Cloning non-transformed sheep B cells.
The capacity to clone B cells and establish permanent B cell lines has greatly facilitated a wide variety of studies characterising the growth, differentiation, and gene expression of murine and human B cells. Similar investigations of B cell biology for other species have been severely restricted by an inability to culture or clone B cells. This is the first report of a method to clone non-transformed sheep B cells using a culture system based on murine CD154 and a combination of human gamma chain-common cytokines. Sheep Peyer's patch B cells were cultured for 120 days and then cloned by limiting dilution culture. The parental B cell culture contained both surface immunoglobulin (sIg)M(+) and sIgG1(+) B cells and both types of B cell were cloned. Clonality was confirmed by PCR analysis of Ig heavy chain (HC) and light chain (LC) expression and DNA sequencing of HC V genes. There was agreement between the PCR and flow cytometric analyses of HC isotype expression on the B cell clones but the available monoclonal antibodies specific for sheep lambda and kappa LC did not react with all clones. Soluble Ig was detected in the culture supernatant of sIgG1(+) clones but not sIgM(+) clones. The B cell clones remained dependent upon CD154 and gamma chain-common cytokine co-stimulation for sustained growth and maintained stable Ig expression. The cloning of non-transformed sheep B cells should provide a valuable tool 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.
Griebel
P J
PJ
Veterinary Infectious Disease Organization, 120 Veterinary road, Saskatoon, Canada. greibelp@sask.usask.ca
Beskorwayne
T
T
Godson
D L
DL
Popowych
Y
Y
Hein
W
W
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods*; D002999:Clone Cells; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D005803:Genes, Immunoglobulin; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007075:Immunoglobulin M / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010581:Peyer's Patches / Q000166:cytology / Q000276:immunology; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D012756:Sheep / Q000235:genetics / Q000276:immunology*
D017931:DNA Primers; D018076:DNA, Complementary; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011947:Receptors, Antigen, B-Cell