By
From the Laboratory of Developmental Immunology, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transgenic mice carrying a 380-kb region of the human immunoglobulin (Ig) light (L) chain
locus in germline configuration were created. The introduced translocus on a yeast artificial chromosome (YAC) accommodates the most proximal Ig
variable region (V) gene cluster, including 15 V
genes that contribute to >60% of
L chains in humans, all J
-C
segments,
and the 3' enhancer. HuIg
YAC mice were bred with animals in which mouse Ig
production was silenced by gene targeting. In the
/
background, human Ig
was expressed by
~84% of splenic B cells. A striking result was that human Ig
was also produced at high levels
in mice with normal
locus. Analysis of bone marrow cells showed that human Ig
and mouse
Ig
were expressed at similar levels throughout B cell development, suggesting that the Ig
translocus and the endogenous
locus rearrange independently and with equal efficiency at the
same developmental stage. This is further supported by the finding that in hybridomas expressing human Ig
the endogenous L chain loci were in germline configuration. The presence of
somatic hypermutation in the human V
genes indicated that the Ig
-expressing cells function
normally. The finding that human
genes can be utilized with similar efficiency in mice and
humans implies that L chain expression is critically dependent on the configuration of the locus.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The light chain component of the Ig protein is encoded
by two separate loci, Ig and Ig
. The proportion of
antibodies containing
or
L chains varies considerably between different species (1); in mice the
/
ratio is 95:5,
compared with 60:40 in humans. Two models exist to account for the dominance of Ig
expression in the mouse.
From the observations that murine Ig
-producing myelomas have rearranged
L chain genes, whereas Ig
-producing cells have the
L chain locus in germline configuration, it was proposed initially that
rearrangement must
occur before
rearrangement can begin (4, 5). Although
the same observation applies for human B cells, the proportions of
- and
-producing cells are similar (4), suggesting
that other factors are involved. The second proposal is that
and
loci are equally available for rearrangement at the
same time, but the mouse
locus is more efficient at engaging the rearrangement process (for review see reference
6). The occasional finding of cells with rearranged
and the
locus in germline configuration may support this (5, 7,
8). Any influence of antigen selection on the biased
/
ratio is discounted by the finding that the ratio is similar in
fetal liver and in cells that have not encountered antigen (9).
L chain V-J rearrangement occurs at the transition from
pre-B-II to immature B cells, where the surrogate L chain
associated with membrane Igµ is replaced by or
(14).
Although the timing of L chain rearrangement is essentially
defined, the processes that activate L chain locus rearrangement are not fully understood. From locus-silencing experiments, it is apparent that
rearrangement is not a prerequisite for
recombination (15), but instead that
and
rearrangements are independent events (16), the activation
of which may be affected by differences in the strength of
the respective enhancers. Targeted deletion of the
3' enhancer in transgenic mice showed that this region is not essential for
locus rearrangement or expression but is required for establishing the
/
ratio (17). A region that
may regulate the accessibility of the human
locus has
been identified ~10 kb downstream of C
7 (18, 19).
Functional analysis using reporter gene assays identified a
core enhancer region flanked by elements that can drastically reduce enhancer activity in pre-B cells (18). Although transfection studies showed that the
and
3' enhancer
regions appear to be functionally equivalent, the core enhancer motifs are flanked by functional sequences that are
remarkably dissimilar. The human Ig
locus on chromosome 22q11.2 is 1.1 Mb in size and typically contains 70 V
genes and 7 J
-C
gene segments (20, 21). Approximately
half of the V
genes and J
-C
1, 2, 3, and 7 are regarded
as functional. The V
genes are organized in three clusters,
with the members of a particular V gene family contained within the same cluster. There are 10 V
gene families,
with the largest, V
III, having 23 members. In human peripheral blood lymphocytes, V gene segments of families I,
II, and III from the J-C proximal cluster A are preferentially rearranged, with the contribution of the 2a2 V
segment (2-14 using a position-based nomenclature; reference
22) being unusually high (23). All
gene segments have
the same polarity that allows deletional rearrangement (24). The diversity of the Ig
repertoire is provided mainly by
V
-J
combination. Additional CDR3 diversity due to N
(nonencoded)1 or P (palindromic) nucleotide additions at
the V to J junction is present in human sequences, although
not as extensively as in IgH rearrangement, but is absent in
sequences from mice (25), where the TdT (terminal
deoxyribonucleotide transferase) activity is downregulated
at the time of L chain rearrangement.
Here we have introduced a 410-kb yeast artificial chromosome (YAC), which contains most of the V genes of
cluster A and all the J
-C
segments in germline configuration, into mice that have one or both endogenous Ig
alleles disrupted. The translocus shows high expression in
both backgrounds, and is able to compete equally with the
endogenous mouse
locus.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The HuIgYAC, Introduction into Embryonic Stem Cells, and Derivation of Transgenic Mice.
Southern Blot Analysis.
Conventional DNA was obtained (33) or high molecular weight DNA was prepared in agarose blocks (34). For the preparation of testis DNA, tissues were homogenized and passed through 70 µM nylon mesh. Pulsed-field gel electrophoresis (PFGE) conditions to separate in the 50-900 kb range were 1% agarose, 180 V, 70 s switch time, and 30 h running time at 3.5°C. Hybridization probes were CHybridoma Production and ELISA Assay.
Hybridomas were obtained from 3-mo-old HuIgFlow Cytometry Analysis.
Cell suspensions were obtained from bone marrow, spleen, and Peyer's patches (PPs). Multicolor staining was then carried out with the following reagents in combinations (illustrated in Fig. 4): FITC-conjugated anti-human
|
Cloning and Sequencing of 5'RACE Products.
Spleen RNA was prepared as previously described (37) and for cDNA preparation 2-3 µg of RNA was ethanol precipitated and air dried. For rapid amplification of 5' cDNA ends (5'RACE) (38) first strand cDNA was primed with oligo(dT)22, and 100 U of Super Script II reverse transcriptase (GIBCO BRL) was used at 46°C according to the manufacturer's instructions with 20 U of placental RNAse inhibitor (Promega). The DNA/RNA duplex was passed through 1 ml G-50 equilibrated with TE (10 mM Tris-HCl, pH 7.8, and 1 mM EDTA) in a hypodermic syringe to remove excess oligo(dT). For G-tailing, 20 U of TdT (Cambio, UK) was used according to standard protocols (39). Double-stranded cDNA was obtained from G-tailed single-stranded cDNA by addition of oligonucleotide Pr1 (see below), 100 µM dNTP, and 2.5 U of Klenow fragment (Cambio), followed by incubation for 10 min at 40°C. After heating the reaction for 1 min at 94°C and extraction with phenol-chloroform, the double-stranded cDNA was passed through G-50 to remove primer Pr1. PCR amplifications, 35 cycles, were carried out in the RoboCycler Gradient 96 Thermal Cycler (Stratagene) using oligonucleotides Pr2 and Pr3. For PCR of PP cDNA 50 cycles were used: 40 cycles in the first amplification and 10 cycles in additional amplifications. Pfu Thermostable Polymerase (Stratagene) was used instead of Taq polymerase to reduce PCR error rates. The amplification products were purified using a GENECLEAN II kit (BIO 101) and reamplified for five cycles with primers Pr2 and Pr4 to allow cloning into EcoRI sites. Oligonucleotide for 5'RACE of V ![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human Ig translocus (Fig. 1) was assembled as a YAC by recombining one
YAC containing about half of the human V
gene segments with three overlapping cosmids containing V
and
J
-C
gene segments and the 3' enhancer (29). This produced a 410-kb YAC accommodating a 380-kb region of
the human
L chain locus containing 15 V
genes regarded as functional, 3 V
s with open reading frames not
found to be expressed, and 13 V
pseudogenes (40). This
HuIg
YAC was introduced into ES cells by protoplast fusion (30) and chimeric mice were produced by blastocyst
injection (31). The ES cell clone used for blastocyst injection showed a 450-kb NotI fragment corresponding to
HuIg
YAC, as identified by PFGE and Southern hybridization with probes to the 3' end of the construct, identifying the C
2+3 regions, and to the left centromeric YAC
arm at the 5' end, identifying the LYS2 gene (data not
shown). Germline transmission was obtained, and PFGE
analysis of testis DNA from one animal is illustrated in Fig.
2. A NotI fragment larger than 380 kb is necessary to accommodate this region of the HuIg
YAC, and the 450-kb
band obtained indicates random integration involving the
single NotI site 3' of J
-C
and a NotI site in the mouse
chromosome. Digests with EcoRI/HindIII and hybridization with the C
2+3 probe further confirmed the integrity
of the transferred HuIg
YAC (Fig. 2). The results indicated that one complete copy of the HuIg
YAC was integrated in the mouse genome.
|
|
|
To assess the human L chain repertoire for the production of authentic human antibodies, the HuIg
YAC
mice were bred with mice in which endogenous Ig
production was silenced by gene targeting (32). In these
/
mice, the mouse Ig
titers are elevated compared with
+/+
strains (32, 41). Serum titrations (Fig. 3) showed that human Ig
antibody titers in HuIg
YAC/
/
mice are between 1 and 2 mg/ml, which is up to 10-fold higher than average
mouse Ig
levels. Interestingly, in the HuIg
YAC/
/
mice, the mouse Ig
production returned to levels similar to that found in normal mice. High numbers of human Ig
+
cells were also identified in flow cytometric analysis of
splenic B cells from HuIg
YAC/
/
mice (Fig. 4 A), with
human
expressed on the surface of ~84% of the B cells
and mouse Ig
+ expressed on <5% (data not shown).
|
Assessment of human Ig production in heterozygous HuIg
YAC+/
+/
mice allowed a detailed comparison of expression and activation of endogenous versus transgenic L chain
loci present at equal functional numbers. Serum analysis (Fig.
3) of mice capable of expressing both human
and mouse
showed similar titers for human and mouse L chains. Human
Ig
levels in HuIg
YAC/
+/+ transgenic mice were similar to those in HuIg
YAC/
+/
mice. Total Ig levels in
HuIg
YAC+/
+/
mice were 1-2 mg/ml, with an average
contribution of ~51% mouse Ig
, 43% human Ig
, and 6%
mouse Ig
. As is also seen in human serum, the analysis of
individual HuIg
YAC/
+/
animals showed there were
variations in the
/
ratios. Three of the HuIg
YAC/
+/
mice produced somewhat higher
levels, whereas in two
mice the human
levels were higher than the Ig
titers. In
HuIg
YAC/
+/
mice, high translocus expression was also
found in B220+ B cells from different tissues, with 38% of
spleen cells expressing human
and 45% expressing mouse
(Fig. 4 A). As illustrated, these values closely resemble the
levels in human volunteers with 34% Ig
+ versus 51% Ig
+
in CD19+ peripheral blood lymphocytes. In HuIg
YAC/
+/+ mice, which carry a wild-type
locus, the levels of Ig
are ~25% and endogenous
levels are ~60% (Fig. 4 A). It is
likely that these differences in expression levels are dependent on the number of active gene loci.
To assess the developmental stage at which the high contribution of the human translocus becomes established, we
examined surface L chain expression by bone marrow cells
of HuIg
YAC/
+/
mice. For this, B cell lineage marker
CD19 and specific antibodies to human
and mouse
were
used in four-color staining with the early B cell markers c-kit
(CD117) and CD25. Fig. 4 B shows that surface L chain expression (human
or mouse
) was detectable on a similar
small proportion of B cells at each of these stages of development, which suggests that human and mouse L chain rearrangements are simultaneous. The specificity of the staining
detecting mouse
and human
on small numbers of early
B cells, which has been reported independently (42), was verified by the absence of similar positive cells in the analysis of
bone marrow from control mice (data not shown).
To further clarify the potential of
the L chain translocus to contribute to the antibody repertoire, we analyzed human and mouse
L chain production using individual hybridoma clones from HuIg
YAC/
+/
mice. Results from two fusions suggest that human
and mouse
L chain-producing cells were present in the
spleen of HuIg
YAC/
/+ mice at similar frequencies.
Furthermore, in the hybridomas the amounts of human Ig
(2-20 µg/ml) or mouse Ig
(4-25 µg/ml) were very similar. To determine whether Ig
rearrangement precedes Ig
, as found in mice and humans (4, 5), the configuration of the endogenous Ig
and the human
translocus were
analyzed in these hybridomas. Southern blot hybridization
of randomly picked hybridoma clones showed that in 11 human Ig
expressers, 7 had the mouse
locus in germline
configuration, 1 clone had mouse Ig
rearranged, and 3 clones had the mouse
locus deleted, whereas in 19 mouse
Ig
expressers, all but 2 had the human Ig
locus in germline configuration. This result suggests that there is no locus
activation bias and further emphasizes that the human
translocus performs with similar efficiency as the endogenous
locus.
The capacity of the human locus to produce a diverse
antibody repertoire is further documented by the V-J rearrangement. Sequences were isolated from spleen and PP
cells by 5'RACE PCR amplification to avoid bias from
specific V gene primers. The use of individual V
genes is
indicated by the triangles in Fig. 1, and shows that a substantial proportion of the V
genes on the translocus are
being used in productive rearrangements, with V
3-1 and
V
3-10 being most frequently expressed. In V
-J
rearrangements, J
2 was used preferentially and J
3 and 1 were
used less frequently, whereas, as expected, J
4, 5, and 6 were not used as they are adjacent to
Cs. Extensive variability due to N or P sequence additions, which is found
in human but not mouse L chain sequences (25, 27, 28),
was not observed. Sequences obtained by RT-PCR from
FACS®-sorted PP germinal center B cells (B220+/PNA+)
revealed that somatic hypermutation is operative in HuIg
YAC mice (Fig. 5). We identified 11 unique V
-J
rearrangements with two or more changes in the V region, excluding the CDR3, which may be affected by V
-J
recombination. The majority of mutations lead to amino acid
replacements, but there was no preferential distribution in
CDR1 and CDR2.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ratio of to
L chain expression varies considerably between different species (1, 43, 44), and in mice
the low
L chain levels are believed to be a result of inefficient activation of the mouse
locus during B cell differentiation (for review see reference 6). The Ig
(~40%) to
Ig
(~60%) ratio in humans is more balanced and suggests
that both
and
play an equally important role in immune responses. This is supported by the finding that the
mouse V
genes are most similar to the less frequently used
distal human V
gene families, whereas no genes comparable to the major contributors to the human V
repertoire
are present in the mouse locus (40). With the HuIg
YAC,
these V
genes are available, and are able to make a significant contribution to the antibody repertoire. The 410-kb
HuIg
YAC translocus accommodates V gene region cluster A containing at least 15 functional V
genes (see Fig. 1).
In humans, cluster A is the main contributor to the
antibody repertoire, with V
2-14 (2a2) expressed most frequently at 27% in blood lymphocytes (23). We also find
expression of V
2-14 in the translocus mice, but the main
contributors to the
L chain repertoire were 3-1 (the V
gene most proximal to the J-C region) and 3-10, both of
which are expressed at ~3% in humans. Although the validity of conclusions about the contribution of different
genes is dependent on the numbers examined, the overexpression of V
3-1 (11 sequences) and V
3-10 (8 sequences) in the 31 sequences obtained may imply that rearrangement or selection preferences are different in mice
and humans. Analysis of recombination signal sequences
(RSS) in mouse L chains showed that
and
RSS differ significantly, and that those genes with the highest similarity to consensus RSS rearrange most frequently (45). The
RSS of V
3-1 and V
3-10 show a 100% match with the
mouse consensus sequence, which may explain their frequent expression in the translocus mice. In addition, most
human V
RSS match the established consensus sequence
significantly better than mouse V
s (21, 45).
We found extensive somatic hypermutation of many rearranged human Ig sequences, indicating that they are
able to participate in normal immune responses. The levels
of mutation in B220+/PNA+ PP cells from HuIg
YAC
translocus mice were similar to what has been reported for
mouse L chains (46). Rather unexpected was the pattern of
somatic hypermutation with similar numbers of silent and
replacement point mutations found in the complementarity-determining and framework regions. Somatic hypermutation is usually associated with a higher level of replacement mutations in CDRs and more silent mutations
in the framework regions, and the distribution observed
here may argue against efficient antigen selection having taken place. Interestingly, however,
L chain sequences
obtained from human peripheral blood lymphocytes also
showed high numbers of mutations in framework 2 (23).
Part of framework 2 lies at the interface of the VL and VH
domains and it has been suggested that this region may be
important for optimal H and
L chain interaction and, in
particular, interaction of the human
L chain and the endogenous mouse H chain (26).
In the mouse, unlike in humans, L chain diversification
due to untemplated nucleotide addition is essentially absent, because TdT expression has been downregulated by
the stage at which L chain rearrangement takes place (28,
47). This concept is challenged by the observation that
mouse L chain rearrangement can occur at the same time as
VH to DJH rearrangements (48) or even earlier (42). Our results also show L chain rearrangement at the pre-B cell stage
with a similar number of human - or mouse
-expressing
B cells also expressing c-kit+ or CD25+ (see Fig. 4). Although the cell numbers are small, the results suggest that
there is no preferential activation of either the human
translocus or the endogenous
locus. However, despite this early activation, there is no accumulation of N or P
nucleotide diversity in the rearranged human
L chains,
unlike rearranged
L chains from human peripheral B cells
(27). The small number (<1%) of human Ig
+/mouse
Ig
+ double positive spleen and bone marrow cells may indicate that haplotype exclusion at the L chain level is less
strictly controlled than is IgH exclusion (49).
In transgenic mice carrying Ig regions in germline configuration on minigene constructs, efficient DNA rearrangement and high antibody expression levels are rarely
achieved. Competition with the endogenous locus can be
eliminated using Ig knockout strains, in which transgene
expression is usually improved (50). Poor transloci expression levels could be a result of the failure of human sequences to work efficiently in the mouse background or, alternatively, of the absence of locus-specific control regions that are more likely to be included on larger transgenic regions (51). Recently we addressed this question
in transgenic mice by the introduction of different sized
minigene- and YAC-based human L chain loci (53). The
result showed that neither the size of the V gene cluster nor
the V gene numbers present were relevant to achieving
high translocus expression levels. The YAC-based loci
contained downstream regions of the human
locus, and it is possible that the presence of an undefined region with
cis-controlled regulatory sequences may have been crucial
in determining expressibility and subsequently L chain choice.
The HuIg
YAC contains equivalent regions from the human Ig
locus, which may promote the use of the translocus
in the L chain repertoire. Hybridomas from HuIg
YAC+/
+/
mice show no evidence for a bias in L chain locus selection during development, as demonstrated by the absence
of rearrangement of the nonexpressed locus. This is in contrast with what is seen in Ig expressing mouse and human
B cell clones (4, 5), and supports the model that
and
rearrangements are indeed independent (15, 54) and that
poor Ig
expression levels in mice may be the result of inefficient signals acting during recombination (16). A possible signal that initiates L chain recombination has been
identified through gene targeting experiments where the 3'
enhancer was deleted (17). In these mice, the
/
ratio was reduced from 20:1 in normal mice down to 1:1, and the
locus was largely in germline configuration in
-expressing
cells, as we also see in the HuIg
YAC+/
+/
hybridoma
clones. The high level of human Ig
expression in the HuIg
YAC+/
+/
mice could be due to the strength of
the downstream enhancer of the human
locus. An analysis of human L chain enhancer activities identified three
synergistic modules at the 3' end of the
locus which constitute a powerful pre-B cell specific enhancer that appears
to be stronger than the corresponding
enhancer (55). Analysis of the mouse
3' enhancer suggests the biased
/
ratio in mice may be a direct result of the differences in locus specific regulation provided by the respective enhancers
(19, 56). The results suggest that strength and ability of the
human 3'
enhancer to function in the mouse background
may be the reason that
and
loci can compete equally at
the pre-B cell stage to initiate L chain rearrangement, resulting in the similar levels of human Ig
and mouse Ig
seen in the HuIg
YAC+/
+/
mice.
![]() |
Footnotes |
---|
Address correspondence to Marianne Brüggemann, Laboratory of Developmental Immunology, Department of Development and Genetics, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK. Phone: 44-1223-496-304; Fax: 44-1223-496-030; E-mail: marianne.bruggemann{at}bbsrc.ac.uk
Received for publication 14 December 1998 and in revised form 8 March 1999.
A.V. Popov and X. Zou contributed equally to this work.We thank Drs. I. Tomlinson, G. Winter, and O. Ignatovich for access to their database of human V sequences and helpful discussions. We are grateful to Drs. D. Melton for provision of the HM-1 ES cells, E. Corps for hybridoma production, B. Goyenechea for help with the Southern hybridization, and N. Miller
for helping with the flow cytometry.
This work was supported by the Biotechnology and Biological Sciences Research Council and the Babraham Institute.
Abbreviations used in this paper ES, embryonic stem; Hu, human; N, nonencoded; P, palindromic; PFGE, pulsed-field gel electrophoresis; PNA, peanut agglutinin; PP, Peyer's patch; RACE, rapid amplification of cDNA ends; RSS, recombination signal sequence(s); RT, reverse transcriptase; TdT, terminal deoxyribonucleotide transferase; YAC, yeast artificial chromosome.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hood, L., W.R. Gray, B.G. Sanders, and W.Y. Dreyer. 1967. Light chain evolution: antibodies. Cold Spring Harbor Symp. Quant. Biol. 32: 133-146 . |
2. |
McIntire, K.R., and
A.M. Rouse.
1970.
Mouse immunoglobulin light chains: alterations of ![]() ![]() |
3. | Arun, S.S., W. Breuer, and W. Hermanns. 1996. Immunohistochemical examination of light-chain expression (lambda/ kappa ratio) in canine, feline, equine, bovine and porcine plasma cells. Zentralbl. Veterinarmed. A. 43: 573-576 [Medline]. |
4. |
Hieter, P.A.,
S.J. Korsmeyer,
T.A. Waldmann, and
P. Leder.
1981.
Human immunoglobulin ![]() ![]() |
5. | Coleclough, C., R.P. Perry, K. Karjalainen, and M. Weigert. 1981. Aberrant rearrangements contribute significantly to the allelic exclusion of immunoglobulin gene expression. Nature. 290: 372-378 [Medline]. |
6. | Selsing, E., and L.E. Daitch. 1995. Immunoglobulin ![]() |
7. |
Berg, J.,
M. McDowell,
H.M. Jäck, and
M. Wabl.
1990.
Immunoglobulin ![]() ![]() |
8. | Abken, H., and C. Bützler. 1991. Re-organization of the immunoglobulin kappa gene on both alleles is not an obligatory prerequisite for Ig lambda gene expression in human cells. Immunology. 74: 709-713 [Medline]. |
9. | Takemori, T., and K. Rajewsky. 1981. Lambda chain expression at different stages of ontogeny in C57BL/6, BALB/c and SJL mice. Eur. J. Immunol. 11: 618-625 [Medline]. |
10. |
McGuire, K.L., and
E.S. Vitetta.
1981.
![]() ![]() |
11. |
Kessler, S.,
K.J. Kim, and
I. Scher.
1981.
Surface membrane
![]() ![]() |
12. |
Lejeune, J.M.,
D.E. Briles,
A.R. Lawton, and
J.F. Kearney.
1982.
Estimate of the light chain repertoire size of fetal and
adult BALB/cJ and CBA/J mice.
J. Immunol.
129:
673-677
|
13. |
Rolink, A.,
M. Streb, and
F. Melchers.
1991.
The ![]() ![]() |
14. | Osmond, D.J., A. Rolink, and F. Melchers. 1998. Murine B lymphopoiesis: towards a unified model. Immunol. Today. 19: 65-68 [Medline]. |
15. |
Zou, Y.R.,
S. Takeda, and
K. Rajewsky.
1993.
Gene targeting in the Ig![]() ![]() ![]() |
16. |
Arakawa, H.,
T. Shimizu, and
S. Takeda.
1996.
Re-evaluation of the probabilities for productive rearrangements on the
![]() ![]() |
17. |
Gorman, J.R.,
N. van der Stoep,
R. Monroe,
M. Cogne,
L. Davidson, and
F.W. Alt.
1996.
The Ig![]() ![]() ![]() |
18. | Glozak, M., and B.B. Blomberg. 1996. The human immunoglobulin enhancer is controlled by both positive elements and developmentally regulated negative elements. Mol. Immunol. 33: 427-438 [Medline]. |
19. | Asenbauer, H., and H.G. Klobeck. 1996. Tissue-specific deoxyribonuclease I-hypersensitive sites in the vicinity of the immunoglobulin C lambda cluster of man. Eur. J. Immunol. 26: 142-150 [Medline]. |
20. | Frippiat, J.P., S.C. Williams, I.M. Tomlinson, G.P. Cook, D. Cherif, D. Le Paslier, J.E. Collins, I. Dunham, G. Winter, and M.P. Lefranc. 1995. Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2. Hum. Mol. Genet. 4: 983-991 [Abstract]. |
21. |
Kawasaki, K.,
S. Minoshima,
E. Nakato,
K. Shibuya,
A. Shintani,
J.L. Schmeits,
J. Wang, and
N. Shimizu.
1997.
One-megabase sequence analysis of the human immunoglobulin ![]() |
22. |
Giudicelli, V.,
D. Chaume,
J. Bodmer,
W. Muller,
C. Busin,
S. Marsh,
R. Bontrop,
L. Marc,
A. Malik, and
M.-P. Lefranc.
1997.
IMGT, the international ImMunoGeneTics database.
Nucleic Acids Res.
25:
206-211
|
23. | Ignatovich, O., I.M. Tomlinson, P.T. Jones, and G. Winter. 1997. The creation of diversity in the human immunoglobulin V(lambda) repertoire. J. Mol. Biol. 268: 69-77 [Medline]. |
24. |
Combriato, G., and
H.-G. Klobeck.
1991.
V![]() ![]() ![]() ![]() |
25. |
Foster, S.J.,
H.-P. Brezinschek,
R.I. Brezinschek, and
P.E. Lipsky.
1997.
Molecular mechanisms and selective influences
that shape the ![]() |
26. | Ignatovich, O. 1998. The creation of diversity in the human
immunoglobulin V![]() |
27. |
Bridges, S.L.,
S.K. Lee,
M.L. Johnson,
J.C. Lavelle,
P.G. Fowler,
W.J. Koopman, and
H.W. Schroeder.
1995.
Somatic
mutation and CDR3 length of immunoglobulin ![]() |
28. |
Victor, K.D.,
K. Vu, and
A.J. Feeney.
1994.
Limited junctional diversity in ![]() |
29. | Popov, A.V., C. Bützler, J.-P. Frippiat, M.-P. Lefranc, and M. Brüggemann. 1996. Assembly and extension of yeast artificial chromosomes to build up a large locus. Gene. 177: 195-201 [Medline]. |
30. | Davies, N.P., A.V. Popov, X. Zou, and M. Brüggemann. 1996. Human antibody repertoires in transgenic mice: manipulation and transfer of YACs. In Antibody Engineering: A Practical Approach. J. McCafferty, H.R. Hoogenboom, and D.J. Chiswell, editors. IRL Press, Oxford. 59-76. |
31. | Hogan, B., R. Beddington, F. Costantini, and E. Lacy. 1994. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 497 pp. |
32. |
Zou, X.,
J. Xian,
A.V. Popov,
I.R. Rosewell,
M. Müller, and
M. Brüggemann.
1995.
Subtle differences in antibody
responses and hypermutation of ![]() ![]() |
33. | Wurst, W., and A.L. Joyner. 1993. Production of targeted embryonic stem cell DNA. In Gene Targeting. A.L. Joyner, editor. IRL Press, Oxford. 33-61. |
34. | Herrmann, B.G., D.P. Barlow, and H. Lehrach. 1987. A large inverted duplication allows homologous recombination between chromosomes heterozygous for the proximal t complex inversion. Cell. 48: 813-825 [Medline]. |
35. | Galfré, G., and C. Milstein. 1981. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 73: 3-46 [Medline]. |
36. | Tijssen, P. 1985. Practice and theory of enzyme immunoassays. In Laboratory Techniques in Biochemistry and Molecular Biology. Volume 15. R.H. Burdon and P.H. Knippenberg, editors. Elsevier, Amsterdam. |
37. | Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159 [Medline]. |
38. | Frohman, M.A., M.K. Dush, and G.R. Martin. 1988. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA. 85: 8998-9002 [Abstract]. |
39. | Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, K. Struhl, and J.A. Smith, editors. 1995. Current Protocols In Molecular Biology. Massachusetts General Hospital, Boston, MA; Harvard Medical School, Boston, MA; University of Alabama, Birmingham, AL; Wiley & Sons, New York. |
40. |
Williams, S.C.,
J.-P. Frippiat,
I.M. Tomlinson,
O. Ignatovich,
M.-P. Lefranc, and
G. Winter.
1996.
Sequence and evolution of the human germline V![]() |
41. |
Chen, J.,
M. Trounstine,
C. Kurahara,
F. Young,
C.-C. Kuo,
Y. Xu,
J.F. Loring,
F.W. Alt, and
D. Huszar.
1993.
B
cell development in mice that lack one or both immunoglobulin ![]() |
42. |
Novobrantseva, T.I.,
V.M. Martin,
R.M. Pelanda,
W. Muller,
K. Rajewsky, and
A. Ehlich.
1999.
Rearrangement
and expression of immunoglobulin light chain genes can precede heavy chain expression during normal B cell development in mice.
J. Exp. Med.
189:
75-88
|
43. | Saitta, M., A. Iavarone, N. Cappello, M.R. Bergami, G.C. Fiorucci, and F. Aguzzi. 1992. Reference values for immunoglobulin kappa and lambda light chains and the kappa/ lambda ratio in children's serum. Clin. Chem. 38: 2454-2457 [Abstract]. |
44. | Hood, L., W.R. Gray, and W.Y. Dreyer. 1966. On the mechanism of antibody synthesis: a species comparison of L-chains. Proc. Natl. Acad. Sci. USA. 55: 826-835 [Medline]. |
45. |
Ramsden, D.A., and
G.E. Wu.
1991.
Mouse ![]() ![]() |
46. |
Gonzalez-Fernandez, A.,
S.K. Gupta,
R. Pannell,
M.S. Neuberger, and
C. Milstein.
1994.
Somatic mutation of immunoglobulin lambda chains: a segment of the major intron hypermutates as much as the complementarity-determining
regions.
Proc. Natl. Acad. Sci. USA.
91:
12614-12618
|
47. | Li, Y.-S., K. Hayakawa, and R.R. Hardy. 1993. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J. Exp. Med. 178: 951-960 [Abstract]. |
48. | Hardy, R.R., C.E. Carmack, S.A. Shinton, J.D. Kemp, and K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173: 1213-1225 [Abstract]. |
49. |
Harada, K., and
H. Yamagishi.
1991.
Lack of feedback inhibition of V![]() |
50. | Brüggemann, M., and M.S. Neuberger. 1996. Strategies for expressing human antibody repertoires in transgenic mice. Immunol. Today. 17: 391-397 [Medline]. |
51. |
Green, L.L., and
A. Jakobovits.
1998.
Regulation of B cell
development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes.
J. Exp. Med.
188:
483-495
|
52. |
Zou, X.,
J. Xian,
N.P. Davies,
A.V. Popov, and
M. Brüggemann.
1996.
Dominant expression of a 1.3 Mb human Ig![]() |
53. |
Xian, J.,
X. Zou,
A.V. Popov,
C.A. Mundt,
N. Miller,
G.T. Williams,
S.L. Davies,
M.S. Neuberger, and
M. Brüggemann.
1998.
Comparison of the performance of a plasmid-based human Ig![]() ![]() |
54. | Nadel, B., P.-A. Cazenave, and P. Sanchez. 1990. Murine lambda gene rearrangements: the stochastic model prevails over the ordered model. EMBO (Eur. Mol. Biol. Organ.) J. 9: 435-440 [Abstract]. |
55. | Asenbauer, H., G. Combriato, and H.-G. Klobeck. 1999. The immunoglobulin lambda light chain enhancer consists of three modules which synergize in activation of transcription. Eur. J. Immunol. 29: 713-724 [Medline]. |
56. | Hagman, J., C.M. Rudin, C. Haasch, D. Chaplin, and U. Storb. 1990. A novel enhancer in the immunoglobulin lambda locus is duplicated and functionally independent of NF kappa B. Genes Dev. 4: 978-992 [Abstract]. |
57. | Eagle, H.. 1955. Propagation in a fluid medium of a human epidermoid carcinoma strain KB. Proc. Soc. Exp. Biol. Med. 89: 362-364 . |
58. |
Taub, R.A.,
G.F. Hollis,
P.A. Hieter,
S. Korsmeyer,
T.A. Waldmann, and
P. Leder.
1983.
Variable amplification of immunoglobulin ![]() |