Cloning non-transformed sheep B cells

The capacity to clone B cells and establish permanent B cell lines has grea tly facilitated a wide variety of studies characterising the growth, differ entiation, and gene expression of murine and human B cells. Similar investi gations of B cell biology for other species have been severely restricted b y an inability to culture or clone B cells. This is the first report of a m ethod to clone non-transformed sheep B cells using a culture system based o n murine CD154 and a combination of human gamma chain-common cytokines. She ep Peyer's patch B cells were cultured for 120 days and then cloned by limi ting dilution culture. The parental B cell culture contained both surface i mmunoglobulin (sIg)M+ and sIgG1(+) B cells and both types of B cell were cl oned. Clonality was confirmed by PCR analysis of Ig heavy chain (HC) and li ght chain (LC) expression and DNA sequencing of HC V genes. There was agree ment between the PCR and flow cytometric analyses of HC isotype expression on the B cell clones but the available monoclonal antibodies specific for s heep lambda and kappa LC did not react with all clones. Soluble Ig was dete cted in the culture supernatant of sIgG1(+) clones but not slgM(+) clones. The B cell clones remained dependent upon CD154 and gamma chain-common cyto kine co-stimulation for sustained growth and maintained stable Ig expressio n. The cloning of non-transformed sheep B cells should provide a valuable t ool for studying sheep B cell biology, establishing Ig HC- and LC-specific monoclonal antibodies, analysing the B cell Ig repertoire, and may be used to produce sheep monoclonal antibodies. (C) 2000 Elsevier Science B.V. All rights reserved.