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Analysis of humanized antibody diversification in rabbits and mice using transgenic Immunoglobulin heavy chain (IgH) mini-loci.

Untersuchung der Diversifikation von Humanisierten Antikörper aus Kaninchen und Mäuse mittels transgene Immunglobulin Schwere kette (IgH) mini-loci.

München, Technische Universität, Fakultät Wissenschaftszentrum Weihenstephan, Diss., 2007, 108 S.
Verlagsversion Volltext
Somatic mutation of rearranged immunoglobulin genes (IgH and IgL) is a key process because it leads to the diversification of the antibody chains, a vital progression in the affinity maturation process whereby antibody-antigen binding is enhanced. Two different mechanisms of somatic mutation have been described and attributed to different species: somatic hypermutaion (in man and mouse) occurs by fixation of individual non-templated nucleotide substitutions, whereas gene conversion (in rabbits, sheep, birds and cattle) occurs by templated substitutions with sequences donated by upstream pseudogenes to the rearranged IgV gene segment. These two processes have been described to be alternative methods employed in revamping a lesion caused by the protein Activation Induced Deanimase (AID) in the rearranged IgV gene segment. The decision which method is thereby used depends on interplay of cis (Ig promoter and enhancer) and trans (genes involved in homologous recombination) elements. In this work a transgenic vector is created using a novel approach, whereby the coding sequences of a rabbit IgH locus are substituted with their human counterparts. In the first part of the study, this transgenic vector is used to generated transgenic mice, and somatic diversification of the humanized antibodies investigated; the question posed being what would be the result of the interaction between rabbit ( a gene converting animal) cis regulatory elements (on the transgenic vector) and the trans elements of the mouse host ( a hypermutating animal)? The results advocate a species specific activity of the Ig cis and trans elements as no somatic hypermutation was observed, albeit successful rearrangement and employment of the translocus. In the second part of the work, a second humanized rabbit transgenic vector was used to generate transgenic rabbits and mice and the somatic diversification of the humanized antibodies investigated. Mice and rabbits transgenic for the same locus proffer a much comprehensible comparison of somatic hypermutation in these animals. The question to be answered in this second part was would the humanized antibodies undergo somatic hypermutation in the transgenic mice and gene conversion in the transgenic rabbits? The results give credit to the hypothesis from the first part: the humanized antibodies from the transgenic mice showed no somatic hypermutation, while those from the transgenic rabbits did undergo effective gene conversion. In total these results argue for a species specific interaction of Ig cis and trans regulatory elements in determining the method of somatic mutation employed.
Unter der somatischen Mutation versteht man das Einfügen von Mutationen in die V-Region von Antikörpergenen einer reifenden B-Zelle. Die Hypermutation kommt bei Mensch und Maus vor und die Genkonversion wird Kaninchen, Schaf, Vogel und Rind zugeordnet. In der vorliegenden Arbeit wurde einen IgH-Mini-Lokus mit humanisierten Genen hergestellt, die noch die Regulationselemente des Kaninchens enthalten. B-Zellen aus transgenen Mäusen und Kaninchen wurden analysiert. In den transgenen Mäusen konnte keine somatische Hypermutation nachgewiesen werden. Die transgenen Kaninchen wiesen Genkonversion auf, was vermutlich auf die kaninchenspezifischen Regulationselemente zurückzuführen ist.
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Publikationstyp Sonstiges: Hochschulschrift
Typ der Hochschulschrift Dissertationsschrift
Schlagwörter Antibody; Gene conversion; somatic hypermutation; AID; Antikörper; Genkonversion; Somatische Hypermutation
Quellenangaben Band: , Heft: , Seiten: 108 S. Artikelnummer: , Supplement: ,
Hochschule Technische Universität
Hochschulort München
Fakultät Fakultät Wissenschaftszentrum Weihenstephan