Cloning of an immunoglobulin variable region gene from mouse embryo.

Abstract

A 4.8-kilobase DNA fragment carrying an immunoglobulin [Ig] gene coding for a mouse .lambda. chain variable region (V.lambda. gene) was enriched about 350-fold from a total endonuclease EcoRI digest of embryonic DNA by a combination of preparative agarose gel electrophoresis of double-stranded DNA and CsCl density gradient centrifugation of R-loops formed with a purified .lambda. chain mRNA. DNA fragments thus enriched for the Ig gene were inserted in vitro in the middle of the genome of the vector phage .lambda.gt Wam 403, Eam 100, Sam 100 by use of the EcoRI cohesive ends. Transfection of CaCl2-treated Escherichia coli 803 [rk-, mk- (lacking restriction and modification systems for K-12)] with such hybrid DNA and subsequent screening of about 4000 plaques by in situ hybridization with purified 125I-labeled .lambda. chain mRNA led to isolation of a clone that carries a V.lambda. gene (.lambda.gtWES-Ig 13). EM of R-loops confirmed the presence of sequences homologous to part of the .lambda. chain mRNA in its 5''-end.