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Divisions of
*
Biosignaling and
Molecular Biology, Research Institute for Biological Sciences, Science University of Tokyo, Chiba, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (11, 12, 13, 14, 15, 16), 
-chain
(17), and H chain (18, 19, 20)-transgenic mice.
In a previous study, Azuma et al. (21) prepared transgenic
mice carrying the chloramphenicol acetyltransferase (CAT)
gene, which was driven by a promoter from
VH17.2.25 (22, 23) and
the intron enhancer (Eµ; Refs. 24 and
25)/matrix attachment region (MAR; Ref. 26).
Somatic hypermutation was detected in CAT but not in the
VH promoter or Eµ/MAR flanking it, although the
frequency of mutation was approximately 1/10 that observed in
endogenous
VH-D-JH,
suggesting that these cis-acting elements were critical for
the induction of hypermutation and that other components such as
C
, C
, or the enhancer flanking 3' of C
(3'E) (27, 28, 29) might be responsible for high-frequency
somatic hypermutation (19, 20, 30). To identify the component(s) important in raising the frequency of hypermutation in the IgH gene, we used a RAG-2-deficient (RAG-2-/-) blastocyst complementation system developed by Chen et al. (31). A series of transgene constructs that differed only in the 3' region flanking Cµ were transfected into embryonic stem (ES) cells that were microinjected into RAG-2-/- blastocysts. The chimeric mice obtained with this system were immunized with a TD Ag. Analyses of the frequency of somatic hypermutation in the VH-D-JH region of transgenes revealed a pivotal role for the DNase I-sensitive (HS) regions 3b and/or HS4 (32, 33, 34) in the induction of high-frequency somatic hypermutation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RAG-2-/- mice (35) with a BALB/c background were obtained from Dr. Y. Shinkai (Kyoto University, Kyoto, Japan) and maintained at this institute. Females 4–12 wk old were used as blastocyst donors. C57BL/6 and BALB/c mice were obtained from the Tokyo Animal Center (Tokyo, Japan).
Construction of IgH transgenes
IgH transgenes carrying human Cµ with different 3'-flanking regions were constructed. A fragment containing the VH17.2.25 promoter (0.55 kb) with KpnI and ApaI sites and another containing Eµ/MAR (1 kb) with XhoI and SalI sites were cloned by PCR from the construct used previously to create CAT-transgenic mice (21). The rearranged VH-D-JH gene (2.0 kb) containing ApaI and XhoI sites also was cloned by PCR with genomic DNA from A6, a hybridoma producing anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) mAb obtained from a C57BL/6 mouse (9, 10). These were cloned into Bluescript SKII. A 6.9-kb XbaI fragment of the human Cµ gene was obtained from a phage clone, CH.H.Igµ-24 (Health Science Research Resources Bank, Japan; Ref. 36). A plasmid containing an XbaI fragment (4.0 kb) of 3'E was obtained from Drs. J. Manis and F. Alt (Harvard Medical School, Boston, MA). Fragments containing HS3b (1.2 kb) and HS4 (1.4 kb) were cloned by PCR with genomic DNA from MPC 11 (32) and the following primers: HS3-S, 5'-TCTAGAACCACATGCGATCTAAGGGATATTGGGG-3'; HS3-anti SpeI, 5'-CAGGACTAGTGATCATTGAGCTCCGGCTCTAAC-3'; HS4-S XbaI, 5'-CTAGTCTAGACTGCAGACTCACTGTTCACCATG-3'; and HS4-anti SpeI, 5'-GTGGACTAGTAAGCTTGGAGTTAGGTGGGTAGG-3', in which the enzyme sites are underlined. These fragments were inserted at the 3' end of 3'E.
Transfection of IgH genes into J558L cells
Linearized IgH gene constructs (30 µg) with NotI
were electroporated into J558L cells with the pSV2-gpt (1.5 µg)
vector, and transfected cells were cultivated in the presence of a
mixture of hypoxanthine, xanthine, and mycophenolic acid
(37). The drug-resistant IgH gene transfectants were
subjected to limiting dilution, and clones were selected on the basis
of Ab production. For the quantitative analysis of Ab production,
several clones of transfectants derived from each transgene construct
were cultured at 2 x 106 cells/ml for
12 h in a total volume of 200 µl IMDM containing 10% FCS in
flat-bottom 96-well trays. The supernatants of various dilutions with
PBS containing 1 mg/ml BSA were analyzed by ELISA with polyvinyl plates
coated with NP9-BSA. Peroxidase (POD)-labeled
anti-mouse -chain Abs (Southern Biotech, Birmingham, AL)
or anti-human µ-chain Abs (Zymed, San Francisco, CA) were used
for detection of bound Abs.
Generation of chimeric mice
The linearized IgH transgene constructs (30 µg) as well as the
EcoRI fragment of pGKneo (1.5 kb, 1 µg) were
electroporated into 1 x 107 E14 cells
(38) with a Gene Pulser (Bio-Rad, Richmond, CA). The
transfected cells then were selected with G418 (150 µg/ml). Colonies
were screened by Southern blotting with the human Cµ gene as a probe
and by PCR using the primers, 5'-GACTCAGGAGGACTCTAGTT-3' and
5'-GGTGTCCCTAGTCCTTCATG-3', which hybridized to DNA sequences
located in the VH17.2.25 promoter
and JH2, respectively. PCR
amplification was performed for 30 cycles of 95°C for 30 s,
65°C for 30 s, and 72°C for 1 min. Copy numbers of integrated
pvehc3'E was estimated by Southern blotting. The copy number of the
other transgenes, pvehc
3'E and pvehc3'EHS3b/4, were estimated by PCR
analysis with DNA from pvehc3'E-transfected ES cells as a standard.
ES cell clones containing the IgH transgene were injected into blastocysts from RAG-2-/- mice (35) and transplanted into uteri of ICR foster mothers. The complementation of the immune system by B and T cells that originated from ES cells in chimeric mice was examined by flow cytometry after staining cells with PE-anti-CD3 Abs or biotin-anti-B220/streptavidin-FITC. The amount of IgM in the sera of chimeric mice was measured by ELISA with plates coated with rat anti-mouse IgM mAb. POD-labeled goat anti-mouse µ-chain Abs were used for detection of bound Abs. For analysis of Abs bearing human Cµ, plates were coated with goat anti-human IgM Ab (Southern Biotech), and POD-labeled anti-human IgM mAb (Zymed) was used for detecting bound Abs. Culture supernatants of J558L transfectants, a human Waldenstrom IgM, and anti-NP IgM mAb, B4-3, were used as control Abs.
Ags, immunization, and Ab production
NP34-chicken -globulin
(NP34-CGG) and NP-BSA with a different NP valence
were prepared as described previously (39). Mice were
administered NP34-CGG (100 µg/mouse) in CFA and
were boosted with the same amount in IFA. Three days after the final
administration of NP34-CGG in PBS, antisera were
collected from the immunized mice. Mice then were sacrificed, and
tissue samples were obtained. For measuring anti-NP Ab production
and affinity maturation of these Abs, polyvinyl plates coated with
NP1-BSA or NP16-BSA were
used. POD-conjugated goat anti-mouse IgG was used for
detection of bound Abs. Of the anti-NP mAbs, F8 was used as a
control for immature Ab and C6 for a maturated mAb control (9, 10).
Flow cytometry
Single-cell suspensions from spleens of NP34-CGG-immunized chimeric mice were depleted of erythrocytes by treatment with 0.83% NH4Cl. In some experiments, T cells also were depleted by treatment with anti-Thy 1 Abs (T24/40 and HO13.4), followed by treatment with rabbit complement. Cells were stained with PE-conjugated anti-CD45R/B220, or FITC-conjugated anti-mouse IgM. The peritoneal cells were stained with biotin-conjugated anti-CD5/FITC-conjugated streptavidin and PE-conjugated anti-CD45R(B220). The CD45R(B220)+IgM-, CD45R(B220)+IgM+, or CD5+CD45(B220)+ cells were fractionated by flow cytometry on a FACSVantage. For control experiments, CD4+ and/or CD8+ cells were obtained from thymus after staining and sorting.
RT-PCR and nucleotide sequence analysis
Total RNA was prepared from cytometrically fractionated spleen
or peritoneal cells using TRIzol (Life Technologies, Rockville, MD)
according to the manufacturer’s instructions. cDNA was prepared from
total RNA using oligo(dT) as a primer and Superscript II reverse
transcriptase (Life Technologies). Each cDNA sample was PCR-amplified
with the VH186.2 primer,
5'-CATGCTCTTCTTGGCAGCAAC-3', and the human Cµ primer,
5'-GCAGCCAACGGCCACGCTGC-3' for transgenes and the
VH186.2 primer and the mouse C1 primer,
5'-GGCCGAATTCCATGGAGTTAGTTTGGG-3', for endogenous mouse Ig genes,
respectively. The reaction protocol consisted of 30 cycles of 95°C
for 1 min, 62°C for1 min, and 72°C for 1 min. PCR products of
transgenes and endogenous IgH genes were analyzed by agarose gel
electrophoresis or ligated into pCR-2.1 with a TA cloning kit (Takara
Shuzo, Kyoto, Japan) and sequenced with M13 (-20) and M13 reverse
primers with a DNA sequencer model 5500 (Hitachi, Tokyo, Japan).
The frequency of mutation was calculated by dividing the total number of mutations by the total number of base pairs sequenced.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The transgene constructs used in this work contain the V region, which is encoded by mouse VH186.2, the dominant VH involved in the response to NP haptens (2). The rearranged mouse VH186.2-DFL16.1-JH2 gene from A6, an anti-NP hybridoma (9, 10), was linked to human Cµ, thereby allowing for easy discrimination between transgene-encoded and endogenous mouse H chains. In addition, the transmembrane exon was removed from Cµ to prevent expression on the cell surface, which might cause skewed B cell development. Transgenes contained only Cµ but not those encoding other H chain isotypes.
All transgene constructs contained, as cis-acting elements,
the VH promoter and Eµ/MAR from the mouse
J-Cµ intron, both of which had been used for CAT transgene
construction (21). A VH
promoter sequence (550 bp) from
VH17.2.25 (22), which
was shorter than that of the CAT transgene (2 kb), was used
in the present experiment. Therefore, differences in the structure of
the transgenes were restricted to the 3'-flanking region (Fig. 1
). One of the constructs, pvehc3'E, lacked the entire untranslated 3'
region and was driven by the VH promoter and
Eµ/MAR. Another construct, pvehc3'E, contained 3'E (29)
in addition to the VH promoter and Eµ/MAR. The
BamHI fragment (4 kb) containing 3'E was used for the gene
construction. The addition of HS3b and HS4 to pvehc3'E gave rise to a
construct referred to as pvehc3'EHS3b/4. HS3b (1.2 kb) and HS4 (1.4 kb)
were obtained from genomic DNA of a B cell lymphoma, MPC11
(32), after amplifying by PCR. 3'E, HS3b, and HS4 were
linked in tandem without any intervening Ig intron sequences.
Endogenous IgH genes contained an additional cis-acting
element, referred to as HS3a (40), with identical
nucleotide sequences to those of HS3b, but this element was in an
inverted form (34). Our transgene constructs did not
contain this cis-acting element.
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Transgene constructs were designed to encode chimeric H chains
possessing the VH region from a mouse anti-NP
mAb and the C region from human Ig, which were expected to assemble
with mouse 1-chains and give rise to
anti-NP Abs. To estimate their transcriptional activity of these
constructs, they were transfected into a mouse myeloma cell line,
J558L, and anti-NP Ab production was measured by ELISA (Fig. 2). All constructs were actively translated in J558L cells and produced
anti-NP Abs by pairing with endogenous
1-chains. Among these constructs, pvehc3'E
showed rather weaker production compared with the others. The addition
of 3'E (pvehc3'E) resulted in a significant increase in Ab production.
However, further addition of HS3b and HS4 seemed to have no effect.
Although little information was available concerning the dependence of
transcription on the copy number of each transfected DNA, we assumed
that the amount of Ab production reflected the transcriptional activity
of the constructs, which did not differ significantly between pvehc3'E
and pvehc3'EHS3b/4.
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To eliminate a possible effect from the positioning of the
integrated transgenes as well as random mouse-to-mouse variation, we
used at least two independently transfected ES clones for
microinjection into the RAG-2-/- blastocysts to
generate chimeric mice. We also used at least two chimeric mice per
transgene construct for analyses of somatic hypermutation. The
transfected ES cell lines and chimeric mice used in this experiment are
shown in Table I.
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A).
However, we were unable to detect Abs bearing human Cµ
encoded by transgenes in preimmune or immune sera of mice immunized
with NP34-CGG (Fig. 3B). As will be
presented elsewhere (A. Terauchi and T. Azuma, manuscript in
preparation), hybridomas producing anti-NP mAbs (11)
were prepared from the pvehc3'EHS3/4 mice immunized with
NP34-CGG. None of these secreted Abs bearing
human Cµ, although some of them synthesized intracellular H chains
having human Cµ. Therefore, it was suggested that the transgenes were
transcribed and translated in B cells of the chimeric mice. However,
the H chain products secreted were under detectable levels. Next, the
Ab response of chimeric mice to NP34-CGG, was
examined by ELISA. Although titers varied, all mice produced
anti-NP IgG Abs, indicating that their immune systems were
responsible for the production of anti-NP Abs (Fig. 3C).
We also examined the ratio of binding of these Abs to
NP1-BSA relative to binding to
NP16-BSA as a measure of affinity maturation. As
shown in Fig. 3D, for a control mAb, F8, which showed no
somatic hypermutation and has an association constant
(Ka) to NP-Cap of 2 x
105 M-1, the ratio was
0.29. In contrast, C6, a well-maturated mAb with a
Ka of 2 x
107 M-1 (10),
had the ratio of around 1. Because all antisera from the chimeric mice
showed ratios similar to that of C6, affinity maturation of anti-NP
Abs proceeded to a similar extent in all chimeric mice after
immunization with NP34-CGG.
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Although immune systems were reconstituted in the chimeric mice,
it was rather difficult to obtain a sufficient number of
PNAhighIgG+ B cells,
known to be germinal center (GC) cells (41), from a single
chimeric mouse. Therefore, we sorted
IgM-B220+ cells among
which we expected to select isotype-switched memory B cells by flow
cytometry with a FACSVantage (BD Biosciences, San Jose, CA)
after immunization with NP34-CGG. Somatic
hypermutation in transgenes was examined by cloning and sequencing of
cDNA prepared by RT-PCR with primers hybridizing to either
VH186.2 or human Cµ. Specific
amplification of transgenes by use of this combination of primers was
confirmed by the facts that PCR products only were observed in spleen
cells from chimeric mice and not in those from C57BL/6 mice immunized
with NP34-CGG (Fig. 4), and that all sequences had identical junctional diversity in CDR3,
which is characteristic of the A6 DNA used for constructing transgenes
(Figs. 5 and 6). Mouse-to-mouse variation in the RT-PCR products of endogenous Ig
genes (Fig. 4) was explained in terms of cross hybridization of the
VH186.2 primer to
VH186.2-related
VH genes, because the
VH186.2 gene was absent in germline
genes of 129/O1a mice (Igha) from which the ES
cells were derived.
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3'E (Fig. 5
A). The frequency of mutation was
estimated to be 0.17% (Table II),
which was in agreement with that observed in
CAT-transgenic mice and confirmed the previous conclusion
that the VH promoter and/or Eµ/MAR were
responsible for the induction of somatic hypermutation
(21). In contrast, we were unable to detect any nucleotide
changes in transgenes from
CD4+/CD8+ thymus cells,
suggesting that the somatic hypermutation machinery did not operate in
these cells and that nucleotide changes attributable to PCR error or to
mutation during generation of chimeric mice were negligible under the
experimental conditions used (data not shown).
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B), although the frequency increased
slightly compared with that of pvehc3'E (Table II). However, it was
still lower than that observed in endogenous
VH genes, suggesting that 3'E did not
contribute significantly to induction of somatic hypermutation.
The addition of HS3b/4 to pvehc3'E resulted in a dramatic increase in
the frequency of somatic hypermutation in
VH186.2-DFL16.1-JH2
genes from the transgene construct pvehc3'EHS3b/4. All PCR clones
sequenced contained nucleotide changes despite the fact that they were
selected randomly. These changes were found with high frequency in the
area around CDR2 and CDR3 (Fig. 6 and Table II).
The distribution and frequency of somatic hypermutation among the
transgene constructs in spleen B cells are compared in Fig. 7. The data obtained from two chimeric mice corresponding to each
transgene construct are shown in this figure and are in good agreement
with each other. A high frequency of hypermutation was apparent in
pvehc3'EHS3b/4. However, mutation was clearly absent in
peritoneal B1 cells from the same chimeric mouse (Fig. 7B).
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shows the nature of substitutions identified in the transgenes. A
comparison of IgH transgenes reveals that transitions and transversions
occur at approximately equal frequency. We also analyzed the occurrence
of somatic hypermutation in the VH186.2
transgene at the location of the sequence referred to as RGYW motif
(A/G, G, C/T, A/T) (42, 43). The germline
VH186.2 gene (294 bp) contains the
RGYW/WRCY motif (93 bp), which corresponds to 32% of the total bp. The
frequency of somatic hypermutation in this motif was found to be 34%,
which would not be significantly higher than that of the other
VH regions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To eliminate the possibility of a
positional effect from the placement of integrated transgenes and
mouse-to-mouse variation, we used at least two chimeric mice per
transgene construct with ES cell transfectants prepared by independent
electroporation. Complementation of the
RAG-2-/- mouse immune system with that of ES
cells was confirmed by the appearance of lymphocytes in peripheral
blood and production of Ab to NP34-CGG. The
cooperation of T and B cells in the immune response to
NP34-CGG was expected to proceed normally,
because affinity maturation of endogenous anti-NP Abs occurred
without any significant difference between mice carrying pvehc3'E and
those carrying pvehc3'EHS3b/4 (Fig. 3C).
To elucidate the mechanisms responsible for somatic hypermutation, it
is essential to identify the cis-acting elements responsible
for its induction (44). Experiments with transgenic mice
carrying -chain or H chain transgenes revealed that Eµ/MAR is
critical for induction of hypermutation (14, 19). IgH gene
exons are not required for the induction, as we previously showed with
transgenic mice carrying the CAT gene driven by the
VH promoter and Eµ/MAR. However, the
rather low frequency of hypermutation in CAT-transgenic mice
suggested that other components are important in raising the frequency
of hypermutation (21). Johnston et al. (45)
created transgenic mice carrying, in addition to Eµ, a 727-bp
fragment of 3'E from Wistar rat genomic DNA. However, the transgene did
not induce a high frequency of hypermutation.
We prepared chimeric mice carrying IgH transgenes with different
cis-acting elements and examined somatic hypermutation after
immunization with NP34-CGG. We used at least two
chimeric mice per transgene and obtained essentially similar results,
suggesting that mouse-to-mouse variation or effects of positioning
of integration sites of transgenes were negligible in our case. In a
prototype transgene, pvehc3'E, we found only a marginal number of
hypermutations and confirmed previous findings that the
cis-acting elements, VH promoter and
Eµ/MAR, were not sufficient for induction of a high frequency of
hypermutation. Addition of a 3'E fragment (4 kb) to pvehc3'E
resulted in an 
30% increase in the hypermutation frequency (Table II). This may be related to an enhanced transcription activity of
pvehc3'E compared with that of pvehc3'E in B cells as observed with
J558L cells (Fig. 2). However, the degree of hypermutation was still
low compared with that of endogenous VH
genes (46, 47). Finally, we introduced HS3b and HS4 to the
3' end of 3'E and found that they greatly enhanced the frequency of
hypermutation. Although we did not determine whether both HS3b and HS4
contributed to the rise in frequency, it is clear that the motifs
responsible for high frequency hypermutation are within an HS3b/4
region of 2.6 kb.
The basic strategy for inducing somatic hypermutation is likely to be
similar for -chain and H chain genes (13, 14, 16, 19, 21, 48, 49). Both genes required an Eµ/MAR as a critical element. The
VH promoter and cis-acting elements
located at the 3' end of C genes were thought to be important for
increasing the hypermutation frequency. With transgenic mice carrying
mutant -chain transgenes, Betz et al. (14) showed that
3'E helped to enhance the frequency of hypermutation. However,
recent experiments with mice lacking 3'E showed that it was not
involved in the induction of hypermutation of -chain genes
(50). The discrepancy between the results of these two
experiments may have arisen from a difference in the size of 3'E
used. Betz et al. (14) used a 1.2-kb fragment to construct
transgenes, and Gorman et al. (51) deleted 808 bp from
3'E. Therefore, the element responsible for a high frequency of
hypermutation may exist outside of 3'E (808 bp) but instead in the
1.2-kb fragment made up of -chain transgenes. Our present findings
suggested that HS3b/4 but not 3'E (HS1, 2) contributed to the
significant enhancement of the hypermutation frequency in H chain
transgenes, although we have no direct evidence of whether HS3b/4 alone
without 3'E was capable of inducing a high frequency of somatic
hypermutation. The validity of our prediction currently is being
examined by preparing chimeric mice carrying either HS3b or
HS4.
3'HS consisting of HS3a, HS1,2, HS3b, and HS4, acts as a locus control
region (32, 52, 53, 54). 3'E corresponds to HS1,2 and was
shown to be an active transcriptional enhancer in B-lineage cells
(47, 55, 56, 57). In fact, pvehc3'E displayed higher
transcriptional activity than pvehc3'E in J558L cells, suggesting
that 3'E augmented the Eµ/MAR effect. However, it is unlikely that
HS3b/4 also acted as enhancer elements in J558L cells because a further
increase in activity was not observed with the addition of HS3b/4 to
pvehc3'E. This transcriptional activity of mutant transgene constructs
in plasma cell lines may not be suitable for measuring the capability
of cis-acting elements to induce hypermutation because
hypermutation occurs only at certain stages of B cell development, such
as the point at which centroblasts appear in the GC
(58, 59, 60). Information on factors that interact with HS3b/4
at GC stage of B cell development may provide further insight into the
mechanism of somatic hypermutation in H chain genes.
It was shown previously that µ-chain genes exhibit a lower frequency
of somatic hypermutation than do their 2a or 2b counterparts
(19). However, our transgene was observed to have a high
degree of somatic hypermutation despite the fact that it contains human
Cµ and that there is a lack of evidence of class-switching between
transgenes and endogenous mouse IgC genes. This high frequency is
likely to be related to the distance between the
VH promoter/Eµ and HS regions. In endogenous Ig
µ-chain genes, the distance was 200 kb, whereas it was less
than 20 kb in the case of pvehc3'EHS3b/4 because 3'E and HS3b/4
directly flanked 3' of Cµ, similar to the situation in class-switched
endogenous Ig genes (32). It was expected that
such a short distance between the VH
promoter/Eµ and HS regions enabled pvehc3'EHS3b/4 to induce a high
frequency of somatic hypermutation.
Because our transgenes lacked membrane exons, they were unable to be
expressed on the B cell surface and to be subjected to Ag selection.
Therefore, results obtained from the transgenic mice revealed the
distribution and frequency of somatic hypermutation without Ag
selection. Hypermutation in pvehc3'EHS3b/4 was frequent around CDR2 and
3 but was less so in CDR1. This is in contrast to the distribution in
endogenous VH186.2, in which somatic
hypermutation accumulated around CDR1 and CDR2 (7, 61, 62). A particularly high frequency of mutation found at position
33 corresponding to the codon TGG has been shown to result from Ag
selection (7) because a nucleotide change from TGG to TTG
gives rise to the replacement of Trp with Leu, accompanied by a 10-fold
increase in affinity. Therefore, the fact that no mutation occurred in
pvehc3'EHS3b/4 at position 33 clearly was explained in terms of the
absence of Ag selection. A high frequency of mutation around CDR3 also
was evident in the transgene. Because CDR3 is at the junction of
VH, D, and
JH segments, complex relationships among
nucleotide sequences in this site, resulting in the addition or
deletion of nucleotides, makes the assignment of somatic hypermutation
difficult, and little information on its frequency in CDR3 was gained
from the analysis of endogenous Ig genes. The results obtained with the
transgene clearly showed that CDR3 is the hypermutation target. As for
the nature of the nucleotide substitutions, transitions and
transversions occur with approximately equal frequency (Table III). In
addition, we were unable to detect a preferential occurrence of somatic
hypermutation in the RGYW motif of the
VH186.2 transgene (data not
shown). We have no clear explanation for the discrepancy between
our finding and those of other investigators (42, 43). We
are currently examining the relationship between the positions of
double-strand breaks and somatic hypermutation with
pve3'EHS3b/4-transgenic lines.
We were unable to detect Abs harboring transgene-encoded µ-chains in
chimeric mice despite the fact that the transgene-encoded anti-NP
Abs were secreted by the transfected J558L cells. Because
transgene-encoded µ-chains were found in the cytoplasm of some
hybridomas, it was anticipated that they were synthesized but not
secreted by Ab-forming cells (AFCs) in chimeric mice (A. Terauchi and
T. Azuma, manuscript in preparation). It is likely that the
transgene products consisting of mouse VH domains
and human Cµ domains are inferior to mouse H chains in assembly with
mouse - or -chains. Therefore, in the absence of competitor H
chains, transgene-encoded H chains were able to assemble with mouse
1-chains, as seen in J558L cells, but not in
the presence of endogenous mouse H chains, as are found in AFCs
of chimeric mice. The inability of transgene-encoded -chains
possessing rat C regions to combine with mouse H chains in the
presence of mouse -chains was shown previously (13),
although the frequency of somatic hypermutation was not influenced by
expression at the protein level (13, 63, 64, 65).
As a result of this study, we are now able to define the minimal elemental unit necessary for the somatic hypermutation of IgH chain genes. The germline transmission of the transgene, pvehc3'EHS3b/4, made it possible to establish a transgenic mouse line carrying this gene. This model will be useful for gaining further insight into the mechanism of somatic hypermutation.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Geriatrics, National Institute for Longevity Science, 36-3 Gengo, Obu, Aichi 474-8522, Japan.
3 Address correspondence and reprint requests to Dr. Takachika Azuma, Division of Biosignaling, Research Institute for Biological Sciences, Science University of Tokyo, Yamazaki 2669, Noda, Chiba 278-0022, Japan. E-mail address: tazuma{at}rs.noda.sut.ac.jp
4 Abbreviations used in this paper: TD, T cell dependent; CAT, chloramphenicol acetyltransferase; Eµ, J-C intron enhancer; MAR, matrix attachment region; 3'E, 3' enhancer; ES, embryonic stem; NP, (4-hydroxy-3-nitrophenyl)acetyl; POD, peroxidase; CGG, chicken -globulin; GC, germinal center; HS, DNase I-sensitive.
Received for publication April 5, 2000. Accepted for publication May 1, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hs4, a novel Ig heavy chain enhancer element regulated at multiple stages of B cell differentiation. Nucleic Acids Res. 23:975. immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc. Natl. Acad. Sci. USA 81:7161. constant region. Mol. Cell Biol. 13:1547. heavy chain enhancer [3'E(hs1,2)] in Vh gene somatic hypermutation. Mol. Immunol. 34:367.[Medline]
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enhancer influences the ratio of Ig versus Ig B lymphocytes. Immunity 5:241.[Medline]
-heavy chain gene products. Mol. Cell. Biol. 6:1903.1 light chains. Proc. Natl. Acad. Sci. USA 88:7933.This article has been cited by other articles:
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