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A Targeted Deletion of a Region Upstream from the J Cluster Impairs Chain Rearrangement In Cis in Mice and in the 103/bcl2 Cell Line

Laurentiu Cocea^*, Annie De Smet^*, Mahasti Saghatchian^*, Simon Fillatreau^*, Laurent Ferradini^*, Stéphane Schurmans, Jean-Claude Weill^*, and Claude-Agnès Reynaud^*

From the ^* Institut National de la Santé et de la Recherche Médicale, Unité 373, Faculté de Médecine Necker, Paris, France; and Institut de Recherches Interdisciplinaires en Biologie Humaine et Nucleare (IRIBHN), Faculté de Médecine, Université Libre de Bruxelles, Bruxelles, Belgium

We have shown previously that a mutation of the KI-KII site immediately 5' to J₁ on the mouse immunoglobulin light chain locus reduces the rearrangement level in cis, although it does not affect transcription. Here we deleted by homologous recombination in mouse embryonic stem cells a 4-kb DNA fragment, located immediately upstream of the KI-KII element, which contains the promoter of the long germline transcript. Analysis of gene-targeted heterozygous mouse splenic B cells showed a strong decrease in rearrangement for the allele bearing the deletion. When both the KI-KII mutation and the 4-kb deletion were present on the same allele, the overall reduction in rearrangement was stronger than with the 4-kb deletion alone underlying the role of these two elements in the regulation of rearrangement. The same deletion was performed by homologous recombination on one allele of the rearrangement- inducible mouse 103/bcl2-hygro^R pre-B cell line, and resulted in a similar reduction in the induction of rearrangement of the mutated allele. This result validates this cell line as an in vitro model for studying the incidence of gene-targeted modifications of the locus on the regulation of rearrangement.

Key Words: regulation of rearrangement • mouse immunoglobulin genes • allelic exclusion • germline promoter • positive regulatory element

L. Cocea was supported by a fellowship from La Fondation pour la Recherche Médicale and Le Rayon Vert, and S. Fillatreau was supported by École Normale Supérieure.

M. Saghatchian's present address is Département d'Anatomo-Pathologie, Institut Gustave Roussy, Villejuif, France, and Simon Fillatreau's is ICAPB, University of Edinburgh, Edinburgh, Scotland, UK.

¹ Abbreviations used in this paper: BCR, B cell receptor; ES, embryonic stem; RT, reverse transcription.

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