PCR-based analysis of the murine immunoglobulin heavy-chain repertoire.
The immunoglobulin heavy-chain repertoire has been mainly analysed by studying the proportion of genes belonging to each of the 14 described families, in terms of the expressed immunoglobulin molecules. Although the proportion of each variable gene family is kept stable throughout adult life and in different mice of the same strain, little information is available on the clonal representation in the repertoire of activated B cells. We describe here a new method that permits an approach to this question by separating the products of a polymerase chain reaction covering the VH-D-JH junction of the immunoglobulin heavy chain gene in a sequencing gel, thereby allowing discrimination of different rearrangements (according to their length) using a given JH and one of the member of a given VH family. Using this method, we show that it is possible to obtain a precise overview of the repertoire of activated B cells, at the mRNA level, as well as the potential repertoire, from a study of the DNA. We also show that this approach permits the detection of an induced immune B cell response and studies of emerging dominant specific B cell clones.
Delassus
S
S
INSERM U277, Département d'Immunologie, Institut Pasteur, Paris, France.
Gey
A
A
Darche
S
S
Cumano
A
A
Roth
C
C
Kourilsky
P
P
D000818:Animals; D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D007114:Immunization; D007143:Immunoglobulin Heavy Chains / Q000032:analysis* / Q000235:genetics; D007158:Immunologic Techniques; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007143:Immunoglobulin Heavy Chains; D012333:RNA, Messenger