DAF Gene Research Articles

The ACE inhibitor captopril inhibits ACN-1 to control dauer formation and aging.

The renin-angiotensin-aldosterone system (RAAS) plays a well-characterized role regulating blood pressure in mammals. Pharmacological and genetic manipulation of the RAAS has been shown to extend lifespan in Caenorhabditis elegans, Drosophila and rodents, but its mechanism is not well defined. Here, we investigate the angiotensin-converting enzyme (ACE) inhibitor drug captopril, which extends lifespan in worms and mice. To investigate the mechanism, we performed a forward genetic screen for captopril-hypersensitive mutants. We identified a missense mutation that causes a partial loss of function of the daf-2 receptor tyrosine kinase gene, a powerful regulator of aging. The homologous mutation in the human insulin receptor causes Donohue syndrome, establishing these mutant worms as an invertebrate model of this disease. Captopril functions in C. elegans by inhibiting ACN-1, the worm homolog of ACE. Reducing the activity of acn-1 via captopril or RNA interference promoted dauer larvae formation, suggesting that acn-1 is a daf gene. Captopril-mediated lifespan extension was abrogated by daf-16(lf) and daf-12(lf) mutations. Our results indicate that captopril and acn-1 influence lifespan by modulating dauer formation pathways. We speculate that this represents a conserved mechanism of lifespan control.

KLF regulation of insulin pathway genes.

Alteration in lipid metabolism can result in fat accumulation in adipose tissues, which may lead to two most important human diseases, obesity and diabetes. A shift in lipid metabolism deregulates signaling pathways which regulates obesity and/or diabetes. In this study, we examined the components of insulin/ TGF-β pathways and their genetic interaction with Krüppel-like transcription factors (KLFs). Their role in energy homeostasis were discussed. We separately created klf/daf genes double mutants by carrying out klfs RNAi on daf-2 (e1391), daf-4 (e1364), daf-7 (e1372); dpy-1 (e1), daf-14 (m77), daf-16 (mgDf50) mutants. And then conducted Oil O Red staining to assay the klf/daf RNAi worms for fat deposits and examine genetic interaction between klfs and daf genes. The results showed that worms bearing klf-1, 2, or 3 and daf-2, or daf-4 mutations deposit large, but similar fat levels as individual mutants. The results suggested that they target the same molecular pathway of fat storage. klf-1, 2 or 3 RNAi /daf-7 worms showed higher fat deposits in klf-1, 2, or 3 RNAi/daf-7 worms than klf-1, 2, or 3 RNAi or daf-7 mutants alone, which showed a functional interaction between klfs and daf-7 in perhaps TGF-β-like pathway. Altogether our study suggests a direct role of klfs in insulin signaling pathway.

PM2.5 induce lifespan reduction, insulin/IGF-1 signaling pathway disruption and lipid metabolism disorder in Caenorhabditis elegans.

Exposure to fine particulate matter (PM), especially PM2.5, can induce various adverse health effects in populations, including diseases and premature death, but the mechanism of its toxicity is largely unknown. Water-soluble components of PM2.5 (WS-PM2.5) were collected in the north of China in winter, and combined in two groups with the final concentrations of 94 μg/mL (CL group, AQI ≤ 100) and 119 μg/mL (CH group, 100 < AQI ≤ 200), respectively. The acute and long-term toxic effects of WS-PM2.5 samples were evaluated in several aspects such as development, lifespan, healthspan (locomotion behavior, heat stress tolerance, lipofucin). DAF mutants and genes were applied to verify the action of IIS pathway in WS-PM2.5 induced-effects. RNA-Sequencing was performed to elucidate the molecular mechanisms, as well as ROS production and Oil red O staining were also served as means of mechanism exploration. Body length and lifespan were shortened by exposure to WS-PM2.5. Healthspan of nematodes revealed adverse effects evaluated by head thrash, body bend, pharyngeal pump, as well as intestinal lipofuscin accumulation and survival time under heat stress. The abbreviated lifespan of daf-2(e1370) strain and reduced expression level of daf-16 and hsp-16.2 indicated that IIS pathway might be involved in the mechanism. Thirty-five abnormally expressed genes screened out by RNA-Sequencing techniques, were functionally enriched in lipid/lipid metabolism and transport, and may contribute substantially to the regulation of PM2.5 induced adverse effects in nematodes. WS-PM2.5 exposure induce varying degrees of toxic effects, such as body development, shorten lifespan and healthspan. The IIS pathway and lipid metabolism/transport were disturbed by WS-PM2.5 during WS-PM2.5 exposure, suggesting their regulatory role in lifespan determination.

ANTI-CRA: A FAMILY STUDY OF A BRAZILIAN FAMILY IN MOLECULAR TIMES

Cromer blood group system consists of 12 high-prevalence and three low-prevalence antigens. The molecular basis for the antigens is known, and the majority is the product of a single nucleotide change in the DAF gene and has been localized to one of the four complement control protein domains on the DAF protein. Antibodies to Cromer antigens are rarely encountered. This case reports a 72 year-oldAfrican descendent Brazilian woman with endometrioid adenocarcionoma had an antibody reactive with all red blood cells (RBCs) and cord RBCs. The direct antiglobulin test (DAT) and autocontrol were negative. She had 2 previous pregnancies and a previous transfusion 20 years ago. Siblings were recruited and a familiar study was performed. Serological and molecular studies were carried out to define the patient‘s phenotype and the antibody specificity. Serological investigation was performed by hemagglutination in gel cards.Antibody identification was performed in the patient's serum with untreated and treated RBCs with papain, trypsin, α-chymotrypsin and 200 mM dithiothreitol (DTT).Antigen typing was performed for the most common antigens with commercial reagents and for high frequency antigens with sera from our collection coming from SCARF. Genomic DNA was used to amplify and sequence CROM exons. The patient‘s serum reacted with all panel cellsincluding phenotypically similar RBCs. Only the autologous control was non-reactive. Crossmatching was negative with RBCs of 2 siblings and positive with the other two. Antibody titration showed a titre of 64.The antigen was sensitive to papain, typsin and α-chymotrypsin-treatment but was resistant to 200 mM DTTwhich suggested the presence of a high-frequency antibody to an antigen of the Cromer system. Sequencing analysis of CROM revealed the patient and her two sisters with negative crossmatching were homozygous for the molecular change c.679G > Cin exon 6, which predict the Cr(a-) phenotype. DNA sequencing of the two other siblings with positive crossmatching showed this molecular change in heterozygous.The antibody was determined to be of anti-Cr a specificity and other underlying antibodies were ruled out through adsorption and elution with R1R1, Jk(b-) and Fy(b-) allogeneic RBCs. This study reported for the first time a rare case of anti-Cr a in a Brazilian family. Due to the complexity of the Cromer system and the scarcity of anti-Cra and Cr(a-) RBCs, molecular analysis was essential for the conclusion of the case. Anti-Cr a is rarely encountered and has already been related to moderate transfusion reaction but its clinical significance can be variable. Although no transfusions were requiredat this time, the family study was essential to find compatible donors.

Impact of using genotyping to predict SERF negative phenotype in Thai blood donor populations.

BackgroundSERF(+) is a high prevalence antigen in the Cromer blood group system that is encoded by a CROM*01.12 allele. The SERF(-) on red cells is caused by a single nucleotide variation, c.647C>T, in exon 5 of the Decay-accelerating factor, DAF gene. Alloanti-SERF was found in a pregnant Thai woman, and a SERF(-) individual was found among Thai blood donors. Since anti-SERF is commercially unavailable, this study aimed to develop appropriate genotyping methods for CROM*01.12 and CROM*01.-12 alleles and predict the SERF(-) phenotype in Thai blood donors.MethodsDNA samples obtained from 1,580 central, 300 northern, and 427 southern Thai blood donors were genotyped for CROM*01.12 and CROM*01.-12 allele detection using in-house PCR with sequence-specific primer (PCR-SSP) confirmed by DNA sequencing.ResultsValidity of the PCR-SSP genotyping results agreed with DNA sequencing; CROM*01.12/CROM*01.12 was the most common (98.42%, 98.00%, and 98.59%), followed by CROM*01.12/CROM*01.-12 (1.58%, 2.00%, and 1.41%) among central, northern, and southern Thais, respectively. CROM*01.-12/CROM*01.-12 was not detected in all three populations. The alleles found in central Thais did not significantly differ from those found in northern and southern Thais.ConclusionThis study is the first to distinguish the predicted SERF phenotypes from genotyping results obtained using in-house PCR-SSP, confirming that the CROM*01.-12 allele frequency ranged from 0.007 to 0.010 in three Thai populations. This helps identify the SERF(-) phenotype among donors and patients, ultimately preventing adverse transfusion reactions.

Heat-killed Lactobacillus spp. cells enhance survivals of Caenorhabditis elegans against Salmonella and Yersinia infections.

This study examined the effect of feeding heat-killed Lactobacillus cells on the survival of Caenorhabditis elegans nematodes after Salmonella Typhimurium and Yersinia enterocolitica infection. The feeding of heat-killed Lactobacillus plantarum 133 (LP133) and Lactobacillus fermentum 21 (LP21) cells to nematodes was shown to significantly increase the survival rate as well as stimulate the expression of pmk-1 gene that key factor for C. elegans immunity upon infection compared with control nematodes that were only fed Escherichia coli OP50 (OP50) cells. These results suggest that heat-killed LP133 and LF21 cells exert preventive or protective effects against the Gram-negative bacteria Salm. Typhimurium and Y. enterocolitica. To better understand the mechanisms underlying the LF21-mediated and LP133-mediated protection against bacterial infection in nematodes, transcriptional profiling was performed for each experimental group. These experiments showed that genes related to energy generation and ageing, regulators of insulin/IGF-1-like signalling, DAF genes, oxidation and reduction processes, the defence response and/or the innate immune response, and neurological processes were upregulated in nematodes that had been fed heat-killed Lactobacillus cells compared with nematodes that had been fed E. coli cells. In this study, the feeding of heat-killed Lactobacillus bacteria to Caenorhabditis elegans nematodes was shown to decrease infection by Gram-negative bacteria and increase the host lifespan. C. elegans has a small, well-organized genome and is an excellent in vivo model organism; thus, these results will potentially shed light on important Lactobacillus-host interactions.

PI3K/Akt pathway restricts epithelial adhesion of Dr+Escherichia coliby down-regulating the expression of decay accelerating factor

The urogenital microbial infection in pregnancy is an important cause of maternal and neonatal morbidity and mortality. Uropathogenic Escherichia coli strains which express Dr fimbriae (Dr+) are associated with unique gestational virulence and they utilize cell surface decay accelerating factor (DAF or CD55) as one of the cellular receptor before invading the epithelial cells. Previous studies in our laboratory established that nitric oxide reduces the rate of E. coli invasion by delocalizing the DAF protein from cell surface lipid rafts and down-regulating its expression. The phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) cell signal pathway plays an important role in host-microbe interaction because many bacteria including E. coli activate this pathway in order to establish infection. In the present study, we showed that the PI3K/Akt pathway negatively regulated the expression of DAF on the epithelial cell surface and thus inhibited the adhesion of Dr(+) E. coli to epithelial cells. Initially, using two human cell lines Ishikawa and HeLa which differ in constitutive activity of PI3K/Akt, we showed that DAF levels were associated with the PI3K/Akt pathway. We then showed that the DAF gene expression was up-regulated and the Dr(+) E. coli adhesion increased after the suppression of PI3K/Akt pathway in Ishikawa cells using inhibitor LY294002, and a plasmid which allowed the expression of PI3K/Akt regulatory protein PTEN. The down-regulation of PTEN protein using PTEN-specific siRNA activated the PI3K/Akt pathway, down-regulated the DAF, and decreased the adhesion of Dr(+) E. coli. We conclude that the PI3K/Akt pathway regulated the DAF expression in a nitric oxide independent manner.

235 PRODUCTION OF PORCINE KNOCK-IN FIBROBLASTS EXPRESSING HUMAN DECAY-ACCELERATING FACTOR on α-1,3-GALACTOSYLTRANSFERASE (GGTA1) GENE LOCUS

Galactose-α1,3-galactose (α1,3-gal) epitope is synthesised at a high concentration on the surface of pig cells by α1,3-galactosyltransferase gene (GGTA1). The α1,3-gal is responsible for hyperacute rejection in pig-to-human xenotransplantation. The generation of transgenic pigs as organ donors for humans is necessary to eliminate the GGTA1 gene that synthesise Galα(1,3)Gal. Knock-in system refers to the integration a foreign gene at a specific target, disrupting the target gene by inserting the transgene. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as complement inhibitor to protect self cells from complement attack. The generation of GGTA1 knockout pigs expressing DAF is necessary to use as organ donors for humans. In this study, we established cell lines of GGTA1 knockout expressing DAF from pig ear fibroblasts to apply somatic cell nuclear transfer. A GGTA1-DAF knock-in vector for positive-negative selection (PNS) consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the diphtheria toxin A fragment (DT-A) gene as a negative selectable marker gene. The DAF gene was subcloned in the GGTA1 gene exon 4 and the endogenous start codon was replaced by that of DAF. The ear fibroblast cell lines were isolated from Chicago miniature pig and used for transfection. Linearized targeting vector DNA was transfected into these cells by electroporation. After selection of G-418 during 11 days, we analysed 242 colonies using PCR and long PCR. We isolated 2 positive colonies for GGTA1 gene disruption. To confirm expression of the hDAF and GGTA1 genes, we isolated total RNA from normal pig cells and the knock-in somatic cell line. A cDNA synthesised using reverse transcriptase progressed to RT-PCR by a pair of DAF-specific primers and α1,3-GT specific primer, respectively. Expression of DAF was detected in DAF knock-in somatic cells, butnot in normal pig cell. Expression of GGTA1 gene was decreased in the knock-in somatic cell line compared with normal pig cell. These cell lines may be used production of α1,3-GT knockout and DAF expression pig for xenotransplantation.

Hypoxia Induces Trimethylated H3 Lysine 4 by Inhibition of JARID1A Demethylase

Histone H3 lysine 4 (H3K4) trimethylation (H3K4me3) at the promoter region of genes has been linked to transcriptional activation. In the present study, we found that hypoxia (1% oxygen) increased H3K4me3 in both normal human bronchial epithelial Beas-2B cells and human lung carcinoma A549 cells. The increase of H3K4me3 from hypoxia was likely caused by the inhibition of H3K4 demethylating activity, as hypoxia still increased H3K4me3 in methionine-deficient medium. Furthermore, an in vitro histone demethylation assay showed that 1% oxygen decreased the activity of H3K4 demethylases in Beas-2B nuclear extracts because ambient oxygen tensions were required for the demethylation reaction to proceed. Hypoxia only minimally increased H3K4me3 in the BEAS-2B cells with knockdown of JARID1A, which is the major histone H3K4 demethylase in this cell line. However, the mRNA and protein levels of JARID1A were not affected by hypoxia. GeneChip and pathway analysis in JARID1A knockdown Beas-2B cells revealed that JARID1A regulates the expression of hundreds of genes involved in different cellular functions, including tumorigenesis. Knocking down of JARID1A increased H3K4me3 at the promoters of HMOX1 and DAF genes. Thus, these results indicate that hypoxia might target JARID1A activity, which in turn increases H3K4me3 at both the global and gene-specific levels, leading to the altered programs of gene expression and tumor progression.

Physiological and molecular biological mechanisms underlying diapause in aquatic invertebrates

The review considers the published data, as well as its own, which demonstrate the abundance and evolutionary conservation of the mechanism of diapause in invertebrates. The ecological reasons for the emergence of diapause in life cycles of hydrobionts are analyzed. The specific physiological features of invertebrate diapausing organisms and the hormonal control of diapause are briefed. The molecular genetic mechanism of diapause is demonstrated by the example of a model species, Caenorhabditis elegans. Recent fundamental discoveries in molecular genetics related to the joint effect of genes and environmental factors on the basic metabolism, choice between the development-diapause alternative, and many other seasonal adaptations in multicellular organisms are discussed. The near discovery of the functional role of daf genes in hydrobionts is postulated. These studies will lead to a deeper understanding of the fine mechanism that underlies photoperiodism and the wider application of diapause phenomenon in theory and practice.

Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.

Decay-Accelerating Factor Suppresses Complement C3 Activation and Retards Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice

Decay-accelerating factor (DAF; CD55) is a membrane protein that regulates complement pathway activity at the level of C3. To test the hypothesis that DAF plays an essential role in limiting complement activation in the arterial wall and protecting from atherosclerosis, we crossed DAF gene targeted mice (daf-1(-/-)) with low-density lipoprotein-receptor deficient mice (Ldlr(-/-)). Daf-1(-/-)Ldlr(-/-) mice had more extensive en face Sudan IV staining of the thoracoabdominal aorta than Ldlr(-/-) mice, both following a 12-week period of low-fat diet or a high-fat diet. Aortic root lesions in daf-1(-/-)Ldlr(-/-) mice on a low-fat diet showed increased size and complexity. DAF deficiency increased deposition of C3d and C5b-9, indicating the importance of DAF for downstream complement regulation in the arterial wall. The acceleration of lesion development in the absence of DAF provides confirmation of the proinflammatory and proatherosclerotic potential of complement activation in the Ldlr(-/-) mouse model. Because upstream complement activation is potentially protective, this study underlines the importance of DAF in shielding the arterial wall from the atherogenic effects of complement.

Understanding the mechanism of the dormant dauer formation of C. elegans: From genetics to biochemistry

Dauer is a dormancy state that may occur at the end of developmental stage L1 or L2 of Caenorhabditis elegans when the environmental conditions are unfavorable (e.g., lack of food, high temperature, or overcrowding) for further growth. Dauer is a nonaging duration that does not affect the postdauer adult lifespan. Major molecular events would include the sensing of the environmental cues, the transduction of the signals into the cells, and the subsequent integration of the signals that result in the corresponding alteration of the metabolism and morphology of the organism. Genetics approach has been effectively used in identifying many of the so-called daf genes involved in dauer formation using C. elegans as the model. Nevertheless, biochemical studies at the protein and metabolic level has been lacking behind in understanding this important life phenomenon. This review focuses on the biochemical understanding so far achieved on dauer formation and dormancy in general, as well as important issues that need to be addressed in the future.

Retrovirus-Mediated Over-Expression of Decay-Accelerating Factor Rescues Crry-Deficient Erythrocytes from Acute Alternative Pathway Complement Attack

Decay-accelerating factor (DAF) and complement receptor 1-related gene/protein y (Crry) are two membrane-bound complement regulators on murine erythrocytes that inhibit C3/C5 convertases. Previously, we found that Crry- but not DAF-deficient erythrocytes were susceptible to alternative pathway complement-mediated elimination in vivo. To determine whether it is a unique activity or a higher level expression of Crry makes it indispensable on murine erythrocytes, we over-expressed DAF on Crry-deficient (Crry(-/-)) erythrocytes by retroviral vector-mediated DAF gene transduction of bone marrow stem cells. DAF retrovirus-transduced erythrocytes expressed 846 +/- 127 DAF molecules/cell (DAF(high)) compared with 249 +/- 94 DAF molecules/cell (DAF(low)) and 774 +/- 135 Crry molecules/cell on control mouse erythrocytes. DAF(high)-Crry(-/-) erythrocytes were significantly more resistant than either DAF(low)-Crry(-/-), DAF(-/-) -Crry(+/+) or wild-type erythrocytes to classical pathway complement-mediated C3 deposition in vitro. Furthermore, increased DAF expression rescued Crry(-/-) erythrocytes from acute alternative pathway complement attack in vivo. Notably, long term monitoring revealed that DAF(high)-Crry(-/-) erythrocytes were still more susceptible than wild-type erythrocytes to complement-mediated elimination as they had a shorter half-life in complement-sufficient mice but survived equally well in complement-deficient mice. These results suggest that both a high level expression and a more potent anti-alternative pathway complement activity of Crry contributed to its indispensable role on murine erythrocytes. Additionally, they demonstrate the feasibility of using stem cell gene therapy to correct membrane complement regulator deficiency on blood cells in vivo.

Discovery, linkage disequilibrium and association analyses of polymorphisms of the immune complement inhibitor, decay-accelerating factor gene (DAF/CD55) in type 1 diabetes.

BackgroundType 1 diabetes (T1D) is a common autoimmune disease resulting from T-cell mediated destruction of pancreatic beta cells. Decay accelerating factor (DAF, CD55), a glycosylphosphatidylinositol-anchored membrane protein, is a candidate for autoimmune disease susceptibility based on its role in restricting complement activation and evidence that DAF expression modulates the phenotype of mice models for autoimmune disease. In this study, we adopt a linkage disequilibrium (LD) mapping approach to test for an association between the DAF gene and T1D.ResultsInitially, we used HapMap II genotype data to examine LD across the DAF region. Additional resequencing was required, identifying 16 novel polymorphisms. Combining both datasets, a LD mapping approach was adopted to test for association with T1D. Seven tag SNPs were selected and genotyped in case-control (3,523 cases and 3,817 controls) and family (725 families) collections.ConclusionWe obtained no evidence of association between T1D and the DAF region in two independent collections. In addition, we assessed the impact of using only HapMap II genotypes for the selection of tag SNPs and, based on this study, found that HapMap II genotypes may require additional SNP discovery for comprehensive LD mapping of some genes in common disease.

Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32

The efficiency of the complement system as an innate immune defense mechanism depends on a fine control that restricts its action to pathogens and prevents non-specific damage to host tissues. Genetic and functional analyses have shown that this critical control of complement activation may be impaired in atypical hemolytic uremic syndrome (aHUS) patients. Mutations in HF1, MCP or FI have been found in aHUS patients, but incomplete penetrance of the disease in individuals carrying these mutations is relatively frequent and no genetic defect has yet been found in a majority of aHUS patients. We report here the identification of a specific SNP haplotype block, spanning the MCP gene in the regulators of complement activation gene cluster, which is over-represented in aHUS patients and strongly associates with the severity of the disease. Linkage disequilibrium analyses suggest that this SNP haplotype also includes the CR1, DAF and C4BP genes. Initial studies identified two SNPs in the haplotype that influence the transcription activity of the MCP promoter in transient transfection experiments. Notably, the SNP haplotype block was found to be particularly frequent among patients who carry mutations in HF1, MCP or FI. These findings and the identification of aHUS patients carrying mutations in two complement regulatory genes provide an important insight into the etiology of aHUS. Together, they suggest that complement regulatory molecules act as a protein network and that multiple hits, involving plasma- and membrane-associated complement regulatory proteins, are necessary to impair protection to host tissues and to confer significant predisposition to aHUS.

33 PRODUCTION OF TRANSGENIC-CLONED PIGS CARRYING HDAF, GnT-III AND HETEROZYGOUSLY DISRUPTED ±-1,3-GALACTOSYLTRANSFERASE GENES

In pig-to-human xenotransplantation, transplants are rapidly rejected by binding of human natural antibodies to porcine xenoantigen, mostly Gal α-1-3Gal oligosaccharides, and subsequent complement attack. To overcome this rejection, we so far have produced transgenic pigs expressing both human CD55/DAF (decay-accelerating factor, a complement-regulatory protein) and GnT-III (N-acetylglucosaminyltransferase III, a sugar chain modifying enzyme). In the present study, we heterozygously disrupted the α-1,3-galactosyltransferase (GT) gene, which catalyses the biosynthesis of Gal α-1-3Gal epitopes, in the fetal fibroblast cells from the DAF/GnT-III transgenic pigs by homologous recombination, and successfully produced GT-knockout pigs by nuclear transfer. Fibroblast cells isolated from Day 30 fetuses of DAF/GnT-III transgenic pigs were transfected with a GT-targeting vector. The targeting event in drug-resistant colonies was confirmed by PCR analysis, and targeted cells were used as nuclear donors. The reconstructed embryos were electrically activated and transferred to estrus-synchronized recipient pigs. At pregnancy Day 27 of gestation, fetuses were collected and their fibroblast cells were isolated for secondary nuclear transfer. The genomic DNA of live-born piglets produced by the secondary nuclear transfer were analyzed for the presence of DAF and GnT-III genes as well as the heterozygous disruption of the GT gene. From a total of 5.5 × 107 cells transfected with the GT-targeting vector, 2,749 drug-resistant colonies were obtained. Eighteen colonies were judged positive for targeting events by PCR analysis. After transfer of 321 cloned embryos reconstructed with the knockout cells to three recipients, four knockout fetuses were obtained from one recipient. Transfer of 633 cloned embryos reconstructed with the knockout fibroblast cells from one knockout fetus to six recipients gave rise to two live knockout piglets. PCR analysis of genomic DNA confirmed that the cloned piglets carried both DAF and GnT-III transgenes as well as the heterozygously disrupted GT gene.

Skin graft of double transgenic pigs of N-acetylglucosaminyltransferase III (GnT-III) and DAF (CD55) genes survived in cynomolgus monkey for 31 days

Background Our previous study reported that cynomolgus monkey did not hyperacutely reject a skin xenograft from a N-acetylglucosaminyltransferase III (GnT-III) transgenic pig. In the present study, we reported on the survival time of skin xenografts in GnT-III, DAF (CD55), and double (D/G) transgenic pigs, and the effect of FK506 thereon. Material and methods Skin from GnT-III, DAF and D/G transgenic pigs were transplanted to cynomolgus monkeys. Under general anesthesia, full thickness skin defects (1.5×1.5 cm each) were made on the back of the monkey. Pig abdominal skin, obtained using an electric dermatome, was cut into pieces and transplanted onto the monkey wounds and fixed. In addition, skins of GnT-III and D/G pigs were also transplanted to cynomolgus monkeys that had been treated intramuscularly with FK506 at a dose of 0.5 mg/kg/day for 14 days after transplantation. Grafts were observed and photographed each day and skin graft biopsies were done on days 3, 5, 7, 10, 11, 14, 21, 28 and 31 after transplantation. Graft rejection was assessed histologically, based on our previous criteria for skin allografts. Results Even in the immuno-suppressive drug free condition, skin xenografts of GnT-III, DAF and D/G transgenic pigs were not hyperacutely rejected in early phase after transplantation by the cynomolgus monkey. The pattern of these xenograft rejections was histologically similar to those for rat allograft rejections. Most of the GnT-III, DAF and D/G pig skin grafts remained nearly intact up to day 5, but slight lymphocyte infiltration was noted on day 7 (grade 1). On day 9, while the GnT-III skin showed moderate lymphocyte and eosinophilic infiltration, the DAF and D/G pig skin grafts showed complete epidermal separation (grade 3). On the other hand, in the case of cynomolgus monkeys treated with FK506, the GnT-III skin showed complete epidermal separation (grade 3) on day 21. In addition, one of the D/G skin graft was intact on day 21 and moderate lymphocyte infiltration and intraepidermal blister formation (grade 1) was finally seen on day 31. Conclusion Our data show the possibility that both the DAF and GnT-III double transgenic pig skin xenografts can be used in place of human skin allografts in cases of severe burns.

Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter.

Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.

Characterization of glycosylphosphatidylinositol-anchored decay accelerating factor (GPI-DAF) and transmembrane DAF gene expression in wild-type and GPI-DAF gene knockout mice using polyclonal and monoclonal antibodies with dual or single specificity.

Decay-accelerating factor (DAF, CD55) is a glycosylphosphatidylinositol (GPI)-linked membrane inhibitor of complement activation. While human and other mammalian species contain only one DAF gene, two distinct DAF genes, referred to as GPI-DAF and transmembrane (TM)-DAF, respectively, have been identified in the mouse. Using several independently generated monoclonal and polyclonal antibodies, either with dual or single specificity for GPI-DAF and TM-DAF gene products, we have examined the expression of the two DAF genes in tissues of the wild-type and a strain of knockout mouse whose GPI-DAF gene has been inactivated. By fluorescence-activated cell sorting (FACS) analysis, we found that DAF protein is present on the wild-type mouse erythrocytes and lymphocytes but no signal was detectable on the same cells of GPI-DAF gene knockout mice. Both T and B lymphocytes and splenic macrophages express the GPI-DAF gene but the expression level is higher on B lymphocytes than on T lymphocytes. Within the T cell population, both CD4+ and CD8+ T cells are positive. DAF protein was detected by immunohistochemistry at high levels on wild-type mouse spermatids and mature sperm. In contrast, only mature sperm stained positive in the GPI-DAF gene knockout mouse testis, suggesting that GPI-DAF but not the TM-DAF gene is expressed on spermatids. Examination of the fetoplacental unit at the day 7.5 stage revealed that GPI-DAF but not the TM-DAF gene is expressed in the maternal decidua cells surrounding the trophoectoderm of the embryo. No DAF expression was detected on trophoblast or the embryo proper. These findings suggest that although the TM-DAF gene is irrelevant on mouse blood cells, the two DAF genes may have different roles in germ cell development and/or mature sperm function. Because complement receptor 1-related gene/protein y (Crry) has been shown to be expressed on early mouse embryos, the complete lack of GPI-DAF and TM-DAF gene expression in early mouse development may explain the observed sensitivity of Crry-deficient embryos to maternal complement attack.