Cloning of a gamma 2b gene encoding anti-Pseudomonas aeruginosa H chains and its introduction into the germ line of mice.
A complete, functional gamma 2b gene (pVCM) was cloned from a mouse hybridoma (VD93) with antibody activity to Pseudomonas aeruginosa. DNA sequencing of the VDJ region of pVCM determined that the VH gene was a member of the J558 family rearranged to JH2. Upon transfection into myeloma cells the gamma 2b gene gave rise to high levels of gamma 2b mRNA and gamma 2b protein. The gamma 2b protein had the same IEF pattern as the parent hybridoma protein VD93 and the antibodies formed from a combination of the pVCM gamma 2b chains and the myeloma lambda-chains bound weakly to P. aeruginosa. However, the hybrid antibodies did not discriminate between the serotypes 2 and 3, whereas the parent protein was specific for serotype 3. Transgenic mice were produced with the pVCM gamma 2b gene which expressed the gamma 2b mRNA (both membrane and secreted forms) only in lymphoid organs. However, contrary to expectations, the gamma 2b mRNA levels were higher in T cells than in B cells in three different transgenic lines. The serum of the transgenic mice had no activity to P. aeruginosa indicating the importance of L chains for the conformation of the Ag binding site. These gamma 2b transgenic mice provide a convenient tool for the study of feedback inhibition of Ig gene rearrangement.
Tsang
H
H
Department of Molecular Genetics and Cell Biology, University of Chicago, IL 60637.
Pinkert
C
C
Hagman
J
J
Lostrum
M
M
Brinster
R L
RL
Storb
U
U
D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D003001:Cloning, Molecular; D005803:Genes, Immunoglobulin; D005854:Germ Cells / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000302:isolation & purification; D007142:Immunoglobulin gamma-Chains / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D011550:Pseudomonas aeruginosa / Q000276:immunology*; D012313:RNA / Q000235:genetics; D014162:Transfection
D000907:Antibodies, Bacterial; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007142:Immunoglobulin gamma-Chains; D012313:RNA