Harnessing Gene Conversion in Chicken B Cells to Create a Human Antibody Sequence Repertoire

Schusser, Benjamin; Yi, Henry; Collarini, Ellen J.; Izquierdo, Shelley; Harriman, William; Etches, R. J.; Leighton, Philip A.

doi:10.1371/journal.pone.0080108

Cited by 34 publications

(22 citation statements)

References 38 publications

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“…More recently, we have described birds carrying an immunoglobulin heavy chain gene that has been inactivated by replacing the J region with a selectable marker cassette composed of β-actin-GFP and CAG-puro R (Schusser et al, 2013a). The cassette introduced into these birds also has an attP site (i.e., the docking site for phi-31 integrase) to target human-derived immunoglobulin transgenes into this location (Schusser et al, 2013b). These birds have now been made and will be described in a future publication.…”

Section: Transgenic Chickensmentioning

confidence: 99%

Inserting random and site-specific changes into the genome of chickens

Collarini¹,

Leighton²,

Pedersen³

et al. 2015

Poultry Science

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During the past decade, modifications to the chicken genome have evolved from random insertions of small transgenes using viral vectors to site-specific deletions using homologous recombination vectors and nontargeted insertions of large transgenes using phi-31 integrase. Primordial germ cells (PGC) and gonocytes are the germline-competent cell lines in which targeted modifications and large transgenes are inserted into the genome. After extended periods of in vitro culture, PGC retain their capacity to form functional gametes when reintroduced in vivo. Rates of stable germline modification vary from 1×10(-5) for nontargeted insertions to 1×10(-8) for targeted insertions. Following transfection, clonally derived cell lines are expanded, injected into Stage 13-15 Hamburger and Hamilton embryos, and putative chimeras are incubated to term in surrogate shells. Green fluorescent protein (GFP) is incorporated into transgenes to reveal the presence of genetically modified PGC in culture and the extent of colonization of the gonad during the first week posthatch. If the extent of colonization is adequate, cohorts of putative chimeras are reared to sexual maturity. Semen is collected and the contribution from donor PGC is estimated by evaluating GFP expression using flow cytometry and PCR. The most promising candidates are selected for breeding to obtain G1 heterozygote offspring. To date, this protocol has been used to (1) knockout the immunoglobulin heavy and light chain genes and produce chickens lacking humoral immunity, (2) insert human V genes and arrays of pseudo V genes into the heavy and light immunoglobulin loci to produce chickens making antibodies with human V regions, (3) insert GFP into nontargeted locations within the genome to produce chickens expressing GFP, and (4) insert Cre recombinase into the genome to produce chickens that excise sequences of DNA flanked by loxP sites.

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Section: Transgenic Chickensmentioning

confidence: 99%

Inserting random and site-specific changes into the genome of chickens

Collarini¹,

Leighton²,

Pedersen³

et al. 2015

Poultry Science

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“…The inactivated immunoglobulin locus can be reconstructed with components encoding human V regions to produce chickens with human‐chicken chimeric immunoglobulin light chains. In combination with a similarly reconstructed heavy chain, we can design chickens from which antibodies with fully human antibody binding domains can be recovered.…”

Section: Discussionmentioning

confidence: 99%

Expression of heavy chain‐only antibodies can support B‐cell development in light chain knockout chickens

Schusser

Collarini²,

Pedersen³

et al. 2016

Eur J Immunol

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“…Germ cell derivation and culture were performed as previously described [34,42,43]. Rederived knock out cell lines IgL KO 229-92 and IgH KO 472-138 were transfected with the SynVL and SynVH constructs, respectively, using phiC31 integrase as described [44].…”

Section: Germ Cell Culturementioning

confidence: 99%

Expression of human lambda expands the repertoire of OmniChickens

et al. 2020

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Most of the approved monoclonal antibodies used in the clinic were initially discovered in mice. However, many targets of therapeutic interest are highly conserved proteins that do not elicit a robust immune response in mice. There is a need for non-mammalian antibody discovery platforms which would allow researchers to access epitopes that are not recognized in mammalian hosts. Recently, we introduced the OmniChicken ® , a transgenic animal carrying human VH3-23 and VK3-15 at its immunoglobulin loci. Here, we describe a new version of the OmniChicken which carries VH3-23 and either VL1-44 or VL3-19 at its heavy and light chain loci, respectively. The Vλ-expressing birds showed normal B and T populations in the periphery. A panel of monoclonal antibodies demonstrated comparable epitope coverage of a model antigen compared to both wild-type and Vκ-expressing OmniChickens. Kinetic analysis identified binders in the picomolar range. The Vλ-expressing bird increases the antibody diversity available in the OmniChicken platform, further enabling discovery of therapeutic leads.

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Harnessing Gene Conversion in Chicken B Cells to Create a Human Antibody Sequence Repertoire

Cited by 34 publications

References 38 publications

Inserting random and site-specific changes into the genome of chickens

Inserting random and site-specific changes into the genome of chickens

Expression of heavy chain‐only antibodies can support B‐cell development in light chain knockout chickens

Expression of human lambda expands the repertoire of OmniChickens

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