International Immunology, Vol 10, 1683-1692, Copyright © 1998 by Oxford University Press
KJ Seidl, A Bottaro, A Vo, J Zhang, L Davidson and FW Alt
Germline CH transcripts initiate from a promoter upstream of a non- coding
I exon, proceed through the switch (S) region and terminate downstream of
the associated CH exons. To elucidate the role of germline transcription in
Ig heavy chain class switch recombination (CSR), we used gene targeting in
embryonic stem (ES) cells to replace most of the Igamma2b exon from
immediately 3' of the majority of transcription initiation sites to beyond
its donor splice site with a PGK-neo(r)gene inserted in the same
transcriptional orientation as the endogenous unit. The mutation was
introduced into both alleles of ES cell lines (referred to as
gamma2-b(N/N)) and the neo(r) gene was deleted (referred to as Igamma2b-/-)
by the loxP/Cre method. These mutations were assayed for effects on CSR in
B cells derived via RAG-2- deficient blastocyst complementation.
Igamma2b-/- B cells lacked ability to switch to IgG2b both in vivo and in
vitro, and, correspondingly, showed no germline transcription through the
Igamma2b exon, Sgamma2b or the Cgamma2b region. In contrast, Igamma2b(N/N)
B cells switched at normal levels to IgG2b and showed substantial
transcription through the Sgamma2b and Cgamma2b regions. Taken together,
these results show that the Igamma2b sequences, per se, are not necessary
for mediating CSR since a transcribed PGK-neo(r) gene can replace its
function. However, the deleted portion of the Igamma2b exon and splice
donor site apparently contain sequences necessary for efficient germline
gene transcription and thus for CSR to IgG2b.
ARTICLES
An expressed neo(r) cassette provides required functions of the 1gamma2b exon for class switching
Howard Hughes Medical Institute and The Children's Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
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