electrophoresis (1% agarose gel in 0.56TBE using a LKB Pulsaphor 2015 equipment with 5 s switch time for 27 h followed by 10 s switch time for 6 h at 140 V and 128C) was performed.…”

Section: Discussionmentioning

confidence: 99%