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Analysis of Homozygous Mutant Chimeric Mice: Deletion Of Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2551-2555, March 1993 Genetics Analysis of homozygous mutant chimeric mice: Deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production (B-cell deficiency/immunoglobulin gene rearrangement/gene targeting/embryonic stem cells) AYA JAKOBOVITS*, GERMAN J. VERGARA, JACQUELINE L. KENNEDY, JOANNA F. HALES, RYAN P. MCGUINNESS, DENISE E. CASENTINI-BOROCZ, DANIEL G. BRENNER, AND GILLIS R. OTTEN Cell Genesys, Inc., 322 Lakeside Drive, Foster City, CA 94404 Communicated by Ronald W. Davis, November 18, 1992 ABSTRACT Using a recently described method for effi- MATERIALS AND METHODS ciently deriving homozygous targeted alleles in embryonic stem cells, we produced chimeric mice whose tissues were derived Targeting-Vector Construction. Plasmid pUC18-JH, con- partially from embryonic stem cells bearing homozygous de- taining a 6.1-kb EcoRI genomic fragment of the mouse Ig letion of the mouse immunoglobulin heavy-chain joining (JH) heavy-chain (Igh) locus (8), was digested with Xho I and Nae region. Characterization of these chimeric mice indicated that I to delete the 2296-bp fragment containing the entire JH of development at region and the DQ52 gene segment, which was replaced by a homozygous JH deletion leads to arrest B-cell 1150-bp Xho I-BamHI fragment of pMClNeopolA (9, 10) to an early stage, resulting in a total lack ofperipheral B cells and form the targeting vector pmHAJ. The unique pUC-derived serum IgM. These results were confirmed in mice containing Nde I site was used for linearization. the homozygous JH deletion in their germ line. This novel Transfection and Selection of Heterozygous and Homozy- B-cell-deficient mouse strain provides a tool for studying the gous AJH-Targeted ES Cell Lines. About 2 x 107 E14TG2a ES recombination and expression of exogenous immunoglobulin cells (11) were electroporated (240 V, 500 ,uF, Bio-Rad Gene genes introduced into the mouse germ line. Pulser) with the linearized targeting vector (50 ,ug/ml). G418 (200 ,g/ml) was added after 24 hr. G418-resistant ES colo- Generation of mice with specific mutations in the immuno- nies were picked and analyzed in pools of four by PCR globulin (Ig) loci, via gene targeting in mouse embryonic stem analysis (45 cycles of 1 min, 94°C; 2 min, 55°C; 3 min, 72°C) (ES) cells, can be a powerful approach for elucidating the using two primers, 5'-ACGGTATCGCCGCTCCCGAT-3' processes of Ig gene assembly and expression and their role and 5'-AGTCACTGTAAAGACTTCGGGTA-3', located 120 in B-cell development. Such mice could obviate the limita- bp 5' of the BamHI site in the neomycin-resistance gene and tions associated with studies of transformed cell lines (1) or 161 bp 3' of the insertion site within the Igh gene, respec- mice carrying Ig transgenes in a background of functional tively. PCR products were analyzed by electrophoresis. endogenous Ig genes (2). Clones from PCR-positive pools were analyzed individually. One valuable model system would be a mouse in which the To generate a AJH-homozygous mutant ES cell line, ES 110-1 process of Ig gene assembly is completely arrested. The cells (5 x 103 per 100-mm plate) were selected in G418 (1400 failure of SCID (3) and RAG-deficient (4, 5) mice to properly ,ug/ml) for 10 days (12); 260 colonies survived (20% of the assemble Ig genes and to produce antibodies is due to mutant expected survival in G418 at 200 jig/ml). genes that act in trans to impair rearrangement of the Ig and Chimeric Mice. Generation of chimeric mice and their T-cell receptor genes. Thus, reconstitution of B-cell devel- breeding were carried out as described (13). opment with unrearranged Ig transgenes cannot be studied in Flow Cytometry of Peripheral Blood and Bone Marrow these mutant hosts. Cells. Peripheral blood mononuclear cells (PBMCs) and bone Our strategy to impair Ig gene assembly was to mutate a marrow cells were purified on Ficoll/Hypaque (Accurate), crucial cis-acting sequence involved in this process. As stained with monoclonal antibody (mAb), and analyzed on a joining of the diversity (D) and heavy-chain joining (JH) gene FACScan (Becton Dickinson). mAbs were phycoerythrin segments is thought to be a prerequisite for heavy-chain (PE)-anti-B220 (Pharmingen), PE-anti-Thy-1.2 (5a8, Caltag), variable region (VH)-D rearrangement, light-chain gene re- and fluorescein isothiocyanate (FITC)-anti-Ly-9.1, -IgMa, or of _IgMb (Pharmingen). The anti-Ly-9.1 mAb stained >99% of arrangement, and B-cell differentiation (1, 6, 7), disruption lymphocytes from the 129/J mouse strain and 0-1% of this event by gene targeting might be sufficient to prevent Ig lymphocytes from the B6 strain. The anti-IgM allotype re- heavy-chain gene assembly and therefore block B-cell de- agents stained 90-97% of B cells in the appropriate strain and velopment and antibody production, thus providing a suitable <1% of B cells in the inappropriate strain. host for introduction of unrearranged Ig transgenes. To identify an Ly-9.1- IgMb- CD43+ B220+ B6-derived To this end we deleted the JH region from one allele by gene subpopulation (see Fig. 5A), the DK207 chimeric bone mar- targeting in ES cells. Furthermore, from single allele-targeted row cells were treated with anti-CD43 (mAb S7, provided by ES cells, we selected a homozygous mutant ES cell clone J. D. Kemp, University of Iowa), followed by FITC-F(ab')2 which gave rise to chimeric mice with a homozygous mutant goat anti-rat IgG (FGAR) (Caltag, South San Francisco, CA). B lineage, for evaluating the effect ofthe JH deletion on B-cell Purified rat IgG (Cappel) was then added to saturate the free development. In addition, chimeric mice were bred to gen- FGAR binding sites. Cells were then incubated with PE-anti- erate mice heterozygous and homozygous in their germ line B220, biotinylated anti-Ly-9.1, and biotinylated anti-IgMb in for the JH deletion (AJH). Abbreviations: AJH, JH deletion; ES cell, embryonic stem cell; Igh, The publication costs of this article were defrayed in part by page charge immunoglobulin heavy chain; mAb, monoclonal antibody; PBMC, payment. This article must therefore be hereby marked "advertisement" peripheral blood mononuclear cell. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2551 2552 Genetics: Jakobovits et al. Proc. Natl. Acad. Sci. USA 90 (1993) A. Targeting Vectorr (pmHAJ) _) FIG. 1. Scheme for deletion ofthe mouse JH region by gene targeting in ES cells. (A) --# | pMCNeopA | pmHAJ targeting vector. (B) Genomic struc- X~~~~~~'*NNX N ture of the mouse JH region and flanking B. Wild type ES cellIgenome X X sequences. The four JH genes and the DQS2 - _ are struc- 521 segment indicated. (C) Predicted U _ X J) TZ t ture of the targeted locus. Arrowheads indi- li1111 t 11 _F cate the approximate positions of the prim- D Jl iTJ2 J3 J4 ers used for the PCR screening. Hybridiza- Prot,e c Probean - tion probes used: probe a, EcoRI-Pst I C. Targeted ES cell genome i 4038bp -1 -<------- -1 fragment, 3' of the insertion site; probe b, _ 578bp* -, 9'17p Xho I-BamHI fragment of the neo gene; 0' ;: u probe c, Xho I-Nae I fragment spanning the L; sizes detected I ~ ~ I II I I JH region. Expected fragment I_. II by probes a, b, and c are shown. The 578-bp ~ i pMlNop1<Probebv 6 F- 3467bp fragment (asterisk) is undetected due to 9libp short overlap with probe c. the presence of excess rat IgG. This was followed by tricolor line, a 917-bp fragment, and an additional 3467-bp fragment streptavidin (Caltag). CD43+ B2201 Ly-9.1+ cells (ES- (Figs. 1 and 2A). Rehybridization with the neo probe (b) DK207-derived) (Fig. SB) were treated similarly except that showed that only the 3467-bp fragment contained neo se- anti-IgMb was omitted. To identify a Ly-9.1- IgMb- Thy-1.2+ quences and demonstrated the absence of additional random B220+ B6 subpopulation (Fig. 5C), we used FITC-anti-Ly- integration events. Deletion ofthe JH region from the targeted 9.1, FITC-anti-IgMb, PE-anti-B220, and biotinylated anti- allele was confirmed with probe c, which detected only the Thy-1.2 followed by tricolor streptavidin. Thy-1.2+ B220+ 4038-bp fragment. These conclusions were confirmed by Ly-9.1+ cells (ES-DK207-derived) (Fig. SD) were treated analysis of Hindlll-digested DNA (data not shown). similarly except that FITC-anti-IgMb was omitted. Purified The effect of a recessive genetic mutation such as AJH can anti-Fcy receptor II (Pharmingen) was added to reduce non- be evaluated in mice homozygous for the mutation. How- specific staining. ever, evaluation of the effect of AJH on Ig expression and Detection ofSerum IgM. Serum IgMa was captured by using B-cell development can be accelerated by AJH-homozygous plastic-immobilized anti-IgMa mAb and detected with biotin- ES cells, which can be used to produce chimeric mice ylated-anti-mouse IgM mAb followed by streptavidin- containing a population of ES-derived lymphoid cells ho- alkaline phosphatase (Pharmingen) and p-nitrophenyl phos- mozygous for the mutant allele. We generated AJH- phate. A405 was measured with a UVmax spectrophotometer homozygous ES cells by subjecting the AJH-heterozygous ES (Molecular Dynamics). 110-1 cell line to elevated levels of G418 (1400 ,g/ml), using a previously described approach (12). Southern blot analysis RESULTS of seven of the surviving colonies indicated that one, ES Generation and Characterization of Heterozygous and Ho- DK207, had lost the native heavy-chain allele and acquired a mozygous AJH-Mutant ES Cells.
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