Elsevier

Molecular Immunology

Volume 39, Issues 5–6, October 2002, Pages 357-365
Molecular Immunology

Generation and characterization of transgenic mice expressing cobra venom factor

https://doi.org/10.1016/S0161-5890(02)00107-4Get rights and content

Abstract

Cobra venom factor (CVF), the anticomplementary protein in cobra venom, activates the alternative complement pathway, eventually leading to complement consumption. Here, we describe the development of a transgenic mouse model for CVF. We generated a DNA construct containing the full-length cDNA for single-chain pre-pro-CVF. Expression of CVF was controlled by the α1-antitrypsin promoter to achieve liver-specific expression. Linearized DNA was microinjected into murine ovary cells (strain CD2F1 (BALB/c×DBA/2J)) and the newborn mice were analyzed for stable integration of CVF DNA. After establishing the transgene, mice were propagated in a BALB/c background. The CVF mRNA was detected in the liver and, in some animals, in the kidney. CVF protein was detected in small amounts in the serum. Serum complement hemolytic activity in CVF-transgenic mice was virtually absent. The concentration of plasma C3 was significantly reduced. The CVF-transgenic animals show no unusual phenotype. They provide an animal model to study the effect of long-term complement depletion by continued activation, as well as the role of complement in host immune response and pathogenesis of disease.

Introduction

The human complement system (for review, see Kinoshita, 1991, Muller-Eberhard, 1988) is an integral component of the immune system, but is also involved in the pathogenesis of various diseases, e.g. autoimmune diseases (Wang et al., 2000), Alzheimer’s disease (Emmerling et al., 2000), reperfusion injury (Zhou et al., 2000), and hyperacute rejection in organ xenotransplantation (Platt and Saadi, 1999). Thus, inhibition of complement can prolong the survival of discordant and concordant xenografts (Kobayashi et al., 1997, Oberholzer et al., 1999) and inhibit development of collagen-induced arthritis in mouse and rat models (Goodfellow et al., 2000, Wang et al., 1995). Consequently, there has been a surge of interest to investigate complement inhibitors and modulators as potential anti-inflammatory therapeutics (Sahu and Lambris, 2000). Examples include monoclonal antibodies against C5 (Kroshus et al., 1995, Thomas et al., 1996) and recombinant complement inhibitors, such as the soluble complement receptor 1 (sCR1) (Weisman et al., 1990) and a decay acceleration factor/membrane cofactor protein chimera (Fodor et al., 1995). Several of these complement-inhibiting drugs are currently undergoing pre-clinical and clinical testing (Sahu and Lambris, 2000).

The most widely used reagent for decomplementation is CVF (for review, see Vogel, 1991, Vogel et al., 1996), a 149 kDa glycoprotein from the venom of the Indian cobra (Naja naja). By administration in low dose, CVF is non-toxic, but a powerful activator of the alternative pathway of human and mammalian complement. CVF shares a structural and functional homology with C3b, the activated form of C3 (Fig. 1). Like C3b, CVF binds to factor B of the alternative pathway to form the bimolecular complex CVF,B which is subsequently cleaved by factor D into the activation peptide Ba and the C3/C5 convertase CVF,Bb. In contrast to the short-lived surface-bound C3/C5 convertase C3b,Bb, which is formed during activation of the alternative pathway and exhibits spontaneous decay–dissociation with a half-life of 1.5 min at 37 °C, CVF,Bb is active in solution and exhibits a slow decay–dissociation with a half-life of 7 h at 37 °C. Furthermore, CVF and CVF,Bb are resistant to the regulatory proteins factors H and I, thus leading to continuous complement activation with subsequent consumption of serum complement activity (for review, see Vogel, 1991, Vogel et al., 1996).

CVF can be safely administered to laboratory animals with its only apparent effect being a temporary depletion of complement (Cochrane et al., 1970). Accordingly, numerous studies have been performed in various laboratory animals depleted of serum complement activity by CVF administration to study the involvement of the complement system in host defense and pathogenesis of disease (for review, see Vogel, 1991).

Recently, we have cloned and sequenced the cDNA for CVF (Fritzinger et al., 1994). This work confirmed the extensive homology between CVF and C3. Both molecules are synthesized as single-chain pre-pro-proteins which exhibit approximately 92% identity at the cDNA level in the case of cobra C3. Whereas, pro-C3 is processed into the mature two-chain protein by removal of four arginine residues, pro-CVF is processed in the venom gland into its mature three-chain form by additionally removing the C3a and C3d domains (Fritzinger et al., 1994) (Fig. 1). Surprisingly, recombinant single-chain pro-CVF was found to exhibit the same functional activity without proteolytic processing as the native three-chain molecule (Kock, 1996, Kock et al., 1996).

Here, we describe the development of transgenic mice expressing pro-CVF. CVF-transgenic mice show no unusual phenotype; they provide an animal model to study the effect of long-term complement depletion by continuous activation, as well as the role of complement in host immune response and pathogenesis of disease.

Section snippets

Materials

CVF was purified from lyophilized venom of the Indian cobra (N. naja kaouthia) (Miami Serpentarium Laboratories, Punta Gorda, FL) as described (Vogel and Müller-Eberhard, 1984). Murine C3 was purified from BALB/c serum as described (Van den Berg et al., 1989). CD2F1 and BALB/c mice were from Harlan, Indianapolis, IN, and DBA/2J mice from Charles River, Wilmington, DE.

Antibodies

Polyclonal anti-mouse C3 antibody (developed in goats) was purchased from ICN (Eschwege, Germany). Anti-CVF IgG was generated by

Production of CVF-transgenic mice

The construct used for microinjection was generated by subcloning cDNA encoding the liver-specific α1-antitrypsin promoter into the expression vector pSport1 containing the full-length cDNA for single-chain pre-pro-CVF (Fig. 2). The KpnI/NotI fragment of this construct was microinjected into the prenucleus of murine ovary cells. These cells were then re-implanted into a pseudopregnant female (strain CD2F1, BALB/c×DBA/2J). Tail biopsies were taken from offsprings, digested with BamHI, and

Discussion

We have generated transgenic mice for pro-CVF. Stable integration of the transgene into the mouse genome was shown by Southern blot analysis. In addition to the two anticipated internal BamHI fragments of 1.2 and 1.8 kb, two larger fragments were obtained, presumably spanning the DNA from the internal BamHI sites to the nearest BamHI sites in the mouse genome where the transgene was inserted.

Transcription and expression of the CVF transgene could be demonstrated by RT-PCR using CVF-specific

Acknowledgements

We thank Dr. Patrick Ziegelmüller for the purification of CVF, Mr. Sebastian Schaffner for participating in the cloning procedures, Ms. Katrin Klensang for performing RT-PCR experiments, and Ms. Cordula Heske for excellent technical assistance. Part of this work was supported by a grant from the Federal Ministry for Education and Research of Germany (BMBF/BEO Grant no. 03111892) and from the Deutsche Forschungsgemeinschaft (DFG, SFB 265), both to D. Paul.

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    Present address: BASF AG, 67056 Ludwigshafen, Germany.

    2

    Present address: Ingenium Pharmaceuticals AG, 82152 Martinsried, Germany.

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