Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: a novel selection method for gal alpha 1,3-Gal antigen-deficient cells.

The objective of the present study was to isolate alpha 1,3-galactosyltransferase (GalGT)-gene double knockout (DKO) cells using a novel simple method of cell selection method. To obtain GalGT-DKO cells, GalGT-gene single knockout (SKO) fetal fibroblast cells were cultured for three to nine passages and GalGT-null cells were separated using a biotin-labeled IB4 lectin attached to streptavidin-coated magnetic beads. After 15-17 days of additional cultivation, seven GalGT-DKO cell colonies were obtained from a total of 2.5 x 10(7) GalGT-SKO cells. A total of 926 somatic nuclear transferred embryos reconstructed with the DKO cells were transferred into eight recipient pigs, producing four farrowed, three liveborns, and six stillborns. Absence of GalGT gene in the cloned pigs was confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that alphaGal antigens were not present in the cells of the cloned DKO pigs.

Differential human serum-mediated neutralization of PERV released from pig cells transfected with variants of hDAF.

BACKGROUND: Expression of complement regulatory proteins (CRP) on pig endothelial cells (PEC) is an effective means of avoiding induction of hyperacute rejection by human sera. However, pig endogenous retrovirus (PERV) from PEC transfected with CRP may acquire resistance to human sera. This study investigated a form of transfected CRP that is easily expressed on PERV particles. METHODS: The PEC line was transfected with the Lac Z gene and PERV-B to investigate PERV infectivity using a Lac Z pseudo-type assay. The cDNAs of several modified DAF (CD55) were then transfected into the PEC(Lac Z)/P-B lines using lipofection. DAF expression was verified by FACS analysis. Complement-dependent PEC lysis was tested to verify the complement regulatory function of the expressed DAF. HEK293 cells were incubated with PEC culture supernatants with or without human sera. The inoculated 293 cells were histochemically stained and Lac Z-positive blue foci were counted. The rate of reduction in Lac Z-positive cells resulting from the addition of human serum was then calculated. In addition, to assess the localization of the expressed DAF, flotation sucrose density analysis was performed. RESULTS: While PERV released from PEC expressing delta-short consensus repeat 2 (delta-SCR2) DAF (lacking CRP function) showed no change in resistance to human serum compared to control cells, PERV from cells expressing delta-SCR1 DAF (with CRP function) showed a significant increase in resistance. The DAF-blocking antibody assay indicated that PERV from the DAF transfectants expressed DAF molecules on the surface of the retrovirus. While delta-SCR1 DAF (PI-anchor form) significantly inhibited the reduction of Lac Z-positive cells by human serum, the reduction of Lac Z-positive cells by human serum was less inhibited in the case of transmembrane (TM)-types of DAF-HLA-G, modified influenza hemagglutinin (HA) and MCP (delta-CYT form). However, the reduction in each TM-type DAF was slightly less than that observed in naive and mock cells. The flotation sucrose density analysis of these transfectants indicated that the PI-anchor form of DAF is a raft-associated protein, and most TM-types of DAF are non-raft proteins. CONCLUSION: Induction of resistance to human serum in PERV, depends on the form of the CRP tail. The CRP/TM hybrid that does not associate with lipid rafts, is a suitable form of CRP for gene transduction.

[Allo- and xeno-transplantation].

Organ transplantation is now effective therapies across a wide range of both fatal and non-fatal diseases. The excellent survival and success rates of organ transplantation have led to high levels of demand globally. The demand has outstripped the supply of organs from both deceased donors and from the altruistic living relatives of patients in need. Increasing use, over the past 10 years, of living donation of nonregenerative organs has extended from kidneys to livers, lungs and pancreas in some instances, despite the hope that reliance on living donors could be reduced. And, it is clear that ethically-unacceptable practices occur in a number of countries. The 1991 World Health Organization (WHO) Guiding Principles (GP) have influenced national legislation and professional codes but over the last 10 years many transplantation practices are no longer in line with the GP. The GP will be revised in 2008. While xenotransplantation offers a potential solution to the demand, 3 problems need to be overcome, i.e. inadequate physiological function, rejection of the graft, and the risk of transmitting a serious and/or novel infectious disease to the human recipient and wider public.

Features of a newly cloned pig C1 esterase inhibitor.

The pig cDNA encoding C1 esterase inhibitor (C1-INH) was isolated and the homology of the sequence was compared with that from other animals. The structure of pig C1-INH contains a two disulfide bridge pattern identical to the human C1-INH. In the amino acid sequence of the first Cys-91 to the C-terminal end, the pigC1-INH has a 76.2% homology with the human protein, and the sequence of the reactive site is close to the human. A surface-bound form of pig and human C1-INH, pC1-INH-PI and hC1-INH, respectively, were next constructed. Stable Chinese hamster ovarian tumor (CHO) cell lines and pig endothelial cell (PEC) lines expressing these C1-INH-PI were prepared by transfection. The basic function and the species specificity of pCI-INH were then investigated using these transfectants. pC1-INH and hC1-INH have almost the same suppressive effect on pig, human, dog and rabbit sera in complement-dependent cell lysis, indicating little species specificity.

Prevention of PERV infections in pig to human xenotransplantation by the RNA interference silences gene.

The possibility of preventing the transmission of porcine endogenous retrovirus (PERV) to human cells using short interfering RNAs (siRNA) was investigated. The siRNA for the p30 of PERV gag region was cloned into pSUPER, the polymerase-III H1-RNA gene promoter. A green fluorescence protein (GFP) was also cloned into pSUPER to establish pSXGH. Pig endothelial cells (PEC) were transduced with the LacZ gene by pseudotype infection, and infected with PERV subtype B, resulting in the formation of PEC(LacZ)/PB. The PEC(LacZ)/PB was next transfected with pSXGH-siRNA. The expression of siRNA was provisionally checked by determining the level of expression of GFP. Culture supernatants of infected cells were then inoculated into HEK293 cells. The siRNA clearly destroyed the PERV infectivity of PEC(LacZ)/PB in both transient cell lines and stable clones. Moreover, the decreased levels of mRNA and gag protein were evidenced in the stable clones by real-time PCR and Western blotting, respectively. The final goal of our study was to establish a transgenic pig expressing the siRNA for PERV. The results suggest that siRNA represents a novel approach for controlling PERV infections in clinical xenotransplantation.

Modulation of the leader peptide sequence of the HLA-E gene up-regulates its expression and down-regulates natural killer cell-mediated swine endothelial cell lysis.

BACKGROUND: The inhibitory function of HLA class I molecules, HLA-G1 and HLA-E, on natural killer (NK) cell-mediated cytolysis has previously been reported. In this study, we report on a study of the effects of the co-expression of these molecules on the inhibition of NK cell-mediated cytolysis, using a newly constructed gene. METHODS: Complementary DNA (cDNA) of HLA-G (G1 and G3), HLA-E, and human beta2-microglobulin (hbeta2m) were prepared and transfected into swine endothelial cell (SEC) and Chinese hamster ovarian tumor (CHO) cell. The leader peptide sequences of HLA-G1 and HLA -E genes were changed to VMAPRTLFL or VMAPRTLVL, which corresponds to the original HLA-G1 and HLA-A2. The cell surface expression of the modified genes was evaluated by flow cytometry, and NK cell-mediated cytolysis by human peripheral blood mononuclear cells (PBMC) was assessed. RESULTS: The transfectant with the hbeta2m and HLA-G1 genes showed a clear expression of the HLA-G1 molecule and had an inhibitory effect on NK cell-mediated SEC lysis. Whereas neither the transfectant with the hbeta2m and HLA-E genes, nor that with the hbeta2m and HLA-G3 genes, expressed the HLA molecule on SEC, the transfectant with triple genes, hbeta2m, HLA-E, and HLA-G3, expressed the HLA-E molecule and also inhibited NK-mediated SEC lysis. Conversely, the modification of the leader sequence of the HLA-E gene successfully induced the expression of the HLA-E molecule on the SEC surface. Furthermore, the transfectant expressed both HLA-G1 and HLA-E molecules, thus efficiently enhancing the inhibition of NK-mediated SEC lysis. CONCLUSION: The co-expression of HLA-G1 and HLA-E molecules with the modified genes has potential for use in preventing xenograft rejection, as mediated by human NK cells.

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.

Selective chemokine and receptor gene expressions in allografts that develop transplant vasculopathy.

BACKGROUND: Chemokine systems probably play a role in transplant vasculopathy; however, a comprehensive study of the expression of chemokines and their receptors in this disease has not been performed. METHODS: The expression of all the rat chemokines and chemokine receptor genes for which the nucleotide sequences are known were quantitatively monitored using the fluorescence-based real-time reverse-transcriptase polymerase chain reaction technique, and selected cytokine-receptor pairs were determined using immunohistochemical staining. The analysis covered the whole time course of transplant vasculopathy in 2 different graft models (cardiac and aortic grafts) with 4 different strain combinations of rats. RESULTS: Among the 13 receptor genes examined, the CXCR3, CCR5, and CCR2 genes and those of their corresponding ligands were selectively and strongly induced in grafts that develop transplant vasculopathy. The expression patterns of the receptors were similar in both cardiac and aortic allografts, although their induction and their absolute levels of expression were amplified several fold in the grafted aorta compared with heart grafts. The genes were induced before morphologic changes became apparent and expression was sustained during the whole period of neointimal formation. Interestingly, immunohistochemical staining for CXCR3 showed a unique pattern of expression: we found weak expression on cells in the outer layer of the neointima and adventitia and found the strongest staining in the innermost layer of the neointima. CONCLUSIONS: This study suggested diagnostic as well as potential functional roles of the chemokine-receptor pairs IP10-CXCR3, RANTES-CCR5, and MCP1-CCR2 in rat models of transplant vasculopathy.

Regulation of complement-mediated swine endothelial cell lysis by herpes simplex virus glycoprotein, gC1.

Herpes simplex virus type 1 (HSV-1) encodes several immuno-regulatory proteins that allow it to escape from the human immune system. The regulatory function of a HSV-1 glycoprotein gC (HSV-gC1) molecule on complement-mediated swine endothelial cell (SEC) lysis was investigated. The HSV-gC1 gene was obtained by the PCR method from the HSV-1 genome. The complement-regulatory function of this molecule was analyzed by cytotoxicity assay, using Chinese hamster ovarian tumor (CHO) cell and SEC transfectants and six human serum samples. FACS and Western blot analysis revealed the expression of the HSV-gC1 molecule on the transfectants. The CHO cell transfectants showed significant resistance to cell lysis by the sera that did not contain the anti-HSV-gC1 antibody. The SEC transfectants, however, showed a marked resistance to cell lysis in all cases. The introduction of a viral immune regulator such as HSV-gC1 into the swine cell provides a new approach for successful xenotransplantation.

The possibility of reducing xenoantigen levels with a novel gal 3'-sulfotransferase (GP3ST).

Glycoprotein-3-sulfotransferase (GP3ST) is a key enzyme in downregulating the expression of Galalpha1,3Galbeta1,4GlcNAc-R (the alpha-Gal epitope), via enzymatic competition with an alpha1,3 galactosyltransferase (alpha1,3GT), such as alpha2,6 sialyltransferase (alpha2,6ST). In this study, we report the dominance of GP3ST over alpha1,3GT using transfected pig endothelial cell (PEC) lines. The introduction of the GP3ST gene into PEC suppresses its antigenicity with respect to normal human pooled serum (NHS), including the alpha-Gal epitope and the Hanganutziu-Deicher (H-D) antigen, and, in addition, reduces the susceptibility to NHS in complement-mediated cell lysis. Western and lectin blot analyses of the products of parental PEC and its transfectants indicated that proteins smaller than 66 kDa have a diminished reactivity with NHS and the IB4 lectin. The levels of the alpha-Gal epitope in neutral glycosphingolipids were also decreased in the GP3ST transfectants as detected in thin layer chromatography by immunostaining. These data indicate that GP3ST is very effective in reducing xenoepitope levels.

Co-effect of HLA-G1 and glycosyltransferases in reducing NK cell-mediated pig endothelial cell lysis.

Natural killer (NK) cells play an important role in xenograft rejection. The aim of this study was to evaluate the co-effect of human leukocyte antigen (HLA)-G1 expression and the remodeling of glycoantigens such as the alpha-Gal epitope, Galalpha1,3Galbeta1,4GlcNAc-R, by the introduction of glycosyltransferase genes related to NK cell-mediated direct cytotoxicity. Human peripheral blood mononuclear cells or an NK-like cell line, YT cells, was used as an effector and pig endothelial cells (PEC) as the target. A PEC transfectant with HLA-G1 was first prepared by the transfection of HLA-G1 and human beta2 microglobulin. Several new transfectants were then established by the transfection of glycosyltransferase to the HLA-G1 transfectant. The effect of HLA-G1 on NK cell-mediated PEC lysis was lower than that by the glycosyltransferases. Therefore, in the case of the co-transfectants except for HLA-G1+alpha2,6sialyltransferase, such as HLA-G1+N-acetylglucosaminyltransferase-III and HLA-G1+alpha1,2fucosyltransferase, the effect of HLA-G1 expression on NK-mediated killing appeared to be accounted for by the transfected glycosyltransferase activities and the reduced alpha-Gal expression on the cell surface. However, these transfectants showed significant reductions in direct NK cell-mediated cytotoxicity, compared with the single HLA-G1 transfectant. The results herein suggest that a combination of HLA-G1 and glycosyltransferases has considerable potential for the downregulation of NK cell-mediated cytolysis.

Only the light chain is sufficient for the serine protease function of the membrane bound form of factor I on a xeno-surface.

The cell membrane-bound forms of whole factor I (fI-PI), the light chain of the serine protease (SP) domain (SP-PI), and the light chain plus the COOH-terminal 45 amino acid (AA) of the heavy chain (SP+45-PI) were constructed. Chinese hamster ovary (CHO) cells, expressing these molecules were established by transfection of cDNA and confirmed by flow cytometry. Amelioration of complement-mediated cell lysis and complement fragment deposition on the cell surface by the transfectant molecules was tested in each CHO cell by means of a lactate dehydrogenase (LDH) assay and flow cytometry, respectively. A highly expressed fI-PI blocked human complement-mediated cell lysis by approximately 84% of the cells. CHO cell transfectants with SP-PI also showed a clear inhibition in cell lysis by human serum, whereas CHO cell transfectants with SP+45-PI showed no inhibition. In addition, fI-PI and SP-PI, but not SP+45-PI, suppressed C5b-9 deposition on CHO cell surface. These data indicate that the last 45 amino acid of the heavy chain, including a disulfide bridge area, did not participate in the serin protease function of factor I. The results suggest that SP-PI has potential for use in clinical xenotransplantation.

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 x 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.

Involvement of position-147 for HLA-E expression.

HLA-E functions as an inhibitory signaling molecule of natural killer (NK) cell-mediated cytolysis. However, the cell surface expression of HLA-E molecules is quite restricted because of the limited repertoire of binding peptide sequences, such as signal peptides of other HLA molecules, especially on xenogeneic cells. In this study, we successfully determined that position-147 is an important amino acid position for cell surface expression by producing point substitutions. For further studies concerning transplantation therapy, the point substitution, Ser147Cys, that resulted in a single atom change, oxygen to sulfur, designated as HLA-Ev(147), led to a much higher expression on the human and pig cell surface and a greater inhibitory function against human NK cells than wild type HLA-E in an in vitro model system of pig to human xenotransplantation. Consequently, HLA-Ev(147) might be a promising alternative gene tool for future transplantation therapy such as xenotransplantation.

Molecular cloning of pigGnT-I and I.2: an application to xenotransplantation.

Xenotransplantation is one of the most attractive solutions for the current worldwide shortage of organs. The knocking out of alpha1,3-galactosyltransferase in pigs resulted in a drastic reduction in xenoantigenicity. However, more recent studies indicate that other xeno-antigens, so-called non-Gal antigens, will also need to be downregulated. In this study, pig N-acetylglucosaminyltransferase I (GnT-I), a key enzyme that initiates the biosynthesis of hybrid- and complex-type N-linked sugar chains, was isolated and the pigGnT-I.2 specific for the O-linked sugar chain was also isolated. Point mutants, pigGnT-I(123) and pigGnT-I(320), were subsequently constructed. While pigGnT-I(123) shows an indistinct dominant negative effect for endogenous GnT-I in pig cells, pigGnT-I(320) had a drastic effect. In addition, in the case of pig cell transfectants with pigGnT-I(320), cell surface carbohydrate structures were significantly altered and its antigenicity to human serum was reduced. Consequently, pigGnT-I(320) appears to be potentially useful in xenotransplantation by remodeling the carbohydrate structures on pig cells.

Effect of tandem forms of DAF(CD55) on complement-mediated xenogeneic cell lysis.

BACKGROUND: It is difficult to produce a transgenic animal with high expression of decay-accelerating factor (CD55: DAF) or other molecules. The purpose of this study was to assess the effect of tandem forms of DAF on a xenogeneic cell membrane against human complement. METHODS: cDNAs of the delta-Short Consensus Repeat (SCR) 1-DAF, the double-DAF, the triple-DAF, and the tetra-DAF with a FLAG-tag were established. Chinese hamster ovary (CHO) cell lines and a pig endothelial cell (PEC) line expressing these molecules were established. The amelioration of complement-mediated lysis by the transfectant molecules on these cells was examined. The CHO cell transfectants were also incubated with normal human serum, and the amount of C3 deposited was determined by FACS analysis. RESULTS: Stable CHO cells and PEC transfectants, in which each molecule was clearly expressed, and Western blots showed that each band corresponded to the expected molecular weight. The extent of amelioration of complement-mediated lysis by these four molecules was then examined. A clear tendency was found, as follows: The higher the tandem number of DAF, the greater was the effect on cytotoxicity. Additional experiments focusing on triple-DAF and tetra-DAF did not indicate any significant difference in complement-mediated lysis. Consistent with the complement-regulatory ability, the inhibitory effect of the deposition of C3 fragments by these molecules was closely related to the degree of amelioration. CONCLUSION: These data indicate that tandem DAF, especially a triple-DAF, is a very effective form for protecting against complement activation.

A study of the xenoantigenicity of neonatal porcine islet-like cell clusters (NPCC) and the efficiency of adenovirus-mediated DAF (CD55) expression.

BACKGROUND: The pig pancreas is considered to be the most suitable source of islets for xenotransplantation in patients with type I diabetes. The objective of this study was to assess the antigenicity of neonatal porcine islet-like cell clusters (NPCC), including the Galalpha1-3Galbeta1-4GlcNAc-R (alpha-Gal) and Hanganutziu-Deicher (H-D) antigens, and the pathway involved in human complement activation. The efficiency of expression of human decay-accelerating factor (DAF: CD55) on NPCC by adenoviral transduction was also examined, and the functional capacity of DAF was also estimated. METHODS: The deposition of human natural antibodies, immunoglobulin (Ig)G and IgM, and the expression of alpha-Gal and H-D antigens on NPCC were investigated by FACS analysis. The downregulation in the antigenicity to human natural antibodies, including the alpha-Gal and H-D antigens on NPCC by treatment with tunicamycin, PDMP and neuraminidase were also examined. In addition, complement-mediated islet lysis was examined using factor D-deficient and C1-deficient sera. An adenovirus encoding DAF under the control of the cytomegalovirus promoter, Ad.pCMV-DAF, was then constructed, and used for transducing NPCC. The amelioration of complement-dependent cytotoxicity of the NPCC by the transduced DAF was assessed as an in vitro hyperacute rejection model of a pig to human xenograft. RESULTS: The NPCC clearly expressed the alpha-Gal epitope, and the human natural antibodies, IgG and IgM, and the anti-H-D antibody also reacted with the NPCC. Treatment of NPCC with tunicamycin led to a drastic reduction in the extent of deposition of IgG, indicating the importance of N-linked sugars on the islets, presumably related to alpha-Gal expression on N-linked sugars. Neuraminidase treatment indicated the presence of, not only the H-D antigen, but also other sialic acid antigens which reacted with the human natural antibody, especially IgG. The complement deposition of factor B on NPCC was clear, and the alternative pathway-mediated NPCC killing accounted for approximately 30% of that by the total complement pathway. On the other hand, approximately 90% of the NPCC could be transduced to express DAF by the adenovector, Ad.pCMV-DAF. The expressed DAF showed an approximately 50-62% suppression in complement-dependent NPCC lysis. CONCLUSION: The origin of the antigenicity of NPCC is mainly N-linked sugars including alpha-Gal and sialic acid antigens, and NPCC expressed the transduced molecule in high efficiency by the adenovector.

A novel strategy for preventing PERV transmission to human cells by remodeling the viral envelope glycoprotein.

BACKGROUND: Porcine endogenous retrovirus (PERV) released from pig cells is a main problem associated with clinical xenotransplantation. In a previous study, we demonstrated that the high mannose type of N-glycan of the envelope glycoprotein is closely related to PERV infectivity with respect to human cells. In this study, we addressed the effects of reducing the high mannose type of N-glycan on PERV infectivity. METHODS: Pig endothelial cells (PEC) were transduced with the LacZ gene by a pseudotype infection to produce PEC(Z). The PEC(Z)s were then further infected with PERV subtype B (PERV-B) to produce PEC(Z)/PB. The PEC(Z)/PBs were next transfected with the alpha 1,2 mannosidase Ib (Man Ib), N-acetylglucosaminyltransferase I (GnT-I) or alpha-mannosidase II (Man II) gene in order to reduce the levels of high mannose type of N-glycan. HEK293 cells were inoculated with the PERV in each of the culture supernatants. The inoculated cells were histochemically stained and the LacZ-positive cells were counted. RESULTS: In experiment I, PERV transmission from the PEC(Z)/PB with GnT-I or Man II to HEK 293 cells was significantly reduced in comparison with control PEC(Z)/PB, while the PEC(Z)/PB with Man Ib was not. However, in experiment II, PERV transmission from the PEC(Z)/PB with ManIb to HEK 293 cells was also significantly reduced in comparison with control PEC(Z)/PB. CONCLUSION: The transfection of these genes to pig cells is effective in reducing the susceptibility of human cells to PERV infection. The results suggest that this represents a potentially useful strategy for further decreasing the likelihood of PERV infections.

Microarray-based gene expression profiling of retransplanted rat cardiac allografts that develop cardiac allograft vasculopathy.

We studied the expression of 9,906 genes in retransplanted rat cardiac allografts that developed cardiac allograft vasculopathy (CAV) with the use of DNA microarray and real-time reverse transcriptase-polymerase chain reaction. Although only a slight difference in the timing of the retransplantation induced the later development of CAV, 1,067 genes were differentially expressed in the allografts 1 day after retransplantation. Thus, the development of CAV was determined by a robust difference in gene expression soon after retransplantation, controlled by a slight difference in retransplantation timing. In contrast, only 26 genes showed significant upregulation in the later phase of CAV development, and the time-course of the induction of 16 genes was associated with CAV progression. Of these genes, 8 were induced in 2 different aortic allograft combinations, and the time-course of the induction was correlated with the development of transplant vasculopathy. Microarray-based gene expression profiling has the potential to elucidate the mechanism of experimental chronic cardiac allograft rejection.