- Email: [email protected]
188 Establishment of murine monoclonal antibodies against murine urokinase receptor raised in urokinase receptor knock-out mice
54
SESSIONX/V: Emerging Technologies Posters
188
189
ESTABLISHMENT OF MURINE MONOCLONAL ANTIBODIES A G A I N S T M U R I N E UROKINASE R E C E P T O R RAISED IN U R O K I N A S E R E C E P T O R K N O C K - O U T MICE.
EPITOPES OF COMPONENTS OF THE PLASMINOGEN ACTIVATION SYSTEM ARE RE-EXPOSED IN FORMALIN-FIXED PARAFFIN SECTIONS BY DIFFERENT RETRIEVAL TECHNIQUES
Ebbe Ronne, Gunilla H. Hansen, Niels Behrendt, Helene Solberg, Jmgen K. Larsen and Keld Dano. Finsen Laboratory, Rigshospitalet, DK-2100 Copenhagen O, Denmark.
Cilia Ferrier, Winny van Geioof, Michael Kramer', Dirk Ruiter, Goos van Muijen Department of Pathology, University Hospital Nijmegen, The Netherlands * Institute for Immunology, Laboratory for lmmunopathology, University Hospital Heidelberg, Germany
The urokinase plasminogen activator receptor (uPAR) plays a central role during cancer invasion and metastasis by localizing and focalizing the uPA mediated proteolytic activity to the cell surface.We have previously raised monoclonal antibodies to the human uPAR (Ronne, E. et al (1991) FEBS Lett. 288, 233-236) and used them for different analysis of human uPAR. None of the antibodies showed any cross-reactivity to murinc uPAR. In order to study the murine uPAR further we immunized uPAR knock-out mice ( Bugge, T.H. et ai (1995) J. Biol. Chem. 270, 16886-16894) with human soluble uPAR, based on the assumption that it might be possible to raise antibodies to epitopes highly conserved between human and mouse. From one fusion we obtained 22 stable hybridomas. Two of the hybridomas produced antibodies that cross-react with the murin uPAR as analysed by immunoblotting using Tx-114 detergent phase extract from a murine cell line. Further analysis of the antibodies showed that the react with an epitop on domain 2+3 of uPAR and only under non-reducing conditions of the antigen. The antibodies also showed useful for immunoaffiuity purification of soluble murin uPAR as well as staining of uPAR on cells as demonstrated by flow cytometry analysis. In conclusion, we have demonstrated that it is possible to raise antibodies to highly conserved epitopes between human and mouse uPAR by immunizing uPAR knock-out mice with human uPAR. The antibodies generated might be a useful tool for demonstrating the occurence and amount ofmurine uPAR in different biological samples but further studies are needed.
The presence of the five components of the plasminogen activation (PA)-system, i.e. uPA, tPA, PAI-1, PAI-2 and uPAR, can well be studied by immunohistochemistry. This technique informs on the quality of expression by the different cell types. Reliable immunohistochemistry on paraffin sections allows retrospective studies using archival material. As counts for many antigens, formalin-fixation and paraffin-embedding interferes with the aceessability of epitopes of the PA-components for the specific antibodies. We performed a sytematic analysis of the sensitivity and specificity of immunohistochemical stainings on routinely processed (formalin-fixed and paraffin-embedded) tissues. For each of the five PA-components, a great number of antibodies were tested and the influence of different antigen retrieval regimens (six microwavemediated pretreaments and two pretreatments by proteolytic digestion) on immunoreactivity were investigated. In the first stage, stalnings were performed on positive and negative control tissues. Then, with selected antibodies and modes of pretreatment, frozen and paraffin sections from the same cancer lesions were stained and staining patterns were compared. For each component, only one or a few of the tested antibodies gave optimal staining on paraffin sections (highly comparable with the staining profiles on frozen sections) in case of combination with a specific pretreatment of the section. For PAI-1, and in a lesser degree also for tPA, an underrepresentation of stromal-cell staining in paraffin material was found while tumor cells showed good staining. For uPA, PAI-2 and uPAR, consistently-representative staining results were obtained on paraffin sections.
190
191
CHARACTERIZATION AND TARGETING OF THE MURINE c%ANTIPLASMIN GENE
TARGETED DISRUPTION OF MOUSE PROTEIN C INHIBITOR GENE
H.R. Lijnen I M. Dewerehin2,K. Okeda ~'3,A. Belayew ~, D. Collen L'2. Center for Molecular and Vascular Biology, KU Leuven, and 2Center for Transgene Technology and Gene Therapy, Flanders lntemniversity Institute for Biotechnology, Leuven, Belgium and Department of Physiology, Kinki University, Osaka, Japan
Institute
The physiological consequences of targeted disruption of the et2-antiplasmin (¢t2-AP) gene will be studied in mice. Therefore, the routine ct2-APgeae was characterized and targeted gene inactivation was achieved by homologous recombination in embryonic stem (ES) cells. A mouse 129 genomic library in P1 phage was screened by PCR using sets of primers annealing in exon 2, 3 or 10 of a2-AP. DNA containing routine ~-AP sequences was digested with HindIII and the fragments were subcloned in the HindIII site of plasmid pUG19. Purified recombinant plasmid DNA was characterized by restriction mapping and necleotide sequencing. A homolegous sequence of 8.3 kb in total was introduced in the parental pPNT vector yielding the targeting vector pPNT.~-AP. The linearized targeting vector was electroporated into 129 R1 ES ceils and targeted clones were used for morula aggregation. Compared to the human gene, exons 2 through 9 in the murine gone have identical size and intron-exon boundaries obeying the GT/AG rule. The 5 ' boundary of exon l0 is identical in both genes while the 3' non-coding region is extended by 64 bp in the human gene. Introns 2, 3, 6 and 8 have similar sizes, whereas introns 1, 5, 7 and 9 are 2- to 6-fold smaller and intrun 4 is 2fold larger in the mouse gene. The open reading frame of the mouse ~x2-AP gene encodes a 491 amino acid protein. The organisation of the routine ct2-AP 5"flanking region differs from that of the human gene and contains a simple repeat sequence (TGG)n about 360 nt upstream of exon 1. The functionality of the cloned murine 5'flanking region was confirmed in both murine and human hepatocyte cell lines using lueiferase reporter constructs containing a 2.0 kb 5'flanking fragment comprising the (TGG)n repeat region. In the targeting vector pPNT.ctz-AP,the neomycin resistance expression cassette replaces a 7 kb ganomic fragment comprising exon 2 (with the start codon) through part of exon l0 (with the stop codon). Four correctly targeted ES clones were obtained and used for morula aggregation yielding chimeric offspring. At the present, one male chimera transmitted the inactivated allele through the germline generating beteruzygous ~-AP deficient progeny. These will be intercrossed to obtain homozygous t~-AP deficient mice.
Uhrin, P., Hilpert, M., Zechmeister-Machhart, M., Geiger, M. and Binder, B.R. for
Vascular
Biology
and
Thrombosis
Research,
Schwarzspanierstrasse 17, A- 1090 Vienna, Austria Protein C inhibitor (PCI) is a serine protease inhibitor that inhibits a variety of plasmatic and extravascular serine proteases. So far, however, the physiological role of PCI has not been elucidated. It is therefore our aim to study the effect o f the knock out of PCI in respect to the tissue damage, bemostatie system, kallikrein - kinin system and the reproduction system, as well. 129/Sv mouse genomic library in the Lambda FIX II vector was screened with mouse PCI cDNA probe and five independent clones were isolated. They were checked for additional rearrangements by a series of restriction digestions. To disrupt the PCI gene, the targeting vector pGNA in which a cassette containing a neomycin (neo) gene and the lac Z with its own ATG was used. The 3" region of the mouse PCI gene (containing part of the sequence ofintron 2 and exon III) and the 5" mPCI region containing the promoter, untranslated exon I, intron t and part of the exon II sequence coding the first 15 aminoacids of mPCI as well as a fragment encoding thymidine kinase gene was inserted into this vector. The targeted vector has been successfully tested for homologous recombination in CCE embryonic stem (ES) cells. Three PCR positive clones were obtained and correct homologous recombination was confirmed by Southern blotting of purified genomic DNA using appropriate restriction enzymes and probes. Transgenic mice will be produced by injection of targeted feeder dependent ES cells, harbouring a homologously recombined PCI allele, into the mouse blastocyst.
SESSIONX/V: Emerging Technologies Posters
188
189
ESTABLISHMENT OF MURINE MONOCLONAL ANTIBODIES A G A I N S T M U R I N E UROKINASE R E C E P T O R RAISED IN U R O K I N A S E R E C E P T O R K N O C K - O U T MICE.
EPITOPES OF COMPONENTS OF THE PLASMINOGEN ACTIVATION SYSTEM ARE RE-EXPOSED IN FORMALIN-FIXED PARAFFIN SECTIONS BY DIFFERENT RETRIEVAL TECHNIQUES
Ebbe Ronne, Gunilla H. Hansen, Niels Behrendt, Helene Solberg, Jmgen K. Larsen and Keld Dano. Finsen Laboratory, Rigshospitalet, DK-2100 Copenhagen O, Denmark.
Cilia Ferrier, Winny van Geioof, Michael Kramer', Dirk Ruiter, Goos van Muijen Department of Pathology, University Hospital Nijmegen, The Netherlands * Institute for Immunology, Laboratory for lmmunopathology, University Hospital Heidelberg, Germany
The urokinase plasminogen activator receptor (uPAR) plays a central role during cancer invasion and metastasis by localizing and focalizing the uPA mediated proteolytic activity to the cell surface.We have previously raised monoclonal antibodies to the human uPAR (Ronne, E. et al (1991) FEBS Lett. 288, 233-236) and used them for different analysis of human uPAR. None of the antibodies showed any cross-reactivity to murinc uPAR. In order to study the murine uPAR further we immunized uPAR knock-out mice ( Bugge, T.H. et ai (1995) J. Biol. Chem. 270, 16886-16894) with human soluble uPAR, based on the assumption that it might be possible to raise antibodies to epitopes highly conserved between human and mouse. From one fusion we obtained 22 stable hybridomas. Two of the hybridomas produced antibodies that cross-react with the murin uPAR as analysed by immunoblotting using Tx-114 detergent phase extract from a murine cell line. Further analysis of the antibodies showed that the react with an epitop on domain 2+3 of uPAR and only under non-reducing conditions of the antigen. The antibodies also showed useful for immunoaffiuity purification of soluble murin uPAR as well as staining of uPAR on cells as demonstrated by flow cytometry analysis. In conclusion, we have demonstrated that it is possible to raise antibodies to highly conserved epitopes between human and mouse uPAR by immunizing uPAR knock-out mice with human uPAR. The antibodies generated might be a useful tool for demonstrating the occurence and amount ofmurine uPAR in different biological samples but further studies are needed.
The presence of the five components of the plasminogen activation (PA)-system, i.e. uPA, tPA, PAI-1, PAI-2 and uPAR, can well be studied by immunohistochemistry. This technique informs on the quality of expression by the different cell types. Reliable immunohistochemistry on paraffin sections allows retrospective studies using archival material. As counts for many antigens, formalin-fixation and paraffin-embedding interferes with the aceessability of epitopes of the PA-components for the specific antibodies. We performed a sytematic analysis of the sensitivity and specificity of immunohistochemical stainings on routinely processed (formalin-fixed and paraffin-embedded) tissues. For each of the five PA-components, a great number of antibodies were tested and the influence of different antigen retrieval regimens (six microwavemediated pretreaments and two pretreatments by proteolytic digestion) on immunoreactivity were investigated. In the first stage, stalnings were performed on positive and negative control tissues. Then, with selected antibodies and modes of pretreatment, frozen and paraffin sections from the same cancer lesions were stained and staining patterns were compared. For each component, only one or a few of the tested antibodies gave optimal staining on paraffin sections (highly comparable with the staining profiles on frozen sections) in case of combination with a specific pretreatment of the section. For PAI-1, and in a lesser degree also for tPA, an underrepresentation of stromal-cell staining in paraffin material was found while tumor cells showed good staining. For uPA, PAI-2 and uPAR, consistently-representative staining results were obtained on paraffin sections.
190
191
CHARACTERIZATION AND TARGETING OF THE MURINE c%ANTIPLASMIN GENE
TARGETED DISRUPTION OF MOUSE PROTEIN C INHIBITOR GENE
H.R. Lijnen I M. Dewerehin2,K. Okeda ~'3,A. Belayew ~, D. Collen L'2. Center for Molecular and Vascular Biology, KU Leuven, and 2Center for Transgene Technology and Gene Therapy, Flanders lntemniversity Institute for Biotechnology, Leuven, Belgium and Department of Physiology, Kinki University, Osaka, Japan
Institute
The physiological consequences of targeted disruption of the et2-antiplasmin (¢t2-AP) gene will be studied in mice. Therefore, the routine ct2-APgeae was characterized and targeted gene inactivation was achieved by homologous recombination in embryonic stem (ES) cells. A mouse 129 genomic library in P1 phage was screened by PCR using sets of primers annealing in exon 2, 3 or 10 of a2-AP. DNA containing routine ~-AP sequences was digested with HindIII and the fragments were subcloned in the HindIII site of plasmid pUG19. Purified recombinant plasmid DNA was characterized by restriction mapping and necleotide sequencing. A homolegous sequence of 8.3 kb in total was introduced in the parental pPNT vector yielding the targeting vector pPNT.~-AP. The linearized targeting vector was electroporated into 129 R1 ES ceils and targeted clones were used for morula aggregation. Compared to the human gene, exons 2 through 9 in the murine gone have identical size and intron-exon boundaries obeying the GT/AG rule. The 5 ' boundary of exon l0 is identical in both genes while the 3' non-coding region is extended by 64 bp in the human gene. Introns 2, 3, 6 and 8 have similar sizes, whereas introns 1, 5, 7 and 9 are 2- to 6-fold smaller and intrun 4 is 2fold larger in the mouse gene. The open reading frame of the mouse ~x2-AP gene encodes a 491 amino acid protein. The organisation of the routine ct2-AP 5"flanking region differs from that of the human gene and contains a simple repeat sequence (TGG)n about 360 nt upstream of exon 1. The functionality of the cloned murine 5'flanking region was confirmed in both murine and human hepatocyte cell lines using lueiferase reporter constructs containing a 2.0 kb 5'flanking fragment comprising the (TGG)n repeat region. In the targeting vector pPNT.ctz-AP,the neomycin resistance expression cassette replaces a 7 kb ganomic fragment comprising exon 2 (with the start codon) through part of exon l0 (with the stop codon). Four correctly targeted ES clones were obtained and used for morula aggregation yielding chimeric offspring. At the present, one male chimera transmitted the inactivated allele through the germline generating beteruzygous ~-AP deficient progeny. These will be intercrossed to obtain homozygous t~-AP deficient mice.
Uhrin, P., Hilpert, M., Zechmeister-Machhart, M., Geiger, M. and Binder, B.R. for
Vascular
Biology
and
Thrombosis
Research,
Schwarzspanierstrasse 17, A- 1090 Vienna, Austria Protein C inhibitor (PCI) is a serine protease inhibitor that inhibits a variety of plasmatic and extravascular serine proteases. So far, however, the physiological role of PCI has not been elucidated. It is therefore our aim to study the effect o f the knock out of PCI in respect to the tissue damage, bemostatie system, kallikrein - kinin system and the reproduction system, as well. 129/Sv mouse genomic library in the Lambda FIX II vector was screened with mouse PCI cDNA probe and five independent clones were isolated. They were checked for additional rearrangements by a series of restriction digestions. To disrupt the PCI gene, the targeting vector pGNA in which a cassette containing a neomycin (neo) gene and the lac Z with its own ATG was used. The 3" region of the mouse PCI gene (containing part of the sequence ofintron 2 and exon III) and the 5" mPCI region containing the promoter, untranslated exon I, intron t and part of the exon II sequence coding the first 15 aminoacids of mPCI as well as a fragment encoding thymidine kinase gene was inserted into this vector. The targeted vector has been successfully tested for homologous recombination in CCE embryonic stem (ES) cells. Three PCR positive clones were obtained and correct homologous recombination was confirmed by Southern blotting of purified genomic DNA using appropriate restriction enzymes and probes. Transgenic mice will be produced by injection of targeted feeder dependent ES cells, harbouring a homologously recombined PCI allele, into the mouse blastocyst.