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and 
Light Chain Yeast Artificial Chromosomes1
*
Laboratory of Developmental Immunology and
Laboratory of Molecular Recognition, The Babraham Institute, Babraham, Cambridge, United Kingdom; and
Laboratory of Molecular Biology, Medical Research Council, Cambridge, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and 
light chain transloci in a background in which the
endogenous IgH and loci have been inactivated. The B lymphocyte
population in these translocus mice is restored to about one-third of
normal levels, with preferential (3:1) expression of human over
human . Human IgM is found in the serum at levels between 50 and 400
µg/ml and is elevated following immunization. This primary human Ab
repertoire is sufficient to yield diverse Ag-specific responses as
judged by analysis of mAbs. The use of DH and J segments is
similar to that seen in human B cells, with an analogous pattern of N
nucleotide insertion. Maturation of the response is accompanied by
somatic hypermutation, which is particularly effective in the light
chain transloci. These mice therefore allow the production of
Ag-specific repertoires of both IgM, and IgM, Abs and should
prove useful for the production of human mAbs for clinical
use. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The alternative to these artificial libraries is to create transgenic
animals carrying human Ig loci in germline configuration in the
presence of defective endogenous Ig loci. The aim is that the
introduced human loci function instead of the mouse loci and are
rearranged and expressed to produce a fully human Ab repertoire that
can be exploited to yield human mAbs of desired specificities. Early
efforts using plasmid-based mini loci have revealed the feasibility of
the approach, although the structural diversity of the human Ab
repertoires produced is restricted owing to the limited number of
germline V gene segments included in these miniloci
(4, 5, 6, 7). More recent experiments using yeast artificial
chromosome (YAC)5
technology has demonstrated that large regions of human DNA can be
introduced into the mouse germline, allowing the production of human Ab
repertoires that derive from increased number of germline V gene
segments (8, 9, 10, 11, 12, 13). To date, such repertoires of wholly
human Abs are only composed of human IgH, Abs. However, in humans
(as opposed to mice) a large component of the functional Ab repertoire
is provided by the Ig locus, with Ig-containing Ab accounting for
almost half the serum Ig (14). Here we describe the
production of mice that carry YAC-based human IgH, human Ig, and
human Ig transloci in a background in which endogenous mouse IgH and
Ig chain expression has been inactivated. The introduced human
transloci function to yield a fully human Ab repertoire that can be
exploited to produce human mAbs of desired specificities.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The production of transgenic mice containing either the HuIg
YAC or the HuIg YAC has been described previously (9, 14). The HuIgH YAC (12, 13) was modified so as to
incorporate two copies of a neomycin resistance cassette in the
acentric YAC arm using homologous recombination in yeast as previously
described (15). The modified YAC was transferred into HM-1
ES cells (16) by protoplast fusion (15), and
a clone carrying a complete copy of the IgH YAC, determined by PCR and
Southern blot (data not shown), was used to derive mice following
injection into BALB/c blastocysts and implantation into foster animals
(8, 17). Mice with their endogenous H chain or L chain
loci rendered nonfunctional have been described previously. The
µMT-/- modification (18) inserts
a stop codon and neo cassette into the membrane exon of the IgM C
region, preventing the surface Ig expression during B cell development,
while the MoIg-/- modification
(19) disrupts Ig expression by the insertion of a neo
cassette in the C region. Transgenic animals were crossed with
µMT-/- and MoIg-/-
animals to produce mice expressing human IgM, Ab (referred to as
four-feature mice) or both IgM, and IgM, Ab (referred to as
five-feature mice). The transgenic status of the offspring was
confirmed by Southern hybridization of genomic DNA with probes for the
following: human IgM C region exons 1, 2, and 3; the human C
region; and the human 3 C region.
Analysis of expression of human Ab proteins
Flow cytometric analysis of translocus-derived Ig expression on
the surface of spleen cells was performed using standard techniques. B
lymphocytes were identified using B220-APC (PharMingen, San Diego, CA).
Human IgM was detected using anti-human Igµ-PE (PharMingen).
Human Ig was detected using biotinylated anti-Ig (Zymed,
South San Francisco, CA) followed by streptavidin-PerCP (Becton
Dickinson, Mountain View, CA), and human Ig was detected using
anti-Ig-FITC (Sigma-Aldrich, Poole, U.K.). Mouse Ig was
detected using anti-mouse Ig-FITC (PharMingen). Stained
cells were analyzed with a FACSCalibur flow cytometer (Becton
Dickinson). Data were collected on 100,000 events and were analyzed
using CellQuest software (Becton Dickinson). For sorting, Peyer’s
patches were isolated, and germinal center B cells were stained with
B220-APC and peanut agglutinin-FITC (Sigma-Aldrich). Up to 100,000
double-positive cells were isolated for RNA preparation followed by
RT-PCR of H chain and L chain rearrangements.
ELISAs to determine the serum concentrations of translocus-derived IgM,
Ig, and Ig Ig chains were performed as described previously
(4, 9, 14). Ig concentrations were standardized using
purified human IgM (Sigma-Aldrich) or purified rheumatoid factor Ab
prepared in the Laboratory for Molecular Recognition (Cambridge,
U.K.).
Immunization and preparation of hybridomas
Four- or five-feature mice were initially immunized with 50 µg of Ag in CFA and were boosted at 4 and 8 wk with 50 µg of Ag in IFA. A final boost was given at 12 wk, and 3 days later hybridomas were prepared by fusion of splenocytes with NS/0 myeloma cells using polyethylene glycol 1500. Fusion supernatants were screened for reactivity with the immunogen by ELISA, and selected colonies were expanded for further analysis. Human IgM expression levels and L chain use were determined by ELISA. The specificity of the hybridomas was confirmed by testing for cross-reactivity to unrelated Ags.
RT-PCR and sequencing of Ig rearrangements
Total cytoplasmic RNA was isolated from hybridoma cells or
sorted Peyer’s patch cells using Tripure reagent (Boehringer Mannheim
U.K., Lewes, U.K.), following the manufacturer’s instructions, and
reverse transcribed using oligo-(dT)22 and
Superscript II Reverse Transcriptase (Life Technologies, Paisley,
U.K.). The primers used for PCR amplification of rearranged Ig genes
are listed in Table I
. Rearranged human
Ig H chains were amplified using the family-specific leader primers and
the IgM Constant primer (20), with separate reactions set
up for each leader primer. Rearranged Ig genes were amplified using
the V primer and the C primer (21). Rearranged
genes were amplified using the V2 and V3 in separate reactions
with the C primer (14). The amplified rearrangements
were purified using the QIAquick Prep System and cloned into the pGem-T
vector (Promega U.K., Southampton, U.K.). Recombinant colonies were
screened for appropriate rearrangements by PCR using primers to the
respective framework 1 and framework 4 regions (Table I). Plasmid was
isolated and sequenced using standard M13 forward or reverse sequencing
primers.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The three different transloci are illustrated in Fig. 1. For the human heavy chain translocus,
we used a 240-kb YAC (HuIgH) that contains the core region of the human
IgH locus comprising five VH segments and the
complete DH and JH loci
linked to Cµ-
in correct germline configuration; the isolation and
characterization of this YAC have been previously described
(13). The HuIgH YAC was modified by insertion of a
neomycin resistance gene into the acentric YAC arm and introduced into
embryonic stem cells using the protoplast fusion technique as described
in Materials and Methods. Mice carrying the
HuIg and HuIg transloci have been previously described (9, 14). The 1.3-Mb HuIg YAC (9) contains a
complement of 103 V region gene segments comprising 20 repeats of
five V region genes obtained from the cosmid cos106
(23), attached to the core of the germline locus,
including three V regions, the complete J cluster, C, and the
deleting element. One of the repeated V region segments
(V2D-10) and one from the core region (V7-3) are pseudogenes,
making a complement of 82 functional V segments in the locus. The
410-kb HuIg YAC (24) contains 28 V region gene
segments (with 16 functional segments) and the seven paired J and
C segments (four of which are functional), in the correct germline
configuration.
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YAC and the Mo-/- knockout
(9). The remaining HuIg translocus was introduced by
crossing the four-feature mice with mice carrying the HuIg YAC and
the Mo-/- knockout (14), with
care to retain the µMT-/- knockout. The
strains were bred to carry two alleles of each of the transloci. Test
breeding showed that the three transloci and two knockouts were not
linked, indicating that the integration of the YACs was random and
independent. Cell surface and serum expression of human Igs
Flow cytometric analysis of spleen populations was conducted to
test whether the human transloci were capable of rescuing B cell
development in the µMT-/-,
Mo-/- background. The percentage of B
lymphocytes (B220-positive) in the spleens of nonimmunized four-feature
and five-feature mice maintained in barrier conditions ranged from
5–25%; representative animals are shown in Fig. 2. This is compared with spleens from
normal BALB/c mice, which contain 
40–45% B220-positive cells, and
µMT-/- mice, which contain <2%
B220-positive cells (Fig. 2A). This level of B cell
reconstitution is equal to or greater than what has been reported for
other human Ig mice (6, 11).
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L chain was expressed on the surface of 45–65% of B cells
from spleens of four-feature animals, with 16–25% of B cells
expressing mouse Ig, a : ratio of 3:1 (Fig. 2
B).
In five-feature animals, 10–20% of B cells expressed human L
chain, while 45–60% expressed human , a : ratio of 1:3 (Fig. 2B). The level of mouse Ig+ B cells
in the five-feature animals was <5%. A small number of L chain
double-positive spleen cells were detected, which probably means that
(as described in normal mice (Ref. 14 and references
therein)) there is a low degree of leakage in isotype exclusion at the
light chain loci. Human peripheral B lymphocytes typically have a
: ratio of 3:2, while in normal mice the ratio is typically 19:1.
The high contribution of the HuIg YAC to the Ig repertoire was also
seen in mice containing the HuIg YAC in the presence of a functional
mouse Ig locus (up to 40% of the splenic B cells expressing human
) (14) and is in contrast to the low expression of the
HuIg locus to the Ig repertoire when a functional mouse Ig locus
is present (with up to 15% of B cells expressing human L chain on
the cell surface) (9).
In the four- and five-feature mice, human IgM was present at between 50
and 400 µg/ml (Fig. 3). The level
varied between individual animals and tended to be higher in the
five-feature animals, but was not correlated to the number of B
cells present in the spleen in these animals. The increased serum
IgM levels in five-feature mice may result from higher secretion
by cells expressing human IgM, Ab due to the stronger
transcriptional activity of the Ig 3' enhancer (25).
Normal BALB/c mice maintained under pathogen-free conditions have serum
IgM levels of 500 µg/ml and total IgG levels of 400 µg/ml
(26). The level of human IgM in the translocus mice is
similar to what was found in mice made using plasmid-based miniloci
(6, 27), although these smaller loci contain fewer
germline segments to form a diverse repertoire. The distribution of
human L chain in the serum of five-feature mice paralleled what was
found on the surface of spleen B cells, with Ig levels 3- to 4-fold
lower than human Ig levels (data not shown).
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Following immunization and boosts, the total serum IgM
concentration was elevated, and specific Ab could be detected (Fig. 3).
The titer of specific IgM produced was lower than that seen during
parallel immunization of BALB/c mice (data not shown). Hybridomas
secreting Ag-specific human IgM, Ab were obtained from four-feature
mice following immunization with the Ags listed in Table II. In culture, the hybridomas secreted
2 µg of human IgM/ml of supernatant, compared with levels of 10
µg/ml or more from mouse IgM hybridomas grown in parallel.
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was
expressed by 14 of 16 hybridoma clones, with the remaining clones
expressing mouse Ig. In seven fusions from five-feature animals,
more human Ig-expressing clones were obtained on four occasions,
with from 4- to 8-fold more human Ig-expressing clones isolated. In
the remaining fusions, the ratio of human Ig to human
Ig-expressing clones was almost 1:1. Combined L chain expression
from different loci, i.e., a combination of human with human or
mouse L chains, was not observed. Generation of diversity of Ig rearrangements
The H chain and L chain rearrangements from the specific
hybridomas described above were sequenced to determine the diversity of
the immune response. The H chains showed a very restricted use of the V
region segments, dominated by VH1-2, accompanied
by a highly diverse CDR3 rearrangement (Table II and Fig. 4). The use of the L chain V segments was
not so restricted, and this was coupled with the use of different J
regions. The potential diversity of the response to a single Ag was
shown by the several hybridomas raised against progesterone-BSA
(1451/A5, 1451/B9, 1477/13, and 1477/14), all of which had unique H
chain and L chain rearrangements. To further assess the diversity of
the available Ab repertoire generated in the translocus mice, a total
of 41 H chain rearrangements, 19 L chain rearrangements, and 6
L chain rearrangements were isolated by RT-PCR from hybridomas, spleen
cells, and Peyer’s patch germinal center cells. All the sequences
isolated used translocus-derived segments in a rearrangement that was
in the correct reading frame to produce a functional Ab protein.
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), with a distribution similar to
H chain rearrangements from human B lymphocytes (28). The
use of D segments is of particular interest, as the mouse D locus is
more restricted than the human locus. The mouse locus has only 12 D
segments, divided into two main families, DSP2 and DFL16, together with
the DQ52 segment (29), while the human D locus contains 27
segments, divided into six families along a 40-kb region
(30). D segments could be identified in 39 of the 41
sequences, with 13 of the 27 segments present in the locus found in
rearrangements (Fig. 6). The majority of
the D segments used were from the 5' region of the locus (further from
the J segments), which parallels D segment utilization in human B
lymphocytes (28). The use of H chain J segments is also
similar to what is seen in human rearrangements, with the
JH4 (18 of 41) and JH6 (19
of 41) segments being used most often. Nongermline-encoded N
nucleotides were found in all rearrangements, with 26 of the 39
sequences containing D segments having insertions at both the V-D
(average, 5.2 ± 2.9 bases) and D-J (average, 2.3 ± 2.7
bases) junctions. The number of N nucleotides added in the H chain is
intermediate between the numbers reported for human V-D (average, 7.3
bases) and D-J (6.3 bases) joins and those for mouse V-D (average,
3.2 ± 2.5 insertions) and D-J (average, 2.2 ± 2.3) joins
(30, 31). A similar level of N nucleotide insertion in
rearranged H chain sequences was observed in other strains of
transgenic mice expressing human Ig repertoires (10, 11).
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rearrangements used the V4–1 and V1D-12 segments most
frequently (7 of 19 and 9 of 19, respectively), with V3D-11 and
V1D-13 being used in only a few rearrangements. The other functional
V genes in the construct, V5–2 and V1D-9, were not found in
the sequences examined. The six Ig rearrangements were obtained from
a single animal and show a limited use of the V gene segments, with
V3–19 used in five sequences. Given the high contribution to the B
cell repertoire seen in FACS and serum analysis, it is likely that the
rearrangement of the locus in the five-feature mice is similar to what
is seen in mice in which HuIg YAC is in the presence of a functional
mouse Ig locus (14). Junctional diversity is less
obvious for the L chain rearrangements and was provided mainly
through the use of different J segments. The CDR3 length was
restricted, with 18 of the 19 V-J sequences encoding a 9-aa CDR3,
and the remaining sequence encoding an 8-aa CDR3. The same restriction
has been reported for mouse V-J sequences, where 39 of 41
productive V-J rearrangements encoded a 9-aa CDR3, with the
remaining CDR3 being 10 and 11 aa (32). In contrast, human
V-J CDR3 can range from 7–12 aa, with at least 20% of sequences
encoding CDR3 longer than 9 aa (33). The increased
variation in human L chain sequences is due to the insertion of N
nucleotides at the V-J junction, which is not seen in L chain
rearrangements from mice (32, 33). Little or no N
insertion is found in the translocus-derived L chains either in four-
or five-feature mice or in mice with the HuIg or HuIg YAC in the
presence of a functional mouse H chain locus (14, 21).
This would suggest that the L chain translocus rearranges at the same
developmental stage as the endogenous L chains, at which time terminal
deoxynucleotide transferase activity is reduced. Somatic hypermutation of human Ig rearrangements
The Ig rearrangements were examined for evidence of somatic
hypermutation, which would indicate whether the B lymphocytes
expressing human IgM were able to participate normally in immune
responses. The majority of L chain rearrangements (13 of 19 V-J
and five of six V-J) contained two or more differences from the
germline sequence, with up to 15 point mutations/sequence, while in
contrast only four of 41 H chain rearrangements contained two or more
mutations (Fig. 7). The frequency of
mutations in the L chains was similar to what was observed in mice
carrying the HuIg YAC in the presence of a functional mouse H chain
locus (21). In the paired sequences from 19 hybridomas,
six clones had no mutations in either the H chain or L chain, 10 clones
had an unmutated H chain with a mutated L chain, and three clones had
mutations in both chains. The most mutated H chain sequence came from
the same hybridoma as the most mutated L chain sequence (hybridoma
1431/AB5).
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L chain rearrangements
shows that of the 28 changes in the CDRs, 25 lead to amino acid
replacement, with a replacement:silent ratio near 8:1, while of 50
point mutations in the framework regions, 27 were replacement mutations
in the encoded Ab, with a replacement:silent ratio near 1:1. The bias
in favor of replacement mutations in the CDR regions is associated with
selection-improved Ag binding (34). No such bias was
observed for the mutations in the H chain rearrangements. These results
suggest that the B cells expressing human surface Ig are capable of
participating in an immune response and undergoing affinity maturation,
but that the HuIgHeavy YAC is a poor target for the introduction of
mutations. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reconstitution of B cell population by Ig transloci
The human Ig transloci are introduced into a background in which
the endogenous mouse H chain and L chain loci are nonfunctional,
with only the mouse locus unaltered. While it is clear that there
is significant reconstitution of the B cell repertoire in these mice,
the number of B cells present in the mice is reduced compared with that
in mice with functional endogenous loci (Fig. 2A). The
comparison of several strains carrying different transgene and knockout
combinations suggests that the low B cell pool results from poor
function of the HuIgHeavy YAC in replacing the mouse H chain locus.
The HuIg and HuIg YACs are each capable of substituting for the
mouse Ig locus during B cell development, leading to the complete
restoration of the B cell pool. The knockout of the locus
(19), with a functional mouse H chain locus present,
reduces the B cell population from around 40% of spleen cells to about
15%, with these cells expressing mouse L chains. The introduction
of either the HuIg YAC or the HuIg YAC to this background
restores the B cell population to near normal levels (9, 14). In contrast, the introduction of the HuIgH YAC only
increases the B cell population to 5–25% of that of spleen cells,
from the <2% present with the µMT-/-
modification (Fig. 2A). The failure of the introduced locus
to restore the B cell repertoire is not due to competition with
endogenous H chain loci, which are still able to rearrange in the
µMT-/- background before the functional
defect is manifest. The restoration of the B cell repertoire is also
not complete if a germline configuration H chain translocus is
introduced into a knockout background in which the entire J segment
locus is removed, and no rearrangement of the endogenous alleles is
possible (11, 35). Similarly, the introduction of a larger
germline configuration H chain YAC containing the majority of
functional V regions does not restore the B cell population to a
greater extent than a locus containing five V regions (10, 11).
Analysis of various L chain transgenes indicates that elements in the locus downstream of the C regions may be necessary during the development of the B cell repertoire (21). No H chain translocus has been described that includes these regions in germline configuration, and those transloci that do include downstream elements, such as other C region genes or downstream enhancers (6, 10), place these elements in close proximity to the IgM and IgD domains and do not allow for the presence of any essential regulatory elements in the regions between the C domains.
Expression of translocus and light chains
The distribution of surface L chain in the five-feature animals
further indicates that the structure of the translocus can affect the
contribution to the B cell repertoire. The reconstitution of the B cell
population was similar in both four-feature and five-feature mice (Fig. 2A), yet the total serum IgM levels tended to be higher in
the five-feature mice (Fig. 3). Although the individual transloci can
both substitute for the mouse Ig locus, the contribution of the
HuIg locus appears to be dominant when in direct competition with
the HuIg locus, with a : ratio of 1:3. In human B cells, Ig
is expressed slightly more frequently, with a typical : ratio of
60:40. In the human genome, both and V regions are gathered in
clusters along their respective loci (36, 37), and the
majority of L chain rearrangements ( or ) use V segments from the
proximal cluster, nearer the core of the locus (38, 39).
The dominant HuIg YAC is arranged in the correct germline
configuration (24), whereas the HuIg YAC was produced
by attaching a multimer of five V regions to the core of the human
locus of three V regions, the J and C segments, and the enhancers
(9). Both transloci contain the 3' enhancers in their
correct location, but the format of the Ig YAC may remove other
regulatory elements from upstream of the core of the locus. The absence
of these elements may affect the rearrangement of the translocus
and therefore lower its contribution to the Ig repertoire in the
five-feature animals. As the mouse locus rearranges so poorly, even
when the mouse locus is nonfunctional (9), this
deficiency in HuIg YAC is not evident in the absence of HuIg YAC,
either in the four-feature animals or when HuIg YAC is present with
the functional mouse H chain locus.
Diversity of the translocus-encoded repertoire
The characteristics of the human Ig rearrangements indicate that the formation of the Ab repertoire from the translocus elements follows the same rules as that for the equivalent mouse loci. The similar use of D and J segments suggests that the recognition of recombination signal sequences is all but identical in mouse and man. The insertion of N nucleotides in the H chains, but not the L chains, indicates that the transloci rearrange at the same time as their endogenous counterparts. The major limitation on the potential variability of the repertoire arises from the limited number of V region gene segments present in the HuIgHeavy YAC, apparent by the overexpression of the VH1-2 segment. The Ig rearrangement process is affected by variations both in the promoter of the V region (40) and in the recombination signal sequences (41). Given the small number of V regions in the locus, even a relatively small advantage in rearrangement frequency during B cell development may manifest as a large bias in the utilization of the region in the Ig repertoire (42). The sequencing of the human H chain locus indicates that the segments in the HuIgH YAC differ in both the promoter region and the recombination signals (43), which may account for the observed utilization of VH1-2 in preference to the other segments. The inclusion of more V region segments in the translocus should overcome this limitation.
Somatic hypermutation of translocus-encoded Ig rearrangements
The presence of point mutations in rearranged Ig genes indicates that they are from a B cell that has undergone affinity maturation following exposure to Ag. Although it has been reported that mutations can be present in the H or L chain but not in the partner sequence, the most common state is that both chains contain mutations (44, 45). Here we found frequent mutations in L chain rearrangements, but almost no mutation in the H chains. The contrast was particularly evident in the paired sequences isolated from hybridoma clones. The presence of mutations in the L chain rearrangements confirms that the B cells expressing human Ab are able to participate in germinal center reactions, and the biased distribution of replacement mutations indicates that these mutations were subject to antigenic selection (34). The consistent absence of mutations in the H chain rearrangements, with only four of 41 rearrangements containing two or more point mutations, would indicate that the H chain translocus provides a poor target for the introduction of somatic hypermutation mechanism. Efficient hypermutation of the VH segments has been described in the case of several, but not all, human IgH transloci (6, 10, 13, 46). This variation could indicate a sensitivity to integration position effects, particularly since transloci are unlikely to contain the full complement of IgH locus cis-acting regulatory elements.
Concluding remarks
We have produced mice carrying germline H chain and and L
chain transloci in a background where the mouse H chain and L chain
are nonfunctional. The transloci can substitute for their mouse
counterparts, leading to the formation of diverse repertoires of fully
human Ab. Sequence analysis indicates that the transloci are rearranged
at the same stage as their mouse equivalents, and that the segments are
used as they would be in human cells. The mice we have developed will
be useful in the study of Ig gene rearrangement during B cell
development. They can be used to produce fully human mAb for therapy,
avoiding the adverse reactions that can be induced by rodent
Ab.
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Acknowledgments |
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Footnotes |
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2 Current address: Child Health Research Institute, Womens and Childrens Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia.
3 Current address: Cell Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
4 Address correspondence and reprint requests to Dr. Marianne Brüggemann, Laboratory of Developmental Immunology, The Babraham Institute, Babraham, Cambridge, CB2 4AT, United Kingdom. E-mail address:
5 Abbreviations used in this paper: YAC, yeast artificial chromosome; H chain, heavy chain of Ig; IGF, insulin-like growth factor; L chain, light chain of Ig; PLAP, placental alkaline phosphatase. (The term "four-feature" mice refers to animals carrying the HuIgHeavy and HuIg YACs and the µMT-/- and Mo-/- knockouts. Similarly, the term "five-feature" mice refers to mice with the HuIgHeavy, HuIg, and HuIg YACs with the µMT-/- and Mo-/- knockouts.)
Received for publication July 13, 1999. Accepted for publication September 28, 1999.
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 M. Viau, N. S. Longo, P. E. Lipsky, L. Bjorck, and M. Zouali Specific In Vivo Deletion of B-Cell Subpopulations Expressing Human Immunoglobulins by the B-Cell Superantigen Protein L Infect. Immun., June 1, 2004; 72(6): 3515 - 3523. [Abstract] [Full Text] [PDF]
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