Cre reconstitution allows for DNA recombination selectively in dual-marker-expressing cells in transgenic mice

Cre/LoxP-based DNA recombination has been used to introduce desired DNA rearrangements in various organisms, having for example, greatly assisted genetic analyses in mice. For most applications, single gene promoters are used to drive Cre production for conditional gene activation/inactivation or lineage-tracing experiments. Such a manipulation introduces Cre in all cells in which the utilized promoter is active. To overcome the limited selectivity of single promoters for cell-type-specific recombination, we have explored the ‘dual promoter combinatorial control’ of Cre activity, so that Cre activity could be restricted to cells that express dual protein markers. We efficiently reconstituted Cre activity from two modified, inactive Cre fragments. Cre re-association was greatly enhanced by fusing the Cre fragments separately to peptides that can form a tight antiparallel leucine zipper. The co-expressed Cre fusion fragments showed substantial activity in cultured cells. As proof of principle of the utility of this technique in vivo for manipulating genes specifically in dual-marker-positive cells, we expressed each inactive Cre fragments in transgenic mice via individual promoters. Result showed the effective reconstitution of Cre activates LoxP recombination in the co-expressing cells.     

HLA antibody responses in HLA class I transgenic mice

In a previous report we described how cross-immunizations of pairs of transgenic mice expressing different HLA class I antigens led to the production of antibodies directed exclusively at polymorphic epitopes. This was ascribed to self-tolerance of HLA that prevents immune responses to monomorphic epitopes and focuses responses on polymorphic ones. In the present report we extend our findings and demonstrate that immunizations of class I transgenic mice with HLA transfected mouse fibrosarcoma as well as with human lymphoblastoid cells also preferentially yield antibodies to polymorphic epitopes. This was the case whether or not immunizations were carried out across locus barriers [e.g., Tg (HLA-A *0201) or Tg (HLA-Cw*0301) transgenic mice immunized with HLA-B27 transfectants] or within the same locus [e.g., Tg (HLA-B*1302) transgenic mice immunized with HLA-B27 transfectants or B27-expressing lympho-blastoid cell]. Use of an extended immunization protocol with four or more booster injections favored antibodies of IgG isotype with affinities high enough to lyse normal peripheral blood lymphocytes (PBLs) in complement-dependent cytotoxicity assays and to immunoprecipitate HLA antigens. The specificities covered by the monoclonal antibodies (mAbs) could be either broad or narrow, depending on the genetic distance of the HLA antigens or alleles involved. For instance, a Tg(HLA-B*1302) transgenic mouse immunized with B27 produced both broad B7/B27-specific antibodies, Bw4-specific antibodies, and one antibody reacting with all B alleles except B13 and with some C alleles. On the other hand, a Tg(HLA-B*1302) transgenic mouse immunized with Bw47 transfectants responded narrowly with an antibody to Bw60 and Bw47. Thus it appears that by choosing appropriate recipient mice and closely related or more distant HLA antigens, antibodies of a programmed specificity can be generated. Address correspondence and offprint requests to: U. Hämmerling.     

Functional recombinant human anti-HAV antibody expressed in milk of transgenic mice

Hepatitis A virus (HAV) is a wide spread pathogenic agent and is the common cause of acute Hepatitis A worldwide. Passive immunization of HAV plays an extremely important role in post-exposure prophylaxis with clinical applications often requiring large amounts of antibody. As an alternative to the in vitro production of recombinant proteins, expression of monoclonal antibodies (mAbs) in the milk of transgenic animals is currently used being associated with low production costs and high activity. In this paper, eight founder lines of transgenic mice were generated by co-microinjection of the two cassettes encoding the heavy- and light-chains of a neutralizing anti-HAV antibody, respectively. The expressed heavy- and light-chains of the mAb were correctly assembled and modified in the mammary gland as detected by western blotting. High expression levels of the antibody were achieved during the lactation period and found to be independent of the copy numbers of integrated transgenes. The highest level was up to 32.2 mg/ml. The binding specificity and neutralizing activity of the expressed mAb were assayed by ELISA and neutralizing test, showing that it is capable to neutralize the JN strain of Hepatitis A virus efficiently. Therefore, our results suggest that a large-scale and efficient production of the anti-HAV mAb in the milk of transgenic farm animals would be feasible in the future.     

Plasma membrane-cytoskeleton damage in eye lenses of transgenic mice expressing desmin

Immunocytochemistry of eye lens cells from transgenic mice coexpressing desmin and vimentin reveals that the transgenic desmin expression is not uniform. In the same lens, some epithelial and fiber cells overexpress desmin, while in others the desmin gene seems to be silent. Conversely, the endogenous vimentin is always expressed. The concomitant expression of vimentin and desmin results in the assembly of hybrid intermediate filaments (IFs). Moreover, the overexpression of the transgene generates pleomorphic IF assembly and leads to intermingled filamentous whorls and to accumulation of amorphous desmin. The abnormalities of IF assembly induced by the genetic manipulation are correlated with perturbation of the enucleation process in the lens fibers, changes in cell shape, fiber fusion and extensive internalization of the general plasma membrane and junctional domains. The alterations of lens cells described in this study were observed in all transgenic mice examined. The level of expression of the transgene was paralleled by the degree of damage. Our results indicate that proper expression, assembly and membrane interaction of IFs play an important role in the terminal differentiation of the lenticular epithelium into fiber cells. We anticipate that alterations during these processes may initiate cataract formation.     

利用转基因小鼠筛选全人源炭疽致死因子中和抗体

炭疽是由炭疽芽孢杆菌引起的严重威胁人类健康的传染病。炭疽毒素包括3种蛋白质成分:保护性抗原(PA)、致死因子(LF)和水肿因子(EF)。PA与LF形成致死毒素(LT),与EF形成水肿毒素(ET)。由于致死毒素(LT)在感染者损伤及死亡中发挥主要作用,因此在炭疽感染晚期单纯使用抗生素治疗难以发挥疗效,治疗性中和抗体成为目前最有效的炭疽治疗药物。目前国外获得的炭疽毒素抗体多为炭疽PA抗体,美国FDA已批准瑞西巴库(人源PA单抗)用于吸入性炭疽的治疗。一旦炭疽芽孢杆菌被人为改构或PA中和表位发生突变,针对PA单一表位的抗体将可能失效,因此针对LF的抗体将成为炭疽治疗的有效补充。目前国外已有的LF抗体多为鼠源抗体和嵌合抗体,而全人源抗体可以避免鼠源抗体免疫原性高等缺点。本研究首先用LF抗原免疫人抗体转基因小鼠,利用流式细胞仪从小鼠脾淋巴细胞中分选抗原特异的记忆B细胞,通过单细胞PCR方法快速获得两株具有结合活性的抗LF单抗1D7和2B9。瞬时转染Expi 293F细胞制备抗体,通过毒素中和实验(TNA)发现1D7和2B9在细胞模型中均显示较好的中和活性,并且与PA单抗联合使用时,表现出较好的协同作用。总之,本文利用转基因小鼠、流式分选技术和单细胞PCR技术的优势,快速筛选到全人源LF抗体,为快速筛选全人源单克隆抗体开辟了新的思路与方法。     

Impaired wound healing in transgenic mice overexpressing the activin antagonist follistatin in the epidermis

Recently, we demonstrated a strong upregulation of activin expression after skin injury. Furthermore, overexpression of this transforming growth factor beta family member in the skin of transgenic mice caused dermal fibrosis, epidermal hyperthickening and enhanced wound repair. However, the role of endogenous activin in wound healing has not been determined. To address this question we overexpressed the soluble activin antagonist follistatin in the epidermis of transgenic mice. These animals were born with open eyes, and the adult mice had larger ears, longer tails and reduced body weight compared with non-transgenic littermates. Their skin was characterized by a mild dermal and epidermal atrophy. After injury, a severe delay in wound healing was observed. In particular, granulation tissue formation was significantly reduced, leading to a major reduction in wound breaking strength. The wounds, however, finally healed, and the resulting scar area was smaller than in control animals. These results implicate an important function of endogenous activin in the control of wound repair and scar formation.     

A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice.

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.     

Spontaneous skin damage and delayed wound healing in SOD1-deficient mice

Superoxide dismutase 1 (SOD1) is an important antioxidative enzyme that protects skin from oxidative stress. SOD1 ^?/? mice with a genetic background of b129Sv mice showed facial skin damage after 15 weeks of age. Eyelid swelling occurred as the initial symptom and caused impairment by triggering self-scratching. The period required for wound healing in the back was markedly delayed in 20-week SOD1 ^?/? mice. Oxidative stress markers, 4-hydroxynonenal and thiobarbituric acid-reactive substances, were unexpectedly lower in SOD1 ^?/? mice at day 1 after wounding. The decay rate of electron paramagnetic resonance signal intensity of intravenously injected nitroxide radical indicated that the half-life of the signal intensity was significantly prolonged in the wounded skin of SOD1 ^+/+ mice. However, while the half-life of the signal intensity in control skin was a little longer in SOD1 ^?/? mice, it did not change in wounded skin. Taken together, these data suggest that the skin of SOD1 ^?/? mice is in redox imbalance and prone to damage by wounding.     

Immunogenicity of different stressed IgG monoclonal antibody formulations in immune tolerant transgenic mice

The presence of protein aggregates in biopharmaceutical formulations is of great concern for safety and efficacy reasons. The aim of this study was to correlate the type and amount of IgG monoclonal antibody aggregates with their immunogenic potential. IgG degradation was obtained by freeze-thawing cycles, pH-shift cycles, heating, shaking and metal-catalyzed oxidation. The size, amount, morphology and type of intermolecular bonds of aggregates, as well as structural changes and epitope integrity were characterized. These formulations were injected in mice transgenic (TG) for human genes for Ig heavy and light chains and their non-transgenic (NTG) counterparts. Anti-drug antibody (ADA) titers were determined by bridging ELISA. Both unstressed IgG and freeze-thawed formulation did not induce measurable ADA levels. A mild antibody response was obtained in a fairly small percentage of mice, when injected with shaken, pH-shifted and heated formulations. The metal-catalyzed oxidized IgG formulation was the most immunogenic one, in both ADA titers and number of responders. The overall titers of NTG responders were significantly higher than the ones produced by TG mice, whereas there was no significant difference between the overall number of TG and NTG responders. This study reinforces the important role of protein aggregates on immunogenicity of therapeutic proteins and provides new insight into the immunogenic potential of different types of IgG aggregates. The results indicate that the quality of the IgG aggregates has more impact on the development of an immune response than their quantity or size.     

Immunogenicity of different stressed IgG monoclonal antibody formulations in immune tolerant transgenic mice

The presence of protein aggregates in biopharmaceutical formulations is of great concern for safety and efficacy reasons. The aim of this study was to correlate the type and amount of IgG monoclonal antibody aggregates with their immunogenic potential. IgG degradation was obtained by freeze-thawing cycles, pH-shift cycles, heating, shaking and metal-catalyzed oxidation. The size, amount, morphology and type of intermolecular bonds of aggregates, as well as structural changes and epitope integrity were characterized. These formulations were injected in mice transgenic (TG) for human genes for Ig heavy and light chains and their non-transgenic (NTG) counterparts. Anti-drug antibody (ADA) titers were determined by bridging ELISA. Both unstressed IgG and freeze-thawed formulation did not induce measurable ADA levels. A mild antibody response was obtained in a fairly small percentage of mice, when injected with shaken, pH-shifted and heated formulations. The metal-catalyzed oxidized IgG formulation was the most immunogenic one, in both ADA titers and number of responders. The overall titers of NTG responders were significantly higher than the ones produced by TG mice, whereas there was no significant difference between the overall number of TG and NTG responders. This study reinforces the important role of protein aggregates on immunogenicity of therapeutic proteins and provides new insight into the immunogenic potential of different types of IgG aggregates. The results indicate that the quality of the IgG aggregates has more impact on the development of an immune response than their quantity or size.     

Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in tissue repair has remained controversial. We established transgenic mice with targeted overexpression of TSP-1 in the skin, using a keratin 14 expression cassette. TSP-1 transgenic mice were healthy and fertile, and did not show any major abnormalities of normal skin vascularity, cutaneous vascular architecture, or microvascular permeability. However, healing of full-thickness skin wounds was greatly delayed in TSP-1 transgenic mice and was associated with reduced granulation tissue formation and highly diminished wound angiogenesis. Moreover, TSP-1 potently inhibited fibroblast migration in vivo and in vitro. These findings demonstrate that TSP-1 preferentially interfered with wound healing-associated angiogenesis, rather than with the angiogenesis associated with normal development and skin homeostasis, and suggest that therapeutic application of angiogenesis inhibitors might potentially be associated with impaired wound vascularization and tissue repair.     

Overexpression of biglycan in the heart of transgenic mice: an antibody microarray study

Biglycan, a small leucine rich proteoglycan, is expressed in almost every tissue of the body, mainly in the extracellular matrix of connective tissues. Although there is an increasing amount of data on the biological role of biglycan protein, its function is still poorly understood. We aimed to gather more information about the biological function of biglycan protein in the cardiac tissues, and its role in signal transduction. Therefore, we generated transgenic mice overexpressing the human biglycan protein and analyzed the cardiac protein profile of transgenic offsprings using quantitative real-time (QRT)-PCR and proteomics. QRT-PCR results showed that most members of extracellular matrix were downregulated whereas cadherins, TGF-beta1, and TGF-beta2 were upregulated. Antibody microarrayer experiment revealed that pyk2, RAF-1, Mcl-1, syntrophin, calmodulin, isoforms of NOS protein family (eNOS, nNOS, and iNOS), and synaptotagmin proteins were unambiguously upregulated in the heart of biglycan transgenic mice. In this study we show that biglycan directly or indirectly activates proteins involved in cardiac remodeling (TGF-beta, pyk2), signal transduction (RAF-1, Mcl-1, syntrophin, calmodulin, nNOS p38MAPK and MAP kinases), cardioprotection (NOS family, TGF-beta) and Ca++ signaling (connexin, calmodulin, synaptotagmin). On the basis of the results presented here, we conclude that biglycan is a multifunctional extracellular protein that has a pivotal role in pathological remodeling of cardiac tissue and mediates cardioprotection.     

Overexpression of TIMP-1 under the MMP-9 promoter interferes with wound healing in transgenic mice

We have generated transgenic mice harboring the murine matrix metalloproteinase 9 (MMP-9) promoter cloned in front of human TIMP-1 cDNA. The transgenic mice were viable and fertile and exhibited normal growth and general development. During wound healing the mice were shown to express human TIMP-1 in keratinocytes that normally express MMP-9. However, the healing of skin wounds was significantly retarded with slow migration of keratinocytes over the wound in transgenic mice. In situ zymography carried out on wound tissues revealed total blockage of gelatinolytic activity (i.e., MMP-9 and MMP-2). The results confirm studies with MMP-9 knockout mice showing that MMP-9 is not essential for general development, but they also demonstrate an important role of keratinocyte MMP-9, as well that of other keratinocyte MMPs that are inhibited by TIMP-1, in wound healing. The transgenic mice generated in this study provide a model for the role of MMPs in MMP-9-producing cells in other challenging situations such as bone fracture recovery and cancer invasion.The expert technical assistance of M. Jarva, L. Ollitervo, S. Kangas, and R. Jokisalo is gratefully acknowledged. This work was supported in part by grants from the Finnish Academy of Science, the Swedish Cancer Foundation, the Novo Nordisk Foundation and EC contract QLG1-CT-2000-01131 (K.T.), the Finnish Dental Society Apollonia and the Northern Finland Cancer Foundation (M.P.), as well as the K. Albin Johansson Foundation and the Einar and Karin Stroems Foundation (E.P.)     

Expression of a functional mouse-human chimeric anti-CD19 antibody in the milk of transgenic mice

Human B cell lymphomas are suitable targets for immunotherapy. Clinical trials with mouse-human chimeric B cell-specific monoclonal antibodies (mAbs) have already shown promising results. However, limitations for their use in clinical trials can be the lack of sufficient amounts and high production costs. Expression of mAbs in the mammary gland of transgenic animals provides an economically advantageous possibility for production of sufficient quantities of a promising antibody for clinical trials and beyond. In this paper, we show the feasibility of this approach, by generating transgenic mice expressing mouse-human chimeric anti-CD19 mAbs in their milk. Mouse anti-CD19 variable (V) region genes were combined with human IgG1 heavy (H) and kappa light (L) chain constant (C) region genes and fused to the bovine -lactoglobulin (BLG) promoter in two separate expression cassettes. Co-injection resulted in five transgenic lines. In one of these lines completely assembled chimeric mAbs were secreted into the milk, at an approximate level of 0.5mg/ml. These mAbs were able to bind specifically to the CD19 surface antigen on human B cells.     

Virus-neutralizing monoclonal antibody expressed in milk of transgenic mice provides full protection against virus-induced encephalitis

Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine beta-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis.     

Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgeulc animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein （EGFP） transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation.