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The K BxN Arthritis Model 1 1 UNIT 15 22 1 Paul A Monach Diane Mathis and Christophe Benoist 1 Joslin Diabetes Center Harvard Medical School Boston Massachusetts ABSTRACT Mice expressing both the T cell receptor TCR transgene KRN and the MHC class II molecule Ag7 K BxN mice develop severe inflammatory arthritis and serum from these mice causes a similar arthritis in a wide range of mouse strains due to autoantibodies recognizing glucose 6 phosphate isomerase GPI K BxN transgenic mice have been useful for investigating the development of autoimmunity and the serum transfer model has been particularly valuable in eliciting mechanisms by which anti GPI autoantibodies induce joint specific inflammation This unit describes detailed methods for the maintenance of a K BxN colony induction of arthritis by serum transfer clinical evaluation of arthritis and measurement of anti GPI antibodies Curr Protoc Immunol C 2008 by John Wiley Sons Inc 81 15 22 1 15 22 12 Keywords arthritis r rheumatoid arthritis r glucose 6 phosphate isomerase r GPI r anti GPI r serum transfer r autoantibodies r mouse model r KRN r K BxN INTRODUCTION Mice expressing the transgenic T cell receptor TCR KRN and the MHC class II allele Ag7 K BxN mice uniformly develop severe inflammatory arthritis and serum from these mice reliably causes arthritis in a wide range of recipient strains serum transfer model due to high levels of autoantibodies to the glycolytic enzyme glucose 6 phosphate isomerase GPI This model system has been useful for identifying genetic determinants of arthritis first by crossing the KRN and Ag7 genes to other genetic backgrounds and second and more powerfully by transferring serum into a variety of gene disrupted and inbred strains The serum transfer system allows for the rapid assessment of potential genetic determinants particular to the effector phase of arthritis independent of determinants of the earlier step of breaking T cell tolerance The following series of protocols is designed to allow any laboratory with general experience in mouse experimentation to set up a colony to produce arthritic K BxN mice and conduct experiments using both the transgenic see Strategic Planning and serum transfer see Basic Protocols 2 and 3 models The maintenance of a colony to produce K BxN mice requires two lines for breeders 1 C57Bl 6 or B10 BR mice carrying the KRN transgene heterozygously and 2 NOD Lt or other mice carrying the Ag7 allele homozygously KRN mice are somewhat immunocompromised due to a limited diversity of TCR among T cells resulting from transgene expression but they do not require special conditions for maintenance Mice homozygous for KRN are viable but have reduced breeding performance so depending on the facility the colony overall will sometimes perform better using KRN heterozygotes despite the inefficiencies of having to genotype breeders and of obtaining only 50 KRN offspring KRN mice can be identified by flow cytometry on the basis of a high number of V 6 positive T cells see Basic Protocol 1 or by PCR of genomic DNA see Alternate Protocol The Support Protocol describes a method for measuring anti GPI antibody titers Animal Models for Autoimmune and In ammatory Disease Current Protocols in Immunology 15 22 1 15 22 12 April 2008 Published online April 2008 in Wiley Interscience www interscience wiley com DOI 10 1002 0471142735 im1522s81 C 2008 John Wiley Sons Inc Copyright 15 22 1 Supplement 81 STRATEGIC PLANNING In order to test the effect of a gene on spontaneous arthritis in K BxN transgenic mice it is necessary to first cross the KRN transgene and the H 2g7 locus separately onto the background with the gene to be tested e g a disrupted gene denoted X then breed these offspring to obtain K BxN mice with the gene of interest as outlined in Figure 15 22 1 The first cross generates KRN X and H 2g7 b X animals Since intercrossing these mice would produce a low yield 1 16 of the KRN H 2g7 b X mice of interest it is generally preferable to include a second round of breeding to enrich for the relevant genes Thus KRN X mice are intercrossed to generate Figure 15 22 1 Crossing KRN and H 2g7 into other genetic backgrounds A gene of interest X is rst crossed separately to KRN left side of gure and H 2g7 right side so as to obtain experimental mice equivalent to K BxN on X background at a frequency of 25 in the third cross along with arthritic K BxN X littermates for comparison The breeding scheme will need to be modi ed if X mice are unable to breed resulting in 12 5 experimental mice in the nal cross Mice shown as smaller are those that are not used for subsequent breeding steps or experimentation Gray circles indicate either or homozygotes of KRN or any genotype of X in a setting in which any of these genotypes is not of interest for the next cross 15 22 2 Supplement 81 Current Protocols in Immunology KRN X offspring at a frequency of 1 8 and H 2g7 b X mice are intercrossed g7 to generate H 2g7 X offspring at a frequency of 1 8 Finally KRN X mice g7 are crossed to H 2g7 X mice in order to generate experimental KRN H 2g7 b X mice and control littermates KRN H 2g7 b X KRN H 2g7 b X and KRN H2g7 b X at frequencies of 1 4 each As described above KRN X mice may be obtained in the first intercross at a frequency of 1 16 and could be used for the final cross in order to maximize the number of experimental mice but breeding performance may be reduced and all control littermates will have both KRN and H 2g7 and thus develop arthritis KRN mice can be detected as in Basic Protocol 1 or 2 H 2g7 b and H 2g7 g7 can be distinguished by flow cytometry of whole blood leukocytes using antibodies that distinguish Ag7 from Ab e g monoclonal antibodies 10 2 16 and Y 3P respectively NOTE All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee IACUC and must follow officially approved procedures for the care and use of laboratory animals SCREENING FOR KRN MICE BY FLOW CYTOMETRY This protocol describes a flow cytometric method for confirming expression of the KRN transgene in putative KRN mice In KRN mice most CD4 cells are V 6 compared to 10 of CD4 cells in wild type mice Fluorescein conjugated anti mouse V 6 TCR antibody is used to assess the frequency of V 6 CD4 T cells BASIC PROTOCOL 1 Materials Male mice to be screened for KRN expression on the C57Bl 6 or B10 BR background see Figure 15 22 1 FACS wash buffer see recipe Heparin RBC lysis buffer see

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