Remodeling of the major mouse xenoantigen, Galalpha1-3Galbeta1-4GlcNAc-R, by N-acetylglucosaminyltransferase-III

beta-D-Mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyses the attachment of an N-acetylglucosamine (GlcNAc) residue to mannose in the beta(1-4) configuration in N-glycans, and forms a bisecting GlcNAc. We have generated transgenic mice that contain the human GnT-III gene under the control of the mouse albumin enhancer/promoter [Lee et al., (2003)]. Overexpression of this gene in mice reduced the antigenicity of N-glycans to human natural antibodies, especially in the case of the alpha-Gal epitope, Galalpha1-3Galbeta1-4GlcNAc-R. Study of endothelial cells from the GnT-III transgenic mice revealed a significant reduction in antigenicity, and a dramatic decrease in both complement- and natural killer cell-mediated mouse cell lysis. Changes in the enzymatic activities of other glycosyltransferases, such as alpha1,3-galactosyltransferase, and alpha-6-D-mannoside beta-1,6 N-acetylglucosaminyltransferase V, did not point to any interaction between GnT-III and these enzymes in the transgenic mice, suggesting that this approach may be useful in clinical xenotransplantation.     

Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both human decay-accelerating factor and N-acetylglucosaminyltransferase III

Heterozygous alpha 1,3-galactosyltransferase (GT) gene knockout pigs were produced with transgenic pig fetal cells expressing both human decay-accelerating factor (hDAF) and N-acetylglucosaminyltransferase III (GnT-III). In this study, we assessed the gene targeting efficiency in the transgenic pig fetal cells derived from different fetal tissues such as brain, skin, heart, and liver, or fetal carcass. Targeted cell colonies were selected by hygromycin B. The GT-knockout colonies (KO colonies) were obtained equally from the cells derived from all tissues except liver. Staining with five antibodies against intermediate filaments, all examined KO cell lines stained positive for vimentin with the exception of a colony that stained positive for both vimentin and glial fibrillary acidic protein simultaneously. This is the first study to produce KO cells from the astrocytes. Some of these KO cell lines were used for nuclear transfer (NT) to obtain KO pig fetuses. Fourteen fetuses were obtained from two recipients of the embryo transfer and eight of them had normal ploidy. The cells from the KO pig fetuses were also used for NT to produce cloned KO pigs. Two healthy clone pigs were born. These pigs were determined to have a heterozygous knockout GT gene and the two transgenes. The cells collected from the KO pigs were shown to have similar expression levels of hDAF and GnT-III compared to their original transgenic pigs and less than a half levels of the alphaGal epitopes existed in wild-type pig cells.     

Reduction of the Gal-alpha1,3-Gal epitope of mouse endothelial cells by transfection with the N-acetylglucosaminyltransferase III gene

In order to prevent hyperacute rejection in pig-to-human xenotransplantation, it would be very useful to be able to down-regulate the Gal alpha1-3 Galbeta 1-4 GlcNAc-R (alpha-Gal epitope) in mouse and swine tissues. When the beta-D-mannoside beta-1,4-N-acetylglucosaminyl-transferase III (GnT-III) gene was introduced into mouse aorta endothelial cells (MEC) their susceptibility to complement-mediated cell lysis by normal human serum (NHS) was reduced. Expression of GnT-III also suppressed the antigenicity of MEC to human natural antibodies as shown by binding of Griffonia simplicifolia 1 isolectin (GS1B4 lectin) to the alpha-Gal epitope. Western blot analysis indicated that the reactivity of the glycoproteins of the transfectants to NHS and GSIB4 lectin was reduced to approximately the same extent. Thus GnT-III, a key enzyme involved in the formation of branched N-linked sugars, reduces the expression of xenoantigens, suggesting that this approach may be of value in clinical xenotransplantation.     

Hyperexpression of N-acetylglucosaminyltransferase-III in liver tissues of transgenic mice causes fatty body and obesity through severe accumulation of Apo A-I and Apo B

N-Acetylglucosaminyltransferase (GnT)-III catalyzes the attachment of an N-acetylglucosamine (GlcNAc) residue to mannose in beta(1-4) configuration in the region of N-glycans and forms a bisecting GlcNAc. To investigate the pathophysiological role of dysregulated glycosylation mediated by aberrantly expressed GnT-III, we generated transgenic mice hyperexpressing the human GnT-III in the liver by introducing human GnT-III cDNA under the control of mouse albumin enhancer/promoter. Total five transgenic founder mice (pGnTSVTpA-10, -14, -20, -25, and -51) expressed the human GnT-III in their livers and were characterized by molecular genetic means. The copy number of transgene integrated into the genome of these mice ranged between 1 and 3 copies per haploid genome. Northern and Western blot analyses showed that the transgene is specifically expressed in the liver but not in any other tissues tested. The triglyceride level in GnT-III transgenic mice was significantly decreased, however, no significant differences in the levels of glucose, cholesterol, or albumin were observed between transgenic and nontransgenic mice. Although glutamate oxaloacetic transaminase and glutamic pyruvic transaminase activities of transgenic mice were also higher than those of nontransgenic mice, no differences in total bililubin and total protein were observed between the two animal lines. Large amounts of apolipoprotein (Apo) A-I and Apo B were specifically detected in the intracellular liver of transgenic mice. The accumulation of Apo A-I in hepatocytes may be due to aberrant glycosylation, since glycosylated Apo A-I was not observed in transgenic mice. However, the accumulated Apo B was severely glycosylated. Therefore, it is suggested that highly expressed transgenic GnT-III allowed unknown target proteins to be glycosylated in large amounts, and the resulting target protein(s) disrupted in assembly formation of Apo A-I in the hepatocytes and cause a decrease in the release of lipoproteins and accumulations of Apo A-I and Apo B in the liver. The transgenic mice showed aberrant glycosylation by GnT-III, resulting in numerous lipid droplets in liver tissues and the obesity. These mice showed microvesicular fatty changes with abnormal lipid accumulation in the hepatocytes. Our study provides the basis for future analysis of the role of glycosylation in hepatic pathogenesis. In the transgenic mice, Apo A-I and Apo B were significantly increased compared with levels in nontransgenic liver tissues.     

Characterization of a hypertriglyceridemic transgenic miniature pig model expressing human apolipoprotein CIII

Hypertriglyceridemia has recently been considered to be an independent risk factor for coronary heart disease, in which apolipoprotein (Apo)CIII is one of the major contributory factors, as it is strongly correlated with plasma triglyceride levels. Although ApoCIII transgenic mice have been generated as an animal model for the study of hypertriglyceridemia, the features of lipoprotein metabolism in mice differ greatly from those in humans. Because of the great similarity between pigs and humans with respect to lipid metabolism and cardiovascular physiology, we generated transgenic miniature pigs expressing human ApoCIII by the transfection of somatic cells combined with nuclear transfer. The expression of human ApoCIII was detected in the liver and intestine of the transgenic pigs. As compared with nontransgenic controls, transgenic pigs showed significantly increased plasma triglyceride levels (83 ± 36 versus 38 ± 4 mg·dL(-1), P < 0.01) when fed a chow diet. Plasma lipoprotein profiling by FPLC in transgenic animals showed a higher peak in large-particle fractions corresponding to very low-density lipoprotein/chylomicrons when triglyceride content in the fractions was assayed. There was not much difference in cholesterol content in FPLC fractions, although a large low-density lipoprotein peak was identified in both nontransgenic and transgenic animals, resembling that found in humans. Further analysis revealed markedly delayed clearance of plasma triglyceride, accompanied by significantly reduced lipoprotein lipase activity in post-heparin plasma, in transgenic pigs as compared with nontransgenic controls. In summary, we have successfully generated a novel hypertriglyceridemic ApoCIII transgenic miniature pig model that could be of great value for studies on hyperlipidemia in relation to atherosclerotic disorders.     

Cloning of the transgenic pigs expressing human decay accelerating factor and N-acetylglucosaminyltransferase III

The present paper describes production of cloned pigs from fibroblast cells of transgenic pigs expressing human decay accelerating factor (DAF, CD55) and N-acetylglucosaminyltransferase III (GnT-III) that remodels sugar-chain biosynthesis. Two nuclear transfer protocols were used: a two-step activation (TA) method and a delayed activation (DA) method. Enucleated in vitro-matured oocytes and donor cells were electrically fused in a calcium-containing medium by TA method or in a calcium-free medium by DA method, followed by electrical activation 1-1.5 h later, respectively. In vitro blastocyst formation rates of nuclear transferred embryos reconstructed by TA and DA method were 8% and 14%, respectively. As a result of embryo transfer of the reconstructed embryos made by each method into recipient pigs, both gave rise to cloned piglets. These cloned pigs expressed transgene as much as their nuclear donor cells. In conclusions, (1) pig cloning can be carried out by TA or DA nuclear transfer methods, (2) expression of transgenes can be maintained to cloned pigs from the nuclear donor cells derived from transgenic animals.     

A comparative study on the integration of exogenous DNA into mouse, rat, rabbit, and pig genomes.

Transgenic mammals, from small laboratory rodents to domestic animals, have been successfully produced to date, but their production efficiency within or across species has been variable. This is probably due to the differences in the type of injected DNA and/or technical procedures employed in each laboratory, as well as the reproductive characteristics of the species. Here we report the direct comparison of the efficiencies of producing transgenic mice, rats, rabbits and pigs by one technician using a fusion gene composed of the bovine alpha S1-casein promoter and human growth hormone (hGH) gene. Before the fusion gene was injected into the zygotes, high magnitude centrifugation to visualize the pronuclei was necessary for all of the pig zygotes and one-third of the rabbit zygotes, but not for mouse and rat zygotes. Post-injection survival of the mouse zygotes (67.1%) was lower than those of the rat, rabbit and pig zygotes (89.6 to 100%). The volume change of the pronucleus following DNA injection was the lowest in mice (50% increase), moderate in rabbits (148% increase), and the most prominent in rats (238% increase). The data from only 1 pig zygote indicated a 22% increase in the pronucleus volume by DNA injection. The PCR analyses of the tail DNA of new born offspring indicated that 0.8% (4/493), 4.8% (22/463), 0.8% (3/367) and 0.9% (2/221) of the injected eggs in mice, rats, rabbits and pigs, respectively, developed into transgenic offspring. Some of the founder animals in all four species expressed the transgene in the mammary gland which was confirmed in hGH mRNA by RT-PCR and/or hGH peptide in Witch's milk with ELISA. These results suggest that the maximum volume of DNA solution injectable into the pronucleus is a possible factor explaining the species differences in the production of transgenic animals.     

Production of a reporter transgenic pig for monitoring Cre recombinase activity

The pig is thought to be the most suitable non-human source of organs for xenotransplantation and is widely used as a model of human disease. Using pigs as disease models requires the design of conditional Cre recombinase-loxP gene modifications, which, in turn, requires a Cre-expressing pig with defined patterns of expression controlled by the use of a tissue-specific promoter. In order to monitor Cre recombinant expression in vivo, it is important to create a reporter strain. We have generated reporter a pig that is based on a single vector that drives the ubiquitous expression of the enhanced green fluorescent protein (EGFP). The EGFP gene is expressed only after Cre-mediated excision of loxP-flanked stop sequences. These reporter transgenic pigs will be of great value for monitoring Cre recombinase activity in vivo.     

Construction of a transgenic pig model overexpressing polycystic kidney disease 2 (PKD2) gene

Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease, affecting millions of people worldwide. The progressive growth of cysts in kidneys eventually leads to renal failure in 50 % of patients, and there is currently no effective treatment. Various murine models have been studied to elucidate the disease mechanisms, and much information has been acquired. However, the course of the disease cannot be fully recapitulated using these models. The pig is a suitable model for biomedical research, and pig PKD2 has high similarity to the human ortholog at the molecular level. Here, a mini-pig PKD2 transgenic model was generated, driven by a ubiquitous cytomegalovirus enhancer/promoter. Using somatic cell nuclear transfer, four transgenic pigs with approximately 10 insertion events each were generated. Quantitative real-time PCR and western blotting showed that PKD2 was more highly expressed in transgenic pigs than in wild-type counterparts. Because of the chronic nature of ADPKD, blood urea nitrogen and serum creatinine levels were continuously measured to assess the pig kidney function. The transgenic pigs continue to show no significant alteration in kidney function; it is estimated that 1–2 more years may be required for manifestation of renal cystogenesis in these pigs.     

Grouping of 20 reference strains of Legionella species by the growth ability within mouse and guinea pig macrophages

20 Reference strains of Legionella species, isolated from human, were classified according to their ability to grow within thioglycolate-induced peritoneal macrophages of mice and guinea pigs. Inbred and congenic mice were used to study the effect of the natural resistance genes Lgn1 and Bcg that are expressed phenotypically in the mouse macrophages. The Lgn1 gene controlled the intracellular growth of Legionella pneumophila Philadelphia-1 and Legionella jordanis GIFU 12657, but the Bcg gene did not affect the intracellular growth of any organism examined. Based on these results and the growth ability in guinea pig macrophages, the 20 reference strains were divided into four groups. This grouping will help us to understand a variety of modes of interaction between Legionella species and macrophages.     

Production of Transgenic-clone Pigs by the Combination of ICSI-mediated Gene Transfer with Somatic Cell Nuclear Transfer

The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce transgenic pigs. We also aimed at examining whether transgenic pigs can be cloned from somatic cells of a transgenic pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 transgenic fetuses were obtained. One of the recipients gave birth to three live piglets including one transgenic pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder transgenic pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live transgenic-clones. The transgene copy number and integrity in the founder pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce transgenic pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in transgenic pig production.     

Safety assessment of <Emphasis Type="Italic">sFat</Emphasis>-<Emphasis Type="Italic">1</Emphasis> transgenic pigs by detecting their co-habitant microbe in intestinal tract

Transgenic pigs containing sFat-1 (synthesized fatty acid desaturase-1) gene were produced by DNA microinjection. The meat of these pigs contain ω-3 unsaturated fatty acid which is beneficial to the health of human being. The aim of this study is to assess the effects of expression of sFat-1 in pig on the health of animal themselves and on the safety of environment from the angle of the changes in microbe population of pig intestinal tract. Four F1 male semi-sibling of sFat-1 transgenic pigs and four F1 female semi-sibling of sFat-1 transgenic pigs were used as experimental animals, together with their none transgenic siblings as control animals. For inspection of any change in populations of microbial flora in various parts of intestinal tract and feces of sFat-1 transgenic pigs total aerobe, total anaerobe, main beneficial bacteria and main harmful bacteria were cultured and analyzed. At the same time foreign gene drift was assessed by PCR amplifying foreign fragment in samples of total aerobe and total anaerobe. Results indicated that in comparison with control pig microbe population of various species in different parts of intestinal tract and feces of sFat-1 transgenic pig remained unchanged and foreign gene sequence could not be detected in DNA of total aerobe and total anaerobe. In conclusion, the data of this study suggest that microbe population in intestinal tract and feces of transgenic pig was not influenced by the expression of transferred foreign gene sFat-1 and also foreign gene drifting from animal genome to microbial genome must be a rare incident.     

猪Gli1基因的克隆、表达谱分析及脂肪组织特异性表达载体的构建

Hedgehog(Hh)信号通路对动物脂肪沉积具有抑制作用,并且从果蝇到脊椎动物具有高度保守性,但在家猪研究中鲜见报道。文章选择家猪Hh通路的转录激活因子Gli1进行研究,通过RT-PCR结合RACE技术,首次获得家猪Gli1基因cDNA全长,利用Real-time PCR对家猪Gli1基因在不同组织中的表达丰度进行了分析,并构建了真核表达载体和脂肪组织特异性表达载体。结果表明:猪Gli1基因cDNA全长3 576 bp,基因组序列全长10 715 bp,共12个外显子,编码1 106个氨基酸。生物信息学分析表明,猪Gli1为不稳定亲水性蛋白,不具有跨膜结构域和信号肽序列,但具有锌指结构与核定位序列。对7个物种的Gli1蛋白序列和基因组序列相似性进行分析,发现各物种间序列相似性均在80%以上,说明Gli1在物种间高度保守。组织表达谱分析表明,Gli1仅在成体猪舌组织中表达;在家猪脂肪组织发育进程中,Gli1仅在出生1周的猪脂肪组织中检测到微弱表达,但1月龄及3月龄猪脂肪组织中均检测不到表达,由此推断猪Gli1表达与脂肪组织发育呈负相关。最后,将猪Gli1编码区克隆到真核表达载体pIRES2-EGFP,体外转染实验证明该载体能够正确表达猪Gli1,另外还构建了脂肪组织特异性表达载体,为构建脂肪组织特异性转基因动物奠定基础。     

Reduced sensitivity to human serum inactivation of enveloped viruses produced by pig cells transgenic for human CD55 or deficient for the galactosyl-alpha(1-3) galactosyl epitope

Complement activation mediated by the major xenogeneic epitope in the pig, galactosyl-alpha(1-3) galactosyl sugar structure (alpha-Gal), and human natural antibodies could cause hyperacute rejection (HAR) in pig-to-human xenotransplantation. The same reaction on viruses bearing alpha-Gal may serve as a barrier to zoonotic infection. Expressing human complement regulatory proteins or knocking out alpha-Gal epitopes in pig in order to overcome HAR may therefore pose an increased risk in xenotransplantation with regard to zoonosis. We investigated whether amphotropic murine leukemia virus, porcine endogenous retrovirus, and vesicular stomatitis virus (VSV) budding from primary transgenic pig aortic endothelial (TgPAE) cells expressing human CD55 (hCD55 or hDAF) was protected from human-complement-mediated inactivation. VSV propagated through the ST-IOWA pig cell line, in which alpha-galactosyl-transferase genes were disrupted (Gal null), was also tested for sensitivity to human complement. The TgPAE cells were positive for hCD55, and all pig cells except the Gal-null ST-IOWA expressed alpha-Gal epitopes. Through antibody binding, we were able to demonstrate the incorporation of hCD55 onto VSV particles. Viruses harvested from TgPAE cells were relatively resistant to complement-mediated inactivation by the three sources of human sera tested. Additionally, VSV from Gal-null pig cells was resistant to human complement inactivation. Such protection of enveloped viruses may increase the risk of zoonosis from pigs genetically modified for pig-to-human xenotransplantation.     

A glycomic approach to hepatic tumors in N-acetylglucosaminyltransferase III (GnT-III) transgenic mice induced by diethylnitrosamine (DEN): identification of haptoglobin as a target molecule of GnT-III

A glycomic approach to the identification of target molecules in glycosyltransferase gene targeting mice is a promising strategy to understand the biological significance of glycosyltransferase genes in vivo . In order to understand the biological effects of N -acetylglucosaminyltransferase III (GnT-III) on tumor formation in the liver, diethylnitrosamine (DEN) induced tumor formation in the GnT-III transgenic mice was examined. Our findings show that the incidence of hepatic tumor could be dramatically suppressed. A glycomic approach using two-dimensional gel electrophoresis followed by lectin blot analysis and sequence analysis revealed that haptoglobin, a radical scavenger molecule in serum was heavily glycosylated in hepatic tumor-bearing GnT-III transgenic mice that had been treated with DEN. Immunoprecipitation followed by E 4 -PHA lectin blot analysis also confirmed that the bisecting GlcNAc, a product of GnT-III was added to haptoglobin molecules. Since the use of DEN is known to lead to the production of lipid peroxidation products which facilitate this reaction and haptoglobin is an acute phase reactant, acting as a radical scavenger against hemoglobin or iron stimulated lipid peroxidation, a relationship between the glycosylation of haptoglobin and the suppression of hepatoma development can not be ruled out. This paper is the first report that shows a relationship between the sugar chains of glycoproteins with radical scavenger activity and hepatocarcinogenesis.     

A Glycomic Approach to Hepatic Tumors in N -acetylglucosaminyltransferase III (GnT-III) Transgenic Mice Induced by Diethylnitrosamine (DEN): Identification of Haptoglobin as a Target Molecule of GnT-III

A glycomic approach to the identification of target molecules in glycosyltransferase gene targeting mice is a promising strategy to understand the biological significance of glycosyltransferase genes in vivo. In order to understand the biological effects of N -acetylglucosaminyltransferase III (GnT-III) on tumor formation in the liver, diethylnitrosamine (DEN) induced tumor formation in the GnT-III transgenic mice was examined. Our findings show that the incidence of hepatic tumor could be dramatically suppressed. A glycomic approach using two-dimensional gel electrophoresis followed by lectin blot analysis and sequence analysis revealed that haptoglobin, a radical scavenger molecule in serum was heavily glycosylated in hepatic tumor-bearing GnT-III transgenic mice that had been treated with DEN. Immunoprecipitation followed by E 4 -PHA lectin blot analysis also confirmed that the bisecting GlcNAc, a product of GnT-III was added to haptoglobin molecules. Since the use of DEN is known to lead to the production of lipid peroxidation products which facilitate this reaction and haptoglobin is an acute phase reactant, acting as a radical scavenger against hemoglobin or iron stimulated lipid peroxidation, a relationship between the glycosylation of haptoglobin and the suppression of hepatoma development can not be ruled out. This paper is the first report that shows a relationship between the sugar chains of glycoproteins with radical scavenger activity and hepatocarcinogenesis.     

Transgenic expression of CTLA4-Ig by fetal pig neurons for xenotransplantation

The transplantation of fetal porcine neurons is a potential therapeutic strategy for the treatment of human neurodegenerative disorders. A major obstacle to xenotransplantation, however, is the immune-mediated rejection that is resistant to conventional immunosuppression. To determine whether genetically modified donor pig neurons could be used to deliver immunosuppressive proteins locally in the brain, transgenic pigs were developed that express the human T cell inhibitory molecule hCTLA4-Ig under the control of the neuron-specific enolase promoter. Expression was found in various areas of the brain of transgenic pigs, including the mesencephalon, hippocampus and cortex. Neurons from 28-day old embryos secreted hCTLA4-Ig in vitro and this resulted in a 50% reduction of the proliferative response of human T lymphocytes in xenogenic proliferation assays. Transgenic embryonic neurons also secreted hCTLA4-Ig and had developed normally in vivo several weeks after transplantation into the striatum of immunosuppressed rats that were used here to study the engraftment in the absence of immunity. In conclusion, these data show that neurons from our transgenic pigs express hCTLA4-Ig in situ and support the use of this material in future pre-clinical trials in neuron xenotransplantation.Caroline Martin,Martine Plat, Philippe Brachet, Bernard Vanhove - These authors have contributed equally to the development of this work.     

Adenovirus－mediated expression of pig α（1，3）galactosyltransferase reconstructs Gal α（1,3） Gal epitope on the surface of human tumor cells

Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.     

Antigenicity of penicillin G in the guinea pig

Antigenicity of penicillin G (PCG) was studied in guinea pigs. PCG 5 mg, 10 mg or 25 mg with Freund's complete adjuvant each on days 0, 7 and 21 was injected to a guinea pig: intramuscularly into both thighs and intracutaneously into four locations on the back. A remarkable antigenicity was induced in animals immunized with 25 mg although only low antigenicity in 5 mg and 10 mg. A maximum serum level of the antibody was observed about 2 weeks after last immunization and all of animals immunized with 25 mg died in active systemic anaphylaxis test. As mentioned above, it has been firstly demonstrated that a remarkable antigenicity of PCG can be produced by immunizing with a high dose of 25 mg in the guinea pig model in which PCG itself is used as immunogen.     

A synthetic dDAF (CD55) gene based on optimal codon usage for transgenic animals

The human DAF (CD55) gene was chosen as a representative molecule in a xenotransplantation study. The gene was synthesized in order to adapt its codons to those which are more frequent in mammals, especially pigs, and the expression levels were then examined in Chinese hamster ovarian (CHO) cells, swine endothelial cell (SEC) and transgenic mice. A significant increase in protein production with no detectable mRNA elevation was observed in the transfectants of synthetic DAF (sDAF), compared with the wild-type DAF (wtDAF) and delta-SCR1 wild-type DAF (Delta1wtDAF). Consistent with the in vitro data, the expression of DAF in mice that carry sDAF was higher than Delta1wtDAF in many organs, especially the pancreas. The sDAF showed a high level of expression in SEC and transgenic mice, suggesting that it will be useful in the development of transgenic pigs with high levels of expression.