- Email: [email protected]
Ligand inducible, B cell-specific gene targeting in mice
23 June 1997 - Poster presentations
09:00-18:30/12:Otk14:88
P.2.02
P.2.02.01
B-cell development
Forum lounges
B-cell development
Hlgh expression of a transgenic X2 light chain
results In impaired development of 82 cells but not Bl cells H. Engel ‘, A. Rolink 2, S. Weiss I. ’ Division of Cell Biology and /mmurw/~, GBF; 39124 Braunschweig, Gemany; 2Basel InstiUte for Immunology 4005 Base/, Switzerland Introduction: Developmental stages of B cell ontogeny in murine bone marrow are well characterised. Differential expression of cell surface markers can be correlated with the stages of immunoglobulin (Ig) rearrangement and expression. Here, we investigate the influence of a transgenic light (L) chain, which is derived from the DNP binding myeloma MOPC 315 (Az’5) on B cell ontogeny. To assure expression at the correct developmental stage, the A.$‘15 transgene was controlled by the 3’-enhancer of the K L chain. MaterlaIr and Methods: Mice were used at the age of 8 weeks. Constant numbers of cells from spleen, bone marrow, peritoneum and Peyer’s Patches were stained with a sat of monoclonal antibodies diagnostic for differentiation stages of B cell ontogeny according to standard procedures and analysed on a [email protected] bone marrow cells were analysed for the expression of different genes by FIT-PCR.Lethally irradiated BALE/c mice were reconstituted with 2 x IO6 BM cells from the high expressing transgenic mouseline and analysed four month after reconstitution. Results: Two transgenic mouselines were established. One line (4-5 transgene copies), showed no abnormalities of B cell development in the bone marrow and a normal percentage of B cells in lymphoid organs. Three populations of B cells were present in these mice: Cells, which expressed only a K chain, K/A double expressers and h only cells. The last population was doubled in mice, which were homozygous with respect to the transgene. The other line (about 18 copies) showed ten times elevated A mRNA levels compared to normal mice. Bone marrow analysis revealed a severe block in B cell development at the stage of pre-B-II cells (B220+, CD25+). Bone marrow reconstitution experiments suggested, that only few newly formed B cells leave the bone marrow. In addition, in these transgenic mice, RAG-2 mRNA could be detected in large pre-B-II-cells. The B cell compartment in the periphery of these mice was filled up to about l/5 of the normal number of B cells. Surprisingly, these cells were almost exclusively of the Bl type (CD5+). They canied only Ig’s with the transgenic A2 chain. Endogenous Y or A, chains were beady detectable. Concluelon: High expression of the transgenic 1;’ L chain leads to impaired 82 cell development, whereas Bl cell development can take place. The transgenic L chain appears to interfere with the pre-B-cell receptor. Since development is blocked, it supports the hypothesis that a signal is missing, which is necessary for downregulation of RAG-2 and proliferative expansion of the B cell precursors. This signal obviously is not important for the development of Bl cells. The study also confirms, that allelic exclusion at the L chain loci is not as strictly regulated as at the heavy chain locus.
1P.2.02.02 ( Human thymlc B cells largely overexpress the VH4 Ig gene family. A possible role in the control of tolerance in situ? C. Tonnelle, C. D’Ercole l, V. Depraetere, M. Fougereau. Cents d’lmmuncbgie INSERM. CNRS de Marseille Luminy, Marseille, France, ’ Hbpiral de la Conception Marseille, France Introduction: Human thymus contains a small population of B cells. A number of features have been repotted characteristic ofthymic B cells such as the presence of a CD2+CDlQ+ in human thymic cell population, an increased expression of CD5 at least in the mouse and a lower reactivity to LPS, anti-p antibody or 114, but the origin and functions of these cells remained to be elucidated. Meterlale and Method8: FACS analysis, PCR amplification and sequence of VDJ transctipts were performed on human thymic B cells enriched on CD19 panning. Reeub Human thymic B cells express CD2 and/or CD5, and revealed a constant coexpression of CD19 and immunoglcbulin light chains. In the absence of detectable B precursors, these cells had reached at least the stage of IgM+ immature B lymphocytes. Analysis of lg transcripts by PCR amplification first revealed a strong bias in favor of the VH4 and sequencing of 45 VH6D-J cDNA clones isolated from 2 thymuses indicated that this thymic repertoire significantly differed from that of peripheral blood lymphocytes (PBL). Most genes of the VH4 family were used with various D-J combinations and a relatively high frequency of somatic mutations.
93
Conchdon: This repertoire thus appears clearly selected in the thymus. This is of particular interest since the VH4 gene family is frequently encountered in autoimmunity, a situation in which the number of thymic B cells is largely increased. They could play a major role in the control of tolerance in situ.
1P.2.02.03 1 A role for the ~75 Tumour Necrosis Factor receptor in lvmohocvte develooment and svstemlc infianimatidn * E. Douni, G. Kollias. Department of Molecular Genetics, Hellenic Pasteur Institute,Athens, Hellas Introduction: Tumour Necrosis Factor (TNF) mediates its diverse biological functions binding to two high-affinity receptors, designated p55 and ~75 TNF-R. The p55 TNF-R appears to signal most of the activities of soluble TNF, whereas the ~75 TNF-R appears to primarily transduce signals from transmembrane TNF. In vitro, p75TNF-R signalling has been implicated in the regulation of hemopoietic cell proliferation, differentiation and death. Methods: To define pathological complications upon p75TNF-R overexpression in vivo,we have generated transgenic mouse lines canying an 80 Kb insert spanning the entire human ~75 TNFR gene. Results: In three transgenic mouse lines obtained, patterns of transgene expression were found to be regulated similarly to the endogenous mup75 TNF-R. In a high-level expressing line and at the heterozygous state, transgenic mice develop normally without any apparent pathological phenotype. However, homozygous mice of this line display a multi-inflammatoty disease which leads to organ failure and death. A similar phenotype develops in the heterozygous line in crosses with transgenic mice over-expressing human TNF in their T cells and in a p55TNF-R-independent fashion (e.g. in p55TNF-R knockout mice). In addition to systemic inflammation these mice show a dramatic decrease of 8220 positive cells in the bone manow, spleen and peripheral blood. In contrast, T cell populations remained largely unaffected. Conclusion: Our data suggest that the p75TNF-R can mediate systemic inflammatory events, at least when overexpressed in vivo, and most importantly, that p75TNF-R signalling may be involved in mechanisms regulating lymphocyte development and function.
U
P.2.02.04
Ligand inducible, 6 cell-specific gene targeting in mice
F. Schwenk, P.-O. Angrand’, R. Kiihn. A.F. Stewart’, K. Rajewsky. Institute for Genetics, University of Cologne, Weyertall21, D-50931 Cologne, FRG, ’ Eurqean Molecular Biology Laboratory, Meyarhofsfrasse 1, D-691 17 Heidelberg, FRG Introduction: The bacteriophage Pi derived Cre-IoxP recombination system has been recently used to improve the gene targeting technique in mice. Cre catalyzes site-specific DNA recombination between a 34 bp sequence referred to as IoxP such that a lo&flanked segment is excised from a DNA molecule. Conditional gene modification, restricted to a particular cell type or time point, can be achieved by crossing a mouse strain with a IoxP-flanked target gene to a strain expressing Cre recombinase in a spatially or temporally contrdled manner. We have established a system which allows inducible and B cell-specific gene targeting in mice by selective ligand administration. Method: Posttranslational control of the activity of site-specific recombinases can be achieved by expressing a chime& protein in which the mcombinase is fused to the ligand binding domain (LBD) of steroid hormone receptors. In the absence of hormone the steroid receptor LBD is bound by heat shock proteins which inactivate the recombinase, presumably by stetical hindrance. The recombinase can be activated by the addition of ligand which releases the heat shock proteins from the fusion protein. Results: To derive a system which allows inducible and B cell-specific gene targeting in mice, we fused Cre to the mutant (G521R) LBD of the human estrogen receptor which is unresponsive to its natural ligands but can be activated by synthetic hormones, such as 4-Hydroxytamoxifen. The activity of the Cre-LBD fusion was tested in a fibroblast line containing a IoxP-flanked target and in mice carrying the cretransgene under the control of a B cell-specific promoter. Our results demonstrate that the efficiency of Cm-mediated gene deletion upon administration of synthetic hormone approaches 90% in cultured cells and in B cells expressing the Cre-LBD fusion.
P.2.02.05
Expression of GL7 antlgen during B cell development
Mszl6 Cervenak. Attila Magyar ‘, Gl6tia Ldszl6. Dept.of Immunolog): Edtv& Lo&d Vniversiiy, Gad, ’ Inst. of Human Morphology and Debwkpmental Biology, Semmelweis Vniv Medical School, Budapest, Hungary GL7, a 38 kDa murine cell membrane bound lymphocyte activation antigen, appears on B and T cells after 80 hrs in vitro stimulation, but it is also expressed
09:00-18:30/12:Otk14:88
P.2.02
P.2.02.01
B-cell development
Forum lounges
B-cell development
Hlgh expression of a transgenic X2 light chain
results In impaired development of 82 cells but not Bl cells H. Engel ‘, A. Rolink 2, S. Weiss I. ’ Division of Cell Biology and /mmurw/~, GBF; 39124 Braunschweig, Gemany; 2Basel InstiUte for Immunology 4005 Base/, Switzerland Introduction: Developmental stages of B cell ontogeny in murine bone marrow are well characterised. Differential expression of cell surface markers can be correlated with the stages of immunoglobulin (Ig) rearrangement and expression. Here, we investigate the influence of a transgenic light (L) chain, which is derived from the DNP binding myeloma MOPC 315 (Az’5) on B cell ontogeny. To assure expression at the correct developmental stage, the A.$‘15 transgene was controlled by the 3’-enhancer of the K L chain. MaterlaIr and Methods: Mice were used at the age of 8 weeks. Constant numbers of cells from spleen, bone marrow, peritoneum and Peyer’s Patches were stained with a sat of monoclonal antibodies diagnostic for differentiation stages of B cell ontogeny according to standard procedures and analysed on a [email protected] bone marrow cells were analysed for the expression of different genes by FIT-PCR.Lethally irradiated BALE/c mice were reconstituted with 2 x IO6 BM cells from the high expressing transgenic mouseline and analysed four month after reconstitution. Results: Two transgenic mouselines were established. One line (4-5 transgene copies), showed no abnormalities of B cell development in the bone marrow and a normal percentage of B cells in lymphoid organs. Three populations of B cells were present in these mice: Cells, which expressed only a K chain, K/A double expressers and h only cells. The last population was doubled in mice, which were homozygous with respect to the transgene. The other line (about 18 copies) showed ten times elevated A mRNA levels compared to normal mice. Bone marrow analysis revealed a severe block in B cell development at the stage of pre-B-II cells (B220+, CD25+). Bone marrow reconstitution experiments suggested, that only few newly formed B cells leave the bone marrow. In addition, in these transgenic mice, RAG-2 mRNA could be detected in large pre-B-II-cells. The B cell compartment in the periphery of these mice was filled up to about l/5 of the normal number of B cells. Surprisingly, these cells were almost exclusively of the Bl type (CD5+). They canied only Ig’s with the transgenic A2 chain. Endogenous Y or A, chains were beady detectable. Concluelon: High expression of the transgenic 1;’ L chain leads to impaired 82 cell development, whereas Bl cell development can take place. The transgenic L chain appears to interfere with the pre-B-cell receptor. Since development is blocked, it supports the hypothesis that a signal is missing, which is necessary for downregulation of RAG-2 and proliferative expansion of the B cell precursors. This signal obviously is not important for the development of Bl cells. The study also confirms, that allelic exclusion at the L chain loci is not as strictly regulated as at the heavy chain locus.
1P.2.02.02 ( Human thymlc B cells largely overexpress the VH4 Ig gene family. A possible role in the control of tolerance in situ? C. Tonnelle, C. D’Ercole l, V. Depraetere, M. Fougereau. Cents d’lmmuncbgie INSERM. CNRS de Marseille Luminy, Marseille, France, ’ Hbpiral de la Conception Marseille, France Introduction: Human thymus contains a small population of B cells. A number of features have been repotted characteristic ofthymic B cells such as the presence of a CD2+CDlQ+ in human thymic cell population, an increased expression of CD5 at least in the mouse and a lower reactivity to LPS, anti-p antibody or 114, but the origin and functions of these cells remained to be elucidated. Meterlale and Method8: FACS analysis, PCR amplification and sequence of VDJ transctipts were performed on human thymic B cells enriched on CD19 panning. Reeub Human thymic B cells express CD2 and/or CD5, and revealed a constant coexpression of CD19 and immunoglcbulin light chains. In the absence of detectable B precursors, these cells had reached at least the stage of IgM+ immature B lymphocytes. Analysis of lg transcripts by PCR amplification first revealed a strong bias in favor of the VH4 and sequencing of 45 VH6D-J cDNA clones isolated from 2 thymuses indicated that this thymic repertoire significantly differed from that of peripheral blood lymphocytes (PBL). Most genes of the VH4 family were used with various D-J combinations and a relatively high frequency of somatic mutations.
93
Conchdon: This repertoire thus appears clearly selected in the thymus. This is of particular interest since the VH4 gene family is frequently encountered in autoimmunity, a situation in which the number of thymic B cells is largely increased. They could play a major role in the control of tolerance in situ.
1P.2.02.03 1 A role for the ~75 Tumour Necrosis Factor receptor in lvmohocvte develooment and svstemlc infianimatidn * E. Douni, G. Kollias. Department of Molecular Genetics, Hellenic Pasteur Institute,Athens, Hellas Introduction: Tumour Necrosis Factor (TNF) mediates its diverse biological functions binding to two high-affinity receptors, designated p55 and ~75 TNF-R. The p55 TNF-R appears to signal most of the activities of soluble TNF, whereas the ~75 TNF-R appears to primarily transduce signals from transmembrane TNF. In vitro, p75TNF-R signalling has been implicated in the regulation of hemopoietic cell proliferation, differentiation and death. Methods: To define pathological complications upon p75TNF-R overexpression in vivo,we have generated transgenic mouse lines canying an 80 Kb insert spanning the entire human ~75 TNFR gene. Results: In three transgenic mouse lines obtained, patterns of transgene expression were found to be regulated similarly to the endogenous mup75 TNF-R. In a high-level expressing line and at the heterozygous state, transgenic mice develop normally without any apparent pathological phenotype. However, homozygous mice of this line display a multi-inflammatoty disease which leads to organ failure and death. A similar phenotype develops in the heterozygous line in crosses with transgenic mice over-expressing human TNF in their T cells and in a p55TNF-R-independent fashion (e.g. in p55TNF-R knockout mice). In addition to systemic inflammation these mice show a dramatic decrease of 8220 positive cells in the bone manow, spleen and peripheral blood. In contrast, T cell populations remained largely unaffected. Conclusion: Our data suggest that the p75TNF-R can mediate systemic inflammatory events, at least when overexpressed in vivo, and most importantly, that p75TNF-R signalling may be involved in mechanisms regulating lymphocyte development and function.
U
P.2.02.04
Ligand inducible, 6 cell-specific gene targeting in mice
F. Schwenk, P.-O. Angrand’, R. Kiihn. A.F. Stewart’, K. Rajewsky. Institute for Genetics, University of Cologne, Weyertall21, D-50931 Cologne, FRG, ’ Eurqean Molecular Biology Laboratory, Meyarhofsfrasse 1, D-691 17 Heidelberg, FRG Introduction: The bacteriophage Pi derived Cre-IoxP recombination system has been recently used to improve the gene targeting technique in mice. Cre catalyzes site-specific DNA recombination between a 34 bp sequence referred to as IoxP such that a lo&flanked segment is excised from a DNA molecule. Conditional gene modification, restricted to a particular cell type or time point, can be achieved by crossing a mouse strain with a IoxP-flanked target gene to a strain expressing Cre recombinase in a spatially or temporally contrdled manner. We have established a system which allows inducible and B cell-specific gene targeting in mice by selective ligand administration. Method: Posttranslational control of the activity of site-specific recombinases can be achieved by expressing a chime& protein in which the mcombinase is fused to the ligand binding domain (LBD) of steroid hormone receptors. In the absence of hormone the steroid receptor LBD is bound by heat shock proteins which inactivate the recombinase, presumably by stetical hindrance. The recombinase can be activated by the addition of ligand which releases the heat shock proteins from the fusion protein. Results: To derive a system which allows inducible and B cell-specific gene targeting in mice, we fused Cre to the mutant (G521R) LBD of the human estrogen receptor which is unresponsive to its natural ligands but can be activated by synthetic hormones, such as 4-Hydroxytamoxifen. The activity of the Cre-LBD fusion was tested in a fibroblast line containing a IoxP-flanked target and in mice carrying the cretransgene under the control of a B cell-specific promoter. Our results demonstrate that the efficiency of Cm-mediated gene deletion upon administration of synthetic hormone approaches 90% in cultured cells and in B cells expressing the Cre-LBD fusion.
P.2.02.05
Expression of GL7 antlgen during B cell development
Mszl6 Cervenak. Attila Magyar ‘, Gl6tia Ldszl6. Dept.of Immunolog): Edtv& Lo&d Vniversiiy, Gad, ’ Inst. of Human Morphology and Debwkpmental Biology, Semmelweis Vniv Medical School, Budapest, Hungary GL7, a 38 kDa murine cell membrane bound lymphocyte activation antigen, appears on B and T cells after 80 hrs in vitro stimulation, but it is also expressed