Genetics and expression of kappa-type light chains in Basilea rabbits

Mage, Rose G.; Young-Cooper, Glendowlyn O.; Alexander, Cornelius B.; Kelus, A. S.

doi:10.1007/bf00364645

Cited by 13 publications

(4 citation statements)

References 32 publications

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“…This loss was compensated in homozygous bas/bas rabbits by a huge increase of a "new" L-chain isotype (K2) and of X chains (7,14). This compensation facilitated the characterization of isotypes-experimentally difficult under normal conditions.…”

Section: Discussionmentioning

confidence: 99%

Mutation affecting the expression of immunoglobulin variable regions in the rabbit.

Kelus¹,

Weiß²

1986

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

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We have found a variant of the allotype allele a2 in the rabbit, which presumably arose by mutation, that segregates as expected for an allele at the a locus. This allele is called "ali" and the corresponding rabbit strain is called "Alicia." In heterozygous animals (al/al and ali/a3) the concentration of a2 molecules is lower by a factor of 1000 than in standard a2/a2 homozygotes. In homozygous ali/al individuals the a2 concentration varies with age-i.e., very low in young rabbits and higher in older ones-but it never reaches normal levels. The low level of a2 is compensated by increased amounts of a-negative molecules. Southern blot analysis did not reveal any gross changes in the intron between JH and CI (oining region of immunoglobulin heavy chain and constant region of immunoglobulin ,u chain) or in the number of VH gene segments encoding a locus specificities. We suggest that the ali phenotype is due to a mutation in a control element.The immunoglobulin (Ig) molecules are made up of heavy (H) and light (L) polypeptide chains. In a few species, the amino acid sequences of these chains and their DNA sequences have been determined (1)(2)(3). H chains can be divided into a variable (VH) region, which contributes to the antibodycombining site, and a constant (CH) region, which determines the class of the Ig. The H chains of the rabbit are encoded by a cluster of closely linked genetic loci: a-ms-d-e-f-g, with a few alleles being known at each locus (2, 4). At the DNA level, the order of gene segments appears to be VH-J-C,-CY--CF-Ca (where J = joining) (5). Allotypic determinants of the a locus are present in VH regions of all Ig classes (2). Some 5-30% of the IgG molecules lack a locus allotypic determinants and are called a-negative. These molecules express other allotypic specificities, encoded by the w, x, and y loci closely linked to the a locus (2, 4), and their N-terminal amino acid sequence differs from that of the a-positive molecules (6).We describe here a variant, presumably a mutant, in which the expression of a-positive molecules is remarkably altered. MATERIALS AND METHODSAnimals. Rabbits were bred at the institute and were identical by descent with respect to the H-chain haplotypes.Igs. Normal serum of allotyped rabbits was run over a protein A-Sepharose 4B (Pharmacia, Uppsala, Sweden) column. After extensive washing the bound material was eluted with 1 M acetic acid, neutralized immediately with Tris buffer, and dialyzed against phosphate-buffered saline (PBS). Standard anti-a2 was purified by affinity chromatography on a2 IgG coupled to CNBr-activated Sepharose 4B according to the procedure suggested by the manufacturer (Pharmacia). Ascites fluid of monoclonal 3.11 anti-a2 was obtained from W. van der Loo and used as such. Commercial anti-rabbit Fc was obtained from Jackson Immunochemical (Avondale, PA) and also used without further purification. Double Diffusion in Agarose Gel. Undiluted serum and antiserum were allowed to diffuse overnight at +40C in 1.5% agarose Indubiose A45 (L'Indus...

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Section: Discussionmentioning

confidence: 99%

Mutation affecting the expression of immunoglobulin variable regions in the rabbit.

Kelus¹,

Weiß²

1986

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

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“…The Basilea strain was developed from a mutant rabbit found in a laboratory colony about 12 years ago (11). The spontaneous mutant lost the capacity to produce Klb9 light chains; the other isotype (K2) was unaffected (10). Homozygous Basilea rabbits compensate for this loss by producing elevated levels of chains (8, 11-13).…”

Section: Discussionmentioning

confidence: 99%

“…This report describes our investigations of the molecular basis for the loss of expression of K1 chains in the Basilea mutant.Rabbits are unusual in that they have two constant region r chain (C,) 1 genes (4, 5, 14-16). Although they have not yet been physically linked (4), the ~1 and ~2 genes are transmitted as linked traits in breeding colonies, and thus far no recombinants have been observed (6,7,(9)(10)(11). The rl and r2 genes each have their own J (joining region) clusters (3,5,15).…”

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confidence: 99%

Lack of K1b9 light chains in Basilea rabbits is probably due to a mutation in an acceptor site for mRNA splicing.

Lamoyi¹,

Mage²

1985

The Journal of Experimental Medicine

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Four forms of r immunoglobulin chains, known as the b4, b5, b6, and b9 allotypes, are products of allelic genes at the r 1 locus in domestic rabbit populations (1). A gene (K2) for a second r chain isotype (K2) is apparently present in all rabbits (2-5); one allotypic form of this gene is expressed at low levels in wild rabbits and some rabbits of b9 allotype (6-10). The Basilea strain was developed by Kelus and Weiss (11) from such a b9 rabbit in which an apparent mutation resulted in failure to produce the major type of Ig K light chains (K1). In these rabbits, the majority of Ig bear ~, light chains, and a small proportion bear r chains of the second isotype (K2) (8,(11)(12)(13). To understand the unusual r chain expression in Basilea rabbits, we have undertaken an analysis of the r genes in these animals. This report describes our investigations of the molecular basis for the loss of expression of K1 chains in the Basilea mutant.Rabbits are unusual in that they have two constant region r chain (C,) 1 genes (4, 5, 14-16). Although they have not yet been physically linked (4), the ~1 and ~2 genes are transmitted as linked traits in breeding colonies, and thus far no recombinants have been observed (6, 7, 9-11). The rl and r2 genes each have their own J (joining region) clusters (3, 5, 15). However, it is not known whether both Ca genes associate with the same variable region K chain (V~) gene pool. CK 1 genes show extensive polymorphism. Although the allelic b allotypes were first defined by serological and chemical analyses, the polymorphisms have now been studied by DNA sequencing (reviewed in l, 16-18). The DNA sequence homologies of the portions encoding K1 constant regions (C,1) range from 79% (b5 to b9) to 86% (b4 to b5) (16, 17). The known C~2 sequences are most similar to C,1 b9 (~89% homology) (17, 18). We used probes from the previously characterized cDNA (5, 18) and genomic clones to show, by Southern hybridization and restriction mapping, that both ~ 1 and K2 genes are present in DNA of Basilea rabbits, and that there are no detectable gross insertions, deletions, or other rearrangements in the K1 gene. For a finer analysis, we isolated and sequenced the mutant rlb9 gene. We found a G ~ A change in the highly conserved AG dinucleotide of the 3' acceptor splice site of the J-C intron. This substitution at ~ Abbreviations used in this paper: C, constant region; IVS, intervening sequence; J, joining region; SSC, saline sodium citrate; SDS, sodium dodecyl sulfate; UT, untranslated region; V, variable region; Y, pyrimidine.

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“…For the generation of transgenic rabbits, superovulated females were mated with males carrying the Basilea mutation, a strain naturally deficient in the rabbit kappa light chain. 13 Linearized DNA coding for the fully human heavy chain (FHHC1.1-bcl2), for the fully human light chain construct (FHLC1.1), or an equimolar mixture of human CD79a/b bacterial artificial chromosome (BAC) DNA were microinjected into oocytes. The final configurations of the FHHC1.1-bcl2 and FHLC1.1 constructs are illustrated in Figure 1.…”

Section: Generation Of Transgenic Rabbits Expressing Human Igg Antibomentioning

confidence: 99%

Rabbits transgenic for human IgG genes recapitulating rabbit B-cell biology to generate human antibodies of high specificity and affinity

Ros

Offner

Klostermann

et al. 2020

mAbs

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Transgenic animals incorporating human antibody genes are extremely attractive for drug development because they obviate subsequent antibody humanization procedures required for therapeutic translation. Transgenic platforms have previously been established using mice, but also more recently rats, chickens, and cows and are now in abundant use for drug development. However, rabbit-based antibody generation, with a strong track record for specificity and affinity, is able to include gene conversion mediated sequence diversification, thereby enhancing binder maturation and improving the variance/selection of output antibodies in a different way than in rodents. Since it additionally frequently permits good binder generation against antigens that are only weakly immunogenic in other organisms, it is a highly interesting species for therapeutic antibody generation. We report here on the generation, utilization, and analysis of the first transgenic rabbit strain for human antibody production. Through the knockout of endogenous IgM genes and the introduction of human immunoglobulin sequences, this rabbit strain has been engineered to generate a highly diverse human IgG antibody repertoire. We further incorporated human CD79a/b and Bcl2 (B-cell lymphoma 2) genes, which enhance B-cell receptor expression and B-cell survival. Following immunization against the angiogenic factor BMP9 (Bone Morphogenetic Proteins 9), we were able to isolate a set of exquisitely affine and specific neutralizing antibodies from these rabbits. Sequence analysis of these binders revealed that both somatic hypermutation and gene conversion are fully operational in this strain, without compromising the very high degree of humanness. This powerful new transgenic strategy will allow further expansion of the use of endogenous immune mechanisms in drug development.

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Genetics and expression of kappa-type light chains in Basilea rabbits

Cited by 13 publications

References 32 publications

Mutation affecting the expression of immunoglobulin variable regions in the rabbit.

Mutation affecting the expression of immunoglobulin variable regions in the rabbit.

Lack of K1b9 light chains in Basilea rabbits is probably due to a mutation in an acceptor site for mRNA splicing.

Rabbits transgenic for human IgG genes recapitulating rabbit B-cell biology to generate human antibodies of high specificity and affinity

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