1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Decay-accelerating factor (DAF) is a membrane regulator of C3 activation that protects self cells from autologous complement attack. In humans, DAF is uniformly expressed as a glycosylphosphatidylinositol (GPI)-anchored molecule. In mice, both GPI-anchored and transmembrane-anchored DAF proteins are produced, each of which can be derived from two different genes (Daf1 and Daf2). In this report, we describe a Daf1 gene knock-out mouse arising as the first product of a strategy for targeting one or both Daf genes. As part of the work, we characterize recently described monoclonal antibodies against murine DAF protein using deletion mutants synthesized in yeast, and then employ the monoclonal antibodies in conjunction with wild-type and the Daf1 knock-out mice to determine the tissue distribution of the mouse Daf1 and Daf2 gene products. To enhance the immunohistochemical detection of murine DAF protein, we utilized the sensitive tyramide fluorescence method. In wild-type mice, we found strong DAF labelling of glomeruli, airway and gut epithelium, the spleen, vascular endothelium throughout all tissues, and seminiferous tubules of the testis. In Daf1 knock-out mice, DAF labelling was ablated in most tissues, but strong labelling of the testis and splenic dendritic cells remained. In both sites, reverse transcription-polymerase chain reaction analyses identified both GPI and transmembrane forms of Daf2 gene-derived protein. The results have relevance for studies of in vivo murine DAF function and of murine DAF structure.






bovine serum albumin


complement control protein modules


decay-accelerating factor


enhanced chemiluminescence


haemolysin-sensitized sheep erythrocytes




fluorescein isothiocyanate






horseradish peroxidase


monoclonal antibodies




  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Decay-accelerating factor (DAF) is a complement regulator that functions intrinsically in the membranes of self cells to circumvent the deposition of autologous C3b on their surfaces.1 It acts to accelerate decay-dissociation of the bimolecular C3 convertases ( inline image and inline image), the central amplification enzymes of the cascade.2–4 In humans, DAF is encoded by a single gene on chromosome 1.5 It is unusual structurally in that, in lieu of a conventional transmembrane (TM) polypeptide, it is expressed with a post-translationally added glycosylphosphatidylinositol (GPI) anchor.6,7 Addition of the anchor is directed by a C-terminal signal peptide in the primary translation product of the protein. In humans, DAF activity is important physiologically: its absence in paroxysmal nocturnal haemoglobinuria leads to increased C3b uptake on affected cells,8 its expression by tumours enhances their resistance to immune elimination,9 and its expression by xenografts prolongs their survival.10–12 Additionally it serves as a ligand for activated leucocytes and possibly other cell types which express CD97.13,14 The importance of this second, presumably non-complement-related function is as yet uncharacterized.

By chromatographic fractionation of detergent extracts of murine erythrocyte stroma, Kameyoshi et al.15 isolated murine DAF protein, and with the use of anti-murine DAF antibody in conjunction with expression cloning, Fukuoka et al.16 isolated murine Daf cDNA. In the course of examining compensatory genes expressed in mice deficient in membrane-associated mucin protein (Muc-1), Spicer et al.17 incidentally cloned two different Daf complementary DNAs (cDNAs) and analysed the mouse Daf genome. Two duplicated closely spaced genes were identified, Daf1 and Daf2, which were found, respectively, to give rise to messenger RNAs (mRNAs) encoding GPI-anchored and TM forms of the protein. Independently in the course of analysing murine uterine mRNAs induced by oestrogen, Song et al.18 recovered a Daf cDNA encoding GPI-anchored DAF. Recently, Ohta et al.19 and Spiller et al.20 have raised a number of monoclonal antibodies (mAbs) against murine DAF protein.

In previous studies using polymerase chain reaction (PCR) and Northern blot analyses, Spicer et al.17 showed that mRNAs deriving from the two Daf genes are differentially expressed in various organs and that Daf2 mRNA expression is highest in testis. Because the immunohistochemical detection of murine DAF in tissues has been difficult, the distribution of murine DAF expression at the protein level is not yet characterized. In this communication, we describe the production of a Daf1 gene knock-out mouse employing a Cre/loxP targeting strategy. By using a highly sensitive detection method of immunohistochemical staining, we report the distribution of mouse DAF protein in the tissues of wild-type mice and of the Daf1 knock-out mice. For this work we determined the reactive sites of the available mAbs to murine DAF, using deletion mutants of murine DAF protein produced in yeast.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References


Rat anti-mouse DAF mAbs MD2C6 (2C6), MD3D5 (3D5) and MD1,20 and hamster anti-murine DAF mAbs RIKO2, RIKO3 and RIKO419 were obtained as described.

Construction of targeting vectors

The mouse Daf1 and Daf2 genes were derived from P1 plasmids as previously used (Spicer A., unpublished results). The targeting vectors pNEOUMSLOX(−)TKDT, and pHYGROLOX(+)TKDT containing loxP sites and the Cre recombinase expression vector pBS185 were gifts from Dr Charles Weissman and Dr Z. W. Li (Universitat Zurich, Zurich, Switzerland, and University of California, San Diego, CA).21 The upstream targeting vector, pDAFup, was generated by blunt-end ligating a 3-kb PCR fragment spanning exon 1 to exon 3 of the Daf1 gene and an ∼3 kb PCR fragment spanning exon 3 to exon 5 of the Daf1 gene into the PmeI site and filled-in SalI site, respectively, of pNEOUMSLOX(−)TKDT. The downstream targeting vector, pDAFdown, was constructed by inserting 2·9 kb of PCR sequence spanning exon 3 to exon 5 and 2·5 kb of PCR-sequence encompassing intron 5 of the Daf2 gene into the filled-in SalI site and PmeI site, respectively, of pHYGROLOX(+)TKDT.

Disruption of the Daf1 gene in ES cell lines

The ES cell line GK129 was cultured as described.21 Fifty microgrammes of pDAFup was linearized with SfiI and electroporated into 2 × 107 ES cells at 250 V/500 µF using a gene pulser (Bio-Rad, Hercules, CA). The electroporated cells were incubated with 400 µg/ml G418, 8–9 days after which 800 surviving colonies were picked and expanded in 96-well plates. Ninety-six colonies were analysed by PCR using primers P1 and P2 (see Fig. 1) and positive colonies were analysed by Southern blotting using the upstream 2·0 kb portion of Daf1 gene intron 5 extending to its EcoRI site.


Figure 1. Targeting strategy for inactivation of the mouse Daf genes. The tandem Daf1 and Daf2 genes are shown diagrammatically (not drawn to scale). The black-filled boxes represent exons and the open boxes represent selection marker genes as marked. As the figure shows, the pDAFup construct integrates to the Daf1 gene first to introduce a loxP site and a TK gene within exon 3, then the pDAFdown construct may integrate into its homologous region in either the Daf1 or Daf2 gene together with another loxP site and TK gene. Upon recombination induced by Cre recombinase, the DNA fragment flanked by the two loxP sites is deleted together with the two TK genes to generate either Daf1 or Daf2 knock-out ES cells.

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Following expansion of two positive Neo-loxP clones in ES cell medium containing 400 µg/ml G418, they were co-electroporated with 40 µg of linearized pDAFdown and 20 µg of pBS185. After 24 hr in 400 µg/ml G418, 100 µg/ml of hygromycin (Hph) was added, and after another 96 hr, 2′deoxy-2′fluoro-β-d-arabinofuranosyl-5-iodouracil (FIAU) was added to a final concentration of 0·2 µm. Fifteen days later, clones were isolated. The Cre/loxP recombination in the resulting clones was confirmed by PCR with primers P3 and P4 and the Daf gene structure in each clone was analysed by PCR using primer P5 and gene-specific primers P6 and P7 (see Fig. 1). The sequences of the primers used were: P1, 5′-GTCCACGACCCAAGCTTGGCTGCAGGTC-3′; P2, 5′-GCGGCCCCACAACTTGAGTACTTTAAAGAGGAGAC-3′; P3, 5′-TGCTCCTGAAGTCCACAATTCACAGTCC-3′; P4, 5′-CGAGACTAGTGAGACGTGCTACTTCCATTT-3′; P5, 5′-CGAGATCAGCAGCCTCTGTTCCACA-3′; P6, 5′-CTTGCTTGGGACCTTGGATTT-3′; and P7, 5′-CTTAATTGGGACCTTAGATTC-3′.

Generation of DAF-deficient mice

Karyotyped targeted ES cells were microinjected into C57BL/6 blastocysts, and the resulting male chimeric mice with > 80% agouti phenotype were bred into the C57BL/6 strain. F1 mice carrying the mutation were bred with each other to obtain homozygotes. All animal work was approved and performed under the guidelines set by the Case Western Reserve University Institutional Animal Care and Use Committee.

Expression of different murine DAF mutants in pichia

Murine DAF complement control protein modules (CCPs) in different combinations were expressed using the Pichia pastoris system.22 The coding regions for mouse DAF CCP1-4, CCP2-3, CCP3-4, CCP2-4, CCP1-2,4 and CCP1,3-4 were amplified from mouse Daf1 cDNA with proofreading Vent DNA polymerase (New England Biolabs, Beverly, MA). PCR products were cloned into the EcoRI and XbaI sites of the expression vector pPICZαA. Following sequence confirmations, the resulting expression constructs were transformed into Pichia strain SMD1168. Expression was induced with 1% methanol for 24 hr, and supernatants were harvested.

Identification of CCPs recognized by anti-mouse DAF mAbs

Supernatants containing DAF CCP variants were loaded onto non-reducing 12% SDS–PAGE gels and the electrophoresed proteins were transferred to polyvinyldifluoride (PVDF) membranes. Each of the rat and hamster mAbs along with polyclonal rabbit anti-mouse DAF antibody15 was incubated with the blots and the blots were developed using rabbit anti-rat (DAKO, Carpenteria, CA), goat anti-hamster (Southern Biotechnology, Birmingham, AL), or goat anti-rabbit (DAKO) secondary antibodies [all labelled with horseradish peroxidase (HRP)] followed by enhanced chemiluminescence (ECL) reagent (Amersham Life Sciences, Arlington Heights, IL).

Fluorescence-activated cell sorter (FACS) analyses of DAF levels

Erythrocytes collected from tail veins were incubated for 30 min on ice with 10 µg/ml of rat anti-murine DAF mAb 2C6. Following washing, the cells were secondarily incubated for 30 min on ice with a 1 : 500 dilution of fluorescein isothiocyanate (FITC)-labelled anti-rat immunoglobulin (Dako) and the stained cells were analysed in a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Analyses of in vivo C3 deposition

Erythrocytes (1 × 107) collected from tail veins were incubated for 30 min on ice with a 1 : 400 dilution goat anti-murine C3 antibody (ICN). Following washing, the cells were secondarily incubated for 30 min on ice with 1 : 500 dilution biotin-labelled rabbit anti-goat immunoglobulin G (IgG) antibody (Zymed, San Francisco, CA). Following a second washing, the cells were stained on ice for another 30 min with 1 : 2000 dilution of streptavidin–FITC (Sigma, St Louis, MO). The triple-stained cells were then analysed with controls in a FACScan flow cytometer as above.

RT-PCR identification of mRNAs encoding gene-specific and GPI-and TM-DAF-specific protein

Total RNA was isolated from tissues (spleen, kidney and testis) with TRIzol reagent (Life Technology, Rockville, MD). Reverse transcription-polymerase chain reaction (RT-PCR) was done by standard methods using an oligo-dT primer to synthesize the first strand cDNAs from total RNA. Gene-specific primers P8 (Daf1-specific), 5′-ATGATCCGTGGGCGGGCGCCT-3′, P9 (Daf2-specific), 5′-CCTCAAAACAGCTCCGGCCAA-3′ and P10, 5′-ATCTATGCACCGGGGTGGTGGAC-3′ were designed to amplify from the signal peptide sequence to the end of CCP4 of the Daf1 and Daf2 genes, respectively. PCR with primers P11, 5′-GTGGGCCGCTCTAGGCACCA-3′ and P12, 5′-TGGCCTTAGGGTGCAGGGGG-3′ was included to amplify β-actin cDNA as a control. GPI-specific primer P13, 5′-CATCCTTCCTCTCCTTTCCTTGAA-3′ and TM-specific primer P14, 5′-CTATGTCAAGTAGCCAATGAGTGA-3′ together with Daf2 gene-specific primer P9 were employed to determine which form of mouse Daf mRNA is produced by the Daf2 gene.

Complement and CD97 binding assays

The cDNA encoding the Daf1 gene CCP1,4 product present in the Daf1 knock-out mouse was amplified by RT-PCR of total kidney RNA. It was expressed in Pichia pastoris as described above and following purification on Ni2+–Sepharose, the product was dialysed against 0·1% gelatine 2·5 mm veronal buffer (pH 7·4) containing 146 mm NaCl, 0·15 mm CaCl2 and 0·5 mm MgCl2 (GVB++). Haemolysin-sensitized sheep erythrocytes (EshA) (10 µl, 1 × 109/ml) were incubated at 37° for 30 min with 10 µl of mouse serum in the presence of 10 µl of increasing concentrations of the Daf1 gene CCP1,4 product, native mouse CCP1-4 or bovine serum albumin (BSA). Cells were then HRP-labelled, stained with FITC-labelled goat anti-mouse C3 immunoglobulin and C3 uptake was measured by flow cytometry.

For measuring CD97 binding activity, COS cells were transiently transfected with pcDNA3 containing mouse CD97 (EGF1,2,4) cDNA kindly provided by Dr J. Hamann (CLB, Amsterdam, the Netherlands) using lipofectamine (Life Technologies, Rockville, MD) in six-well culture plates. After 3 days, 1 × 108 erythrocytes from wild-type or knock-out mice were overlaid on the COS cells for 30 min at room temperature. Non-adhering red blood cells were removed by washing three times with phosphate-buffered saline (PBS). The number of erythrocytes remaining attached per high-powered field was counted.

Immunohistochemical analyses of tissues from wild-type and Daf1−/− mice

Mouse tissues were frozen on corks in OCT compound (Sakura Finetech USA, Torrance, CA) in isopentane. Frozen sections (5 µm thickness) cut on a Cryocut 1800 cryostat (Leitz, GMBH, Germany) were picked up on silane-prep slides (Sigma) and air-dried. Sections were fixed for 5 min at 20° in acetone, and endogenous peroxidase quenched by incubation for 30 min in 0·25% H2O2/methanol. Sections were washed in PBS and incubated for 30 min with blocking buffer [TSA Fluorescence system kit (NEN Life Science Products Inc, Boston, MA)]. They were then incubated for 30 min at 20° with 2C6 rat anti-mouse DAF antibody (3 µg/ml) or irrelevant isotype-matched rat IgG2a (PharMingen, San Diego, CA). The treated sections were subsequently washed with TNT (0·1 m Tris, pH 7·5, 0·15 m NaCl, 0·05% Tween-20) buffer and incubated for 30 min with 3 µg/ml HRP-conjugated mouse anti-rat IgG (Jackson ImmunoResearch Labs. Inc., West Grove, PA). After a second wash with TNT buffer, the sections were stained for 6 min at 20° with Tyramide-green (NEN Life Science Products Inc.). The stained sections were examined using an OM6 fluorescence microscope (Olympus Optical Co., Ltd, Tokyo, Japan). In some experiments, biotin anti-mouse/rat I-A class II-OX3 (Immunotech, Coulter Corp, Miami, FL) and Texas-Red-streptavidin (Zymed Lab Inc, San Francisco, CA), or FITC-conjugated anti-mouse CD19 ID3 (PharMingen), hamster anti-mouse CD11c N418 (Endogen Inc, Woburn, MA) and HRP-conjugated anti-rat IgG followed by Tyramide-red (NEN Life Science Products Inc.) were used.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Generation of DAF knock-out ES cells

Because two DAF genes are present in the mouse genome (in ‘head-to-tail’ organization), and independent Daf1 and Daf2 gene knock-outs could occur on different chromosomes, a single Cre/loxP targeting strategy was designed to allow for either single elimination of the Daf1 gene to produce a selective Daf1 knock-out mouse or concomitant elimination of both Daf genes to produce a complete DAF knock-out mouse. An additional advantage of the approach was that it would self-screen for clones (see Discussion). For this system, we first introduced an upstream loxP site into the 5′ end of the Daf1 gene, and then, in conjunction with Cre recombinase, introduced a downstream loxP site able to integrate into the 3′ end of the Daf1 or Daf2 coding region (Fig. 1).

Following the initial step of the procedure, i.e. introduction of the upstream targeting construct, pDAFup, into the cells, eight clones carrying the correct integration were identified by PCR and confirmed by Southern blotting. In the first set of subsequent transfections, i.e. introduction of the pDAFdown targeting vector containing the TK-loxP-Hph cassette (in conjunction with the Cre expression plasmid pBS185), selection with G418, Hph and FIAU yielded seven colonies. PCR (Fig. 2, panel A1) with primers P3 and P4 (see Fig. 1) verified that the Neo and Hph cassettes were brought together after the Cre/loxP rearrangement, indicative of deletion of the region flanked by the two loxP sites. Additional PCR of three of the colonies using primers P6 and P7 (see Fig. 1) specific for exon 6 of the Daf1 and Daf2 genes, respectively, together with primer P5 demonstrated the pDAFdown construct recombined into homologous sequence in the Daf1 gene (Fig. 2 panel A2), and consequently selectively knocked out the Daf1 gene (see Fig. 1).


Figure 2. PCR analysis of recombined ES cells. (a1) PCR with Neo and Hph-specific primers P3 and P4 yielded a ∼500-bp fragment verifying that the two markers were brought together by Cre/loxP recombination. M, 1 kb ladder; C, PCR with the wild-type DNA as template; K/O, recombined DNA as template. (a2) PCR with primers P5, P6 (Daf1-specific) and P7 (Daf2-specific) showing that the pDAFdown construct integrated into exon 5 of the Daf1 gene and that the Daf1 gene thus was selectively inactivated. (a3) RT-PCR with primers P8 and P10 of the Daf1 mRNA product in the Daf1−/− mice. A truncated product corresponding to sequence for CCP1,4 is seen. (b) A diagram of the mouse Daf1 and Daf2 genes is shown. B, BamHI; E, EcoRI; and S, SacI. The position of the 1·5-kb SacI probe is indicated and the hybridized EcoRI and BamHI fragments are shown by the brackets. (c) Southern blot analyses of EcoRI- and BamHI-digested genomic DNA from parental and K/O mice. DNA from wild-type, heterozygous (Daf1+/−) and homozygous (Daf1−/−) knock-out mice were hybridized with the SacI fragment of the Daf1 gene (panel B). The pattern corresponded to the expected deletion from Daf1 exon 3 to exon 5. The high Mr band corresponds to the homologous EcoRI fragment in the Daf2 gene.

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Production and identification of Daf1 knock-out mice

Following injection of the targeted ES cells into blastocysts and breeding of male chimeras, screening of F1 progeny showed that one chimera gave 100% germline-transmission. Intercross of two of the F1s from this chimera generated Daf1−/− knock-out mice. Southern blotting with a 1·5-kb Sac1 fragment of the Daf1 gene confirmed deletion of the Daf1 sequence encompassing exons 3–5 (Fig. 2b,c).

Rather than no Daf1 gene product, as conventionally occurs, RT-PCR with primers P8 and P10, homologous to Daf1 gene CCPs 1 and 4, showed a Daf1 gene-derived mRNA product in the Daf1 knock-out mice (Fig. 2 panel A3). It was 378 bp smaller than normal. Sequencing showed that the transcript lacked exon 3, exon 4 and exon 5 coding for CCP2 and CCP3 of mouse Daf1 gene-derived protein. The mRNA thus arose from transcription through the Neo and Hph cassettes to downstream Daf1 sequence, followed by splicing out of the two selection markers as introns, resulting in the formation of a novel mRNA containing the Daf1-coding regions of CCP1 and CCP4 but lacking the sequence for DAFs two functional CCP2 and CCP3 domains (see Discussion).

Functional assays of the Daf1 residual CCP1-4 protein

Although the novel Daf1 gene mRNA generated in the Daf1 knock-out did not encode DAFs functional CCP2 and CCP3 domains, studies were performed to confirm that the remaining CCP1,4 protein was completely devoid of DAF function. For this purpose, protein corresponding to the mRNA was synthesized in Pichia and the product was examined for its ability to disable murine C3 convertase activity. C3 uptake experiments with EshA and mouse serum (Fig. 3a,b) showed that in contrast to native murine CCP1-4, which inhibited C3b uptake at concentrations as low as 100 ng/ml, the truncated CCP1,4 product exhibited no measurable effect on C3b deposition irrespective of concentration.


Figure 3. Functional analyses of Daf1-derived CCP1,4 protein. (a) and (b), EshA were incubated with mouse serum in the presence of the purified Daf1 CCP1,4 product, purified native CCP1-4 protein, or BSA. (a) FACS analyses showing that C3b deposition on EshA was unaffected by the CCP1,4 product. (b) Dose–response curve of the relative inhibitory activities of the DAF1,4 product and native DAF1-4 as compared to BSA on C3b deposition on EshA. (c) CD97-transfected COS cells were incubated with erythrocytes from wild-type and the Daf1 gene-targeted mice. The number of adherent erythrocytes per high powered field (∼50 COS cell transfectants) was scored.

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Since DAF possesses the additional function of serving as a ligand for CD97, studies were next performed to confirm that the Daf1 gene CCP1,4 product was also completely devoid of this activity. For this analysis, mouse CD97 cDNA was introduced into COS cells and the COS cell transfectants were incubated with erythrocytes either from wild-type or from the Daf1 gene-disrupted mice. Whereas large rosettes formed with the wild-type cells, no rosette formation was observable with Daf1 knock-out cells (Fig. 3c).

Localization of the binding sites of anti-murine DAF mAbs

The production of several different mAbs against murine DAF protein has recently has been reported.19,20 To map the site on mouse DAF where each mAb reacts and thereby identify mAbs specific for different CCPs, murine DAF CCP1-4, and deletion mutants CCP2-3, CCP3-4, CCP2-4, CCP1-2,4 and CCP1,3-4 were expressed using the Pichia expression system. The different CCP-containing products were then probed on Western blots with each of the available mAbs. The studies (summarized in Table 1) showed that rat mAb 2C620 and 3D520 bind to CCP2, rat mAb MD120 binds to CCP4, hamster RIKO219 binds to CCP2-3, and hamster RIKO319 binds to CCP1. In the case of hamster RIKO4,19 Western blotting was faint even at higher mAb concentrations but indicated that it appeared to bind to CCP4. Consistent with the findings that the Daf1 gene in the Daf1 knock-out expresses only CCP1,4, FACS analyses with 2C6 of erythrocytes and splenic suspension cells (Fig. 4a,b) from the Daf1−/− mice showed no signals. The mAb 2C6 was thus adopted for use in detecting Daf2 gene expression in the Daf1 knock-out mice.

Table 1.  Binding sites of murine DAF mAbs on DAF epitopes
SubstrateMonoclonal antibody
  1. Different DAF mAbs were analysed by Western blot with the following DAF mutants: CCP1-4, CCP2-3, CCP3-4, CCP1-2,4 and CCP1,3-4.

  2. +, positive reaction on the Western blot; −, negative reaction on the Western blot.

Binding siteCCP2CCP2CCP4CCP2-3CCP1CCP4

Figure 4. (a) FACS analyses of erythrocytes from wild-type and Daf1−/− mice using 2C6 rat anti-murine DAF mAb. (b) Similar analyses of splenic suspension cells from wild-type and Daf1−/− mice. The unfilled peaks show the staining with isotype-matched non-relevant control mAb. (c) FACS analyses of autologous C3 deposition in vivo on erythrocytes from Daf1−/− and wild-type littermates. The bars show the average values (P < 0·005).

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Analysis of in vivo deposited C3 fragments

To determine if cells from Daf1−/− mice exhibit changes in C3 convertase regulation, erythrocytes from eight knock-out mice and their littermates were examined for in vivo deposited C3 fragments. As shown in Fig. 4c, three-layer flow cytometric analyses of cells (see Materials and methods) from the Daf1−/− animals showed significantly increased staining as compared to the controls (P < 0·005).

Analysis of the tissue distribution of the Daf1 and Daf2 gene products

The immunochemical analyses of DAF protein expression in tissues of wild-type and the Daf1 knock-out mice using frozen tissues and mAb 2C6 are shown in Fig. 5, and the overall results are summarized in Table 2. In wild-type mice, in addition to staining of endothelium in essentially all sites, the most prominent parenchymal labelling was observed in the kidney, spleen, small intestine, lung and testis. In the kidney, strong expression of DAF was present in the glomerular capillary walls and mesangial areas as well as in arterial, arteriolar and capillary intertubular endothelium (Fig. 5a–c); cortical and medullary renal tubules were negative. In the spleen, the white pulp and especially dendritic cells labelled strongly, while red pulp labelled less intensely (Fig. 5d). In the liver, the endothelium within portal and central veins and the arteries was labelled moderately, and sinusoidal endothelium was labelled less intensely (Fig. 5e). Villous, but not crypt, epithelium in the small bowel stained weakly, with the apical portion of the cell labelled much more intensely than the basolateral portion (Fig. 5f). In the lung, tracheal and bronchial epithelium and large vessel endothelium labelled strongly, whereas alveolar capillary endothelium labelled moderately (Fig. 5g). In the testis, seminiferous tubule cells labelled strongly whereas interstitial cells did not label (Fig. 5h). In the heart, colon, stomach and skin, only capillary endothelium showed moderate labelling whereas in the brain, the arachnoid membrane, vascular endothelium and the choroid plexus showed moderate labelling (not shown).


Figure 5. Immunostaining of DAF in wild-type mice and Daf1−/− mice. All tissues were stained with 2C6 mAb. In sections from wild-type mice (a through h), the kidney at ×100 magnification (a), a glomerulus at ×400 magnification (b) and a renal artery at ×400 magnification (c) showed intense labelling. The spleen at ×100 magnification (d) showed heavy staining of the white pulp, with weaker focal staining of the red pulp. The liver at ×100 magnification (e) revealed moderate endothelial staining in larger blood vessels, but weak labelling of sinusoidal endothelium (inset in corner shows negative control). Epithelial staining was detected in the testis at ×100 magnification (f), the villi of the small intestine at ×200 magnification (g) and the bronchi at ×400 magnification (h). In corresponding but not identical sections from Daf1−/− mice stained with H&E (A1–F1) and with 2C6 mAb (A2–F2), the liver at ×100 (A1, A2), a glomerulus at ×400 (B1, B2), intestinal villi at ×200 (C1, C2) and bronchi at ×400 (D1, D2) were completely negative. In contrast, intense staining was seen in the testis at ×400 magnification (E1, E2) and in dendritic cells in the spleen at ×400 magnification (F1, F2). Staining of the other duplicate spleen sections from Daf1−/− mice with 2C6 mAb at ×100 (G) and with CD11c mAb at ×100 (H) showed an identical staining pattern of dendritic cell labelling.

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Table 2.  Summary of tissue staining of mouse DAF
OrganLocationStaining intensity
  • *

    Negative values could reflect levels below the sensitivity of the staining technique.

interfibre capillary+/−
Lunglarge vessel endothelium+++/++
trachea/bronchial epithelium++/+
alveolar capillary+/−
capillary endothelium++
airway epithelium+++
Liverlarge vessel endothelium+++/++
portal vein, central vein, artery+
sinusoidal endothelium+
Spleenred pulp+/−
white pulp+++/++
dendritic cell+
intertubular endothelium++
Smallcap epithelium++
 intestinevillous epithelium+
crypt epithelium*
Colonlarge blood vessel+
cap endothelium+++/++
Post epithelium
Testisseminiferous tubules+++/++
Skinlarge blood vessel+

In the Daf1 knock-out mice almost all tissues were negative, even in microvascular endothelium (Fig. 5A–C). However, the spleen and testis retained strong labelling (Fig. 5D–F). The labelling pattern in the spleen appeared to be selective. To discriminate which cell types were positive, two-colour immunohistochemical studies of tissue sections from wild-type and the Daf1 knock-out mice were performed using major histocompatibility complex (MHC) class II mAb OX3, which recognizes B cells and macrophages (not shown), and with CD11c mAb N418, which identifies dendritic cells.23 The assay (Fig. 5G,H) localized the DAF staining to dendritic (CD11c+) cells. Neither T cells nor B cells in the knock-out mice expressed immunohistochemically detectable DAF (data not shown). As shown in Fig. 6, RT-PCR analyses of whole spleen cells using Daf1- and Daf2-specific primers P8, P9 and P10 (Fig. 6a) identified mRNA deriving from the Daf2 gene. Additional RT-PCR analyses using TM- and GPI-specific primers P13 and P14 together with P9 (Fig. 6b) identified both the GPI- and TM-forms of Daf2 mRNA. The immunohistochemical labelling in the testis resembled that in wild-type animals (Fig. 5D). As with spleen cells, RT-PCR analyses (Fig. 6a,b) showed mRNA deriving from the Daf2 gene and both TM- and GPI-forms of Daf2 mRNA.


Figure 6. (a) RT-PCR analysis of Daf mRNA expression in different tissues in wild-type and Daf1−/− mice. Parallel amplification of β-actin was included as a control. M, 1 kb ladder; 1, Daf1; 2, Daf2; A, β-actin. (b) RT-PCR analyses of Daf2 mRNA in spleen and testis of Daf1−/− mice using GPI-and TM-specific primers P13 and P14 in conjunction with P9. In both sites, the TM form of the transcript is seen. TM, TM-DAF; GPI, GPI-DAF.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In the studies reported here, we used a Cre/loxP-based strategy to delete selectively the Daf1 gene. Due to the high homology of the two Daf genes [Daf1 and Daf2 share 93% identity in their coding regions as well as > 90% homology in their known intronic sequence (intron 1)], the pDAFdown construct can alternatively integrate into the Daf1 gene as well as its homologous region in the Daf2 gene.

Disruption of the Daf1 gene and both of the two Daf genes using conventional homologous recombination methodology would be more complex than the methodology adopted. After selectively disrupting the Daf1 gene, the targeting event for the second Daf gene would not only have to occur on the same chromosome but also avoid the highly homologous Daf1 gene on the other chromosome. Based on available genomic mapping data showing similarity between the genes in known restriction sites it would be difficult to detect the correct integration event. In previous studies, the Cre/loxP system has been used to delete stretches of DNA as large as 200 kb21 and even 3–4 centimorgans.24 Here, we applied the system with two TK genes positioned between the two loxP sites allowing for ‘self’ negative selection. Thus, following Cre/loxP induced deletion, only those clones in which integration of the pDAFup and pDAFdown constructs occurs on the same chromosome are able to survive the combined G418, hygromycin and FIAU selection. Clones in which there is random integration, where integration occurs on different chromosomes, or where no deletion occurs after Cre/loxP-mediated recombination, cannot survive. In our studies, the system was so effective that all seven clones that emerged after selection were confirmed to be knock-outs.

Usually, genes interrupted by inserting a marker into or by making exon deletions of one of their exons do not yield products. Here we describe a case where the inserted markers (i.e. Neo and Hph) within the targeted gene exons are removed as introns during RNA splicing. The interrupted Daf1 gene consequently still was transcribed yielding an RNA product, except that the functional CCP2 and CCP3 coding regions25–29 were deleted by Cre/loxP-mediated homologous recombination.

To verify that the novel Daf1 gene protein retaining CCP1 and CCP4 possessed no activity, functional analyses were performed. To exclude the effects of Crry, a second cell surface C3 regulator in mice (see below), for the assay assessing complement regulatory function, recombinant CCP1,4 protein was produced in yeast and tested in C3b uptake assays in vitro using sensitized Esh. Recombinant native mouse CCP1-4 produced in Pichia was employed as a control. The studies showed no measurable effect of the CCP1,4 product as compared to BSA control. To verify that the CCP1,4 protein was also devoid of CD97-binding activity, COS cells transfected with mouse CD97 were tested for rosettes using assays with erythrocytes from the Daf1-targeted mouse. Again, no activity was detectable not only confirming that the Daf1 gene was inactivated with respect to CD97 binding but extending mAb blocking studies13 indicating that CCPs 2 and 3 are essential for the interaction. Thus, though parts of CCP1 and 4 remain in the Daf1 knock-out, the mouse is null with respect to function. Consistent with this, analyses of erythrocytes from the Daf1−/− animals using a three-layer flow cytometric technique to enhance detection efficiency showed increased in vivo deposition of C3 fragments. This finding is significant since, in contrast to previous studies employing a less sensitive method30, it indicates that in mice, in addition to Crry, DAF function is important physiologically.

Human membrane DAF protein is uniformly GPI-anchored.6 In contrast, similar to findings in mice, DAF in the rat and guinea-pig is additionally expressed in an alternative TM form. Unlike the situation in mice in which two genes are present, the GPI-anchored and TM forms of guinea-pig and rat DAF are generated as a result of alternative splicing from a single gene.31,32 In the guinea-pig, the TM and GPI forms of DAF have been shown to possess similar decay-accelerating activity.33 In the mouse, with the use of CHO cells transfected with the GPI and TM forms of mouse DAF arising from the Daf1 and Daf2 genes, Ohta et al.19 showed that the latter protein also has decay-accelerating activity similar to the former.

Initially the murine Daf1 gene was believed to encode GPI-anchored DAF protein while the Daf2 gene was believed to encode TM-anchored DAF protein.17 However, using isolated genomic clones and Southern analyses, Miwa et al.34 recently have found that the exons that code for the GPI and TM regions of DAF are present in both the Daf1 and Daf2 genes. Additionally, both Miwa et al.34 and Harris et al.35 demonstrated that the Daf2 gene can give rise to a mRNA encoding a GPI-anchored product. Thus, as a result of alternative splicing, both genes can make both forms of DAF, though most GPI-anchored DAF comes from the Daf1 gene while most of TM-anchored DAF is the product of the Daf2 gene.34

A Daf1 knock-out mouse has also been reported by Sun et al.30 but studies of DAF expression at the protein level were not carried out. In studies of the rat at the protein level, Spiller et al. recently showed that the tissue distribution of DAF closely resembles that of human DAF36 in that the protein is expressed in almost all tissues.37 Nishikawa et al. also reported a similar distribution of guinea-pig DAF.38 In the initial studies in mice by Spicer et al. it was found that Daf mRNA is detectable by Northern blot analysis only in the lung, small intestine, lymph nodes, lactating mammary gland and testis.17 Due to low labelling intensity in immunohistochemical analyses, it heretofore has not been possible to assess murine DAF expression at the protein level. Precise characterization of the tissue and cell distribution of mouse DAF protein is essential for the use of the mouse as an experimental model to study in vivo DAF function.

By preparing soluble mouse DAF CCP1-4 and CCP deletion mutants, we were able to localize the reactive sites of six available anti-murine DAF mAbs. The rat mAbs, 2C6, 3D5 and MD1 were found to detect CCP2, CCP2 and CCP4, respectively. The hamster mAbs RIKO2, RIKO3 and RIKO4 detected CCP2-3, CCP1, and CCP4, respectively (Table 1). To allow comparative immunohistochemical analyses of the distribution of DAF in wild-type mice and our Daf1 knock-out mice which contains CCP1 and CCP4, we used 2C6 antibody, which recognizes CCP2.

We exploited the highly sensitive tyramide staining method39 in which tissues secondarily stained with HRP are subsequently stained with fluorescent tyramide hapten. Upon activation of hydrogen peroxide by the HRP, the hapten binds covalently to nearby tyrosine residues. Using this method which enhances sensitivity by combining enzyme and fluorescent-based cytochemical detection, we successfully detected DAF protein in multiple organs in wild-type mice, and found that the tissue distribution of murine DAF expression is basically similar to that of human DAF expression (Fig. 5, Table 2). That DAF protein was not clearly detectable in the skin and in the stomach was unexpected but could reflect levels below that of the sensitivity of the tyramide staining technique. In the spleen, the white pulp labelled more strongly than the red pulp. Double labelling for DAF and either the B-cell and macrophage marker class II MHC, or the dendritic cell marker CD11c, showed that both of these cell types were DAF-positive. It is interesting that in the rat spleen, labelling of the red pulp was stronger than the white pulp36 indicating some differences between the two species. In the testis, germinal epithelium and seminiferous tubules were strongly labelled40. Labelling was much stronger than in human testis, as previously inferred from comparative Northern analyses.17 In the kidney, glomeruli and intertubular capillaries labelled strongly. These results are the same as found in the rat but different from those in humans.37 By Northern analyses, Daf mRNA levels in both the rat and mouse are high in the lung. However, at the protein level, the spleen and kidney of the mouse and rat label stronger for DAF, indicating that Daf mRNA levels do not correlate with steady-state DAF protein levels.

When tissues of the Daf1 knock-out mice were labelled for DAF protein, only the testis and spleen remained positive (Fig. 5, Table 2). In the testis, Daf2 gene-derived protein was found to be present mainly in seminiferous tubules, and in the spleen, it was found to be present in dendritic cells. In both cases, the expression of mRNAs for both the TM and GPI isoforms of the protein was documented by RT-PCR (Fig. 6). The labelling pattern of the testis was the same as that in wild-type mice whereas that of the spleen was cell-type-specific, i.e. dendritic cells. Labelling of the kidney and other organs was completely negative.

Other complement regulators work together with DAF in protecting self cells from autologous complement-mediated injury. A second complement regulator, membrane co-factor protein [MCP (CD46)], co-operates with DAF in circumventing autologous C3 deposition,41,42 while a third, HRF20 (CD59), inhibits the pathway at its critical end-point.43–45 MCP serves as a co-factor for factor I, a serum serine protease which degrades C3b to an inactive form, iC3b. CD59 functions to inhibit the formation of lytic membrane attack complexes (MAC) on the membrane. In the mouse and rat, Crry46–49 is expressed which has actions overlapping both DAF and MCP. It is widely distributed and appears to serve the function of MCP in rodents50–52 as cDNA cloning and tissue analyses have shown that the direct MCP homologue in both species is expressed significantly only in the testis.53–55 The combined activities of these complement inhibitors are essential for protecting self cells from autologous complement-mediated injury. Knowledge of the tissue distribution of DAF in wild-type and Daf1 knock-out mice should permit analyses of the physiological importance of DAF in overall self cell protection in this experimentally relevant species.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This investigation was supported by National Institutes of Health grants R01 AI23598, R01 EY11288, and P30 EY11373. The authors thank Drs C. Weissman, Z. W. Li, H. Okada, B. P. Morgan and J. Hamann for reagents, Sara Cechner for manuscript preparation, and the Histology Core Lab and Visual Science Photography Center at University Hospitals of Cleveland for help in producing graphics.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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