© 2013 John Wiley & Sons A/S
Xenotransplantation 2013: 20: 308–310 Printed in Singapore. All rights reserved doi: 10.1111/xen.12064
XENOTRANSPLANTATION
Literature Update
Xenotransplantation literature update, July–August 2013 Schneider MKJ, Seebach JD. Xenotransplantation literature update, July–August 2013. Xenotransplantation 2013: 20: 308–310. © 2013 John Wiley & Sons A/S.
M arten K.J. Schneider1 and J€org D. Seebach2 1
Division of Internal Medicine, Laboratory of Vascular Immunology, University Hospital Zurich, Zurich, Switzerland, 2Service of Immunology and Allergology, Department of Internal Medicine, University Hospital and Medical Faculty, Geneva, Switzerland Key words: 3-galactose – galactose-a1 – islet cells – pig – xenotransplantation – xenozoonosis Abbreviations: BHV, bioprosthetic heart valves; CMAH, Cytidine monophospho-N-acetylneuraminic acid hydroxylase; F6H8, perfluorohexyloctane; Gal, Gala1,3Gal; GT-KO, a1,3-galactosyltransferase gene-knockout; hRBC, human red blood cells; LTR, long terminal repeat; PERV, porcine endogenous retroviruses; PFD, perfluorodecalin; SPION, superparamagnetic iron oxide nanoparticles; ZFN, zinc finger nuclease Address reprint requests to M arten K.J. Schneider, Division of Internal Medicine, Laboratory of Vascular Immunology, University Hospital Zurich, Raemistrasse 100, LAB C 7, CH-8091 Zurich, Switzerland (E-mail: [email protected]) Received 28 August 2013; Accepted 28 August 2013
Reviews
During the period July–August 2013, two reviews on xenotransplantation were published. Cooper et al. [1] reviewed the potential of genetically engineered pigs in providing an alternative source of organs and cells for transplantation. The authors summarized the hurdles to successful xenotransplantation and the genetically modified pigs currently available for xenotransplantation research. Although considerable progress has been made in this respect, further genetic manipulations of source pigs are necessary, with most of the genes that are likely to be beneficial already identified. Jang et al. [2] reviewed the immunologically significant non-human carbohydrate antigens, including Gala1,3Gal (Gal) antigens, which are responsible for hyperacute immunologic rejection in pig-to-human xenotransplantation. They further discussed the importance of studying human, pig, and a1,3-galactosyltransferase gene-knockout 308
(GT-KO) pig glycoprofiles and of developing adequate pig-to-human glycan databases. Islet and cellular transplantation
Transportation of pig pancreases from distant breeding centers to experienced islet production facilities is associated with impaired islet quality and outcome. Brandhorst et al. [3] compared the protective effect of perfluorohexyloctane (F6H8) with that of perfluorodecalin (PFD) on long-term stored pig pancreases, using the split lobe model to minimize donor variability. Pancreases were dissected into the connecting and splenic lobe, intraductally flushed with University of Wisconsin solution and immersed for 8 to 10 h in either pre-oxygenated F6H8 or PFD. Pancreatic lobes stored in pre-oxygenated F6H8 had a significantly higher intrapancreatic pO2 value compared with pancreata in oxygen pre-charged PFD, which correlated with a higher ATP-to-inorganic
Xenotransplantation literature update phosphate ratio. Compared with PFD, treatment with F6H8 improved the glucose-stimulated insulin response, the viability, and the post-culture survival, but did not influence islet yield or purity. In summary, this study suggests that F6H8 improves the quality of pig islets isolated after prolonged cold ischemia. Mettler et al. [4] evaluated a new method to reduce the transplantation volume of microencapsulated islets by magnetic separation of encapsulated islets from empty capsules. Rat islets were labeled with three different superparamagnetic iron oxide nanoparticles (SPION): dextran-coated SPION, siloxane-coated SPION, and heparin-coated SPION. Labeling with dextran-coated SPION reduced the graft volume by 70%, with a 33% loss of islet-containing capsules. Heparin-coated SPION labeling led to a 46% reduction in graft volume and a 4.5% loss of islet-containing capsules. No purification was achieved with siloxane-coated SPION due to its toxicity to the primary islets. Furthermore, injection of heparin-coated SPIONlabeled encapsulated rat islets into the hamstrings of mice allowed for in vivo imaging after transplantation. In conclusion, magnetic purification of encapsulated islets can reduce the graft volume without significantly impairing their function. Human hepatocyte chimeric mice are useful for research on drug metabolism and toxicity and on intrahepatic pathogens. However, the small body size of these mice restricts the availability of biologic samples for biochemical analyses and makes surgical manipulation difficult. To achieve larger chimeric animals for such studies, Tachibana et al. [5] generated human hepatocyte chimeric rats. Rats at 2 weeks of age were treated with hepatotoxin retrorsine to inhibit hepatocyte proliferation and transplanted 3 days later via the portal vein with syngeneic rat or human hepatocytes under immunosuppression. Rat hepatocytes engrafted and repopulated the liver at ratios of 16 and 48% at 3 and 6 weeks after transplantation, respectively. Human hepatocytes engrafted the rat livers at a repopulation ratio of 2.5% at 3 weeks post-transplantation, comparable with the ratio seen in the humanized chimeric mouse models. In conclusion, xenogeneic human hepatocytes were able to engraft and grow in rat livers for at least 3 weeks post-transplantation. Immunobiology
Kumar et al. [6] investigated whether anti-pig antibody levels correlated with an individual’s geographic location during childhood, age, gender, diet, and history of vaccination. Blood samples of 75 human volunteers of all ABO blood groups
who had lived at least the initial 18 yr of their lives in a specific region of the world were analyzed. Antibody binding to GT-KO pig cells was less than to wild-type cells. With age, a reduction in anti-pig IgM and anti-Gal IgM, but a slight increase in anti-non-Gal IgG, was recorded. Women had higher levels of anti-Gal IgM than men, and blood group A subjects had higher levels of anti-pig IgM and IgG than those of group AB. Typhoid or measles-mumps-rubella vaccination was associated with lower anti-non-Gal IgG or anti-Gal IgG, respectively, whereas influenza vaccination was associated with higher anti-non-Gal IgG. Diet had no influence on antibody levels. Finally, the mean anti-non-Gal and anti-Gal IgG levels in subjects from Middle Eastern countries were significantly higher than in subjects from all other geographic regions, except for Japan and South-East Asia, respectively. The authors conclude that clinical trials of xenotransplantation may be influenced by various factors, including the recipient’s geographic location during childhood, possibly associated with exposure to different microorganisms. Extracorporeal perfusion of porcine livers represents a potential therapeutic approach for patients in fulminant hepatic failure. However, when perfused with human blood, porcine livers consume significant amounts of human red blood cells (hRBC) and platelets. Porcine Kupffer cells are involved in the phagocytosis of hRBC and are able to bind to N-acetylneuraminic acid on hRBC via the lectin sialoadhesin. Waldman et al. [7] examined whether blocking of porcine sialoadhesin with specific monoclonal antibodies (mAb) prevents the recognition and destruction of hRBC. Wild-type pig livers were perfused ex vivo with isolated hRBC for 72 h in the presence of antiporcine sialoadhesin or isotype control mAb. Compared to treatment with isotype control mAb, the addition of anti-porcine sialoadhesin mAb reduced the loss of hRBC over a 72-h period and prolonged pig liver metabolic function throughout the perfusion. This study shows the involvement of sialoadhesin in mediating the destruction of hRBC in extracorporeal porcine liver xenoperfusion. Residual Gal epitopes present after glutaraldehyde fixation of clinical bioprosthetic heart valves (BHV) induce an increase of circulating human anti-Gal antibodies early after BHV implantation. However, a quantitative determination of the xenogeneic Gal epitopes in commercially available BHV has never been performed. Therefore, Naso et al. [8] determined the number of Gal epitopes in seven different models of BHV (five of bovine and two of porcine origin). The porcine EpicTM (St. Jude Medical, St. Paul, MN, USA) valve was the 309
Schneider and Seebach only one where the Gal antigen was completely shielded. In the composite TrifectaTM (St. Jude Medical), valve cusps of bovine pericardial tissue were negative for Gal, while the stent cover strip of porcine pericardium still maintained 30% of active Gal antigens originally present in native tissue. All other tested BHV expressed significant amounts of residual Gal antigens. The authors suggest that the quantitative analysis of Gal epitopes in BHV might become a mandatory quality control tool able to correct, modify, and redirect further research toward the production of Gal-free BHV. Donor animals
The Hanganutziu-Deicher antigen, a glycan containing N-glycolylneuraminic acid at its terminal, is a major non-Gal antigen that is implicated in xenograft rejection. To generate pigs devoid of this non-Gal antigen, Kwon et al. [9] produced zinc finger nuclease-mediated monoallelic/biallelic male and female cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs. CMAH KO pigs had decreased transcript levels of the Hanganutziu-Deicher antigen. There was no presence of N-glycolylneuraminic acid in fibroblasts derived from biallelic CMAH KO pigs. Both monoallelic and biallelic CMAH KO pigs were healthy and showed no signs of abnormality and off-target mutations. The authors conclude that CMAH KO pigs generated on the GT-KO background could serve as an important model for xenotransplantation research. Xenozoonosis
Jung et al. [10] analyzed the promoter activity and methylation status of long terminal repeat (LTR) elements of porcine endogenous retrovirus (PERV) in NIH miniature pigs. LTR in PERV elements showed promoter activity that could affect neighboring functional genes. The methylation status and promoter activities of three LTR structures (PERV-LTR1, LTR2, and LTR3) belonging to the PERV-A family were examined. The PERV LTR3 element exhibited hypomethylation and stronger promoter activity than the other LTR elements in human liver cells. Several transcription factors such as Nkx2-2 and Elk-1 positively influenced the high transcriptional activity of the PERV LTR3 element. Ethics
Experts on xenotransplantation, ethics, and the ophthalmologic society in Korea established the consensus about the conditions for undertaking 310
clinical trials of xenocorneal transplantation in Korea (Kim et al. [11]). The authors reviewed the key ethical requirements and progress of a Korean regulatory framework for such clinical trials. Further, recommendations were provided, which are essentially based on the International Xenotransplantation Association (IXA) islet xenotransplantation consensus statement. These include donor pig source, quality control, required preclinical efficacy, prevention of PERV transmission, patient selection, and informed consent in xenocorneal transplantation. This consensus statement will build the basis for the Korean Food and Drug Administration to discuss final regulatory guidelines for clinical trials in Korea and for the International Xenotransplantation Association to develop international consensus standards on xenocorneal transplantation. References 1. COOPER DK, HARA H, EZZELARAB M et al. The potential of genetically-engineered pigs in providing an alternative source of organs and cells for transplantation. J Biomed Res 2013; 27: 249–253. 2. JANG KS, KIM YG, ADHYA M, PARK HM, KIM BG. The sweets standing at the borderline between allo- and xenotransplantation. Xenotransplantation 2013; 20: 199–208. 3. BRANDHORST H, IKEN M, SCOTT WE III et al. Quality of isolated pig islets is improved using perfluorohexyloctane for pancreas storage in a split lobe model. Cell Transplant 2013; 22: 1477–1483. 4. METTLER E, TRENKLER A, FEILEN PJ et al. Magnetic separation of encapsulated islet cells labeled with superparamagnetic iron oxide nano particles. Xenotransplantation 2013; 20: 219–226. 5. TACHIBANA A, TATENO C, YOSHIZATO K. Repopulation of the immunosuppressed retrorsine-treated infant rat liver with human hepatocytes. Xenotransplantation 2013; 20: 227–238. 6. KUMAR G, SATYANANDA V, FANG J et al. Is there a correlation between anti-pig antibody levels in humans and geographic location during childhood? Transplantation 2013; 96: 387–393. 7. WALDMAN JP, VOGEL T, BURLAK C et al. Blocking porcine sialoadhesin improves extracorporeal porcine liver xenoperfusion with human blood. Xenotransplantation 2013; 20: 239–251. 8. NASO F, GANDAGLIA A, BOTTIO T et al. First quantification of alpha-Gal epitope in current glutaraldehyde-fixed heart valve bioprostheses. Xenotransplantation 2013; 20: 252–261. 9. KWON DN, LEE K, KANG MJ et al. Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs. Sci Rep 2013; 3: 1981. 10. JUNG YD, LEE JR, KIM YJ et al. Promoter activity analysis and methylation characterization of LTR elements of PERVs in NIH miniature pig. Genes Genet Syst 2013; 88: 135–142. 11. KIM MK, LEE JJ, CHOI HJ et al. Ethical and regulatory guidelines in clinical trials of xenocorneal transplantation in Korea; the Korean xenocorneal transplantation consensus statement. Xenotransplantation 2013; 20: 209–218.
Xenotransplantation 2013: 20: 308–310 Printed in Singapore. All rights reserved doi: 10.1111/xen.12064
XENOTRANSPLANTATION
Literature Update
Xenotransplantation literature update, July–August 2013 Schneider MKJ, Seebach JD. Xenotransplantation literature update, July–August 2013. Xenotransplantation 2013: 20: 308–310. © 2013 John Wiley & Sons A/S.
M arten K.J. Schneider1 and J€org D. Seebach2 1
Division of Internal Medicine, Laboratory of Vascular Immunology, University Hospital Zurich, Zurich, Switzerland, 2Service of Immunology and Allergology, Department of Internal Medicine, University Hospital and Medical Faculty, Geneva, Switzerland Key words: 3-galactose – galactose-a1 – islet cells – pig – xenotransplantation – xenozoonosis Abbreviations: BHV, bioprosthetic heart valves; CMAH, Cytidine monophospho-N-acetylneuraminic acid hydroxylase; F6H8, perfluorohexyloctane; Gal, Gala1,3Gal; GT-KO, a1,3-galactosyltransferase gene-knockout; hRBC, human red blood cells; LTR, long terminal repeat; PERV, porcine endogenous retroviruses; PFD, perfluorodecalin; SPION, superparamagnetic iron oxide nanoparticles; ZFN, zinc finger nuclease Address reprint requests to M arten K.J. Schneider, Division of Internal Medicine, Laboratory of Vascular Immunology, University Hospital Zurich, Raemistrasse 100, LAB C 7, CH-8091 Zurich, Switzerland (E-mail: [email protected]) Received 28 August 2013; Accepted 28 August 2013
Reviews
During the period July–August 2013, two reviews on xenotransplantation were published. Cooper et al. [1] reviewed the potential of genetically engineered pigs in providing an alternative source of organs and cells for transplantation. The authors summarized the hurdles to successful xenotransplantation and the genetically modified pigs currently available for xenotransplantation research. Although considerable progress has been made in this respect, further genetic manipulations of source pigs are necessary, with most of the genes that are likely to be beneficial already identified. Jang et al. [2] reviewed the immunologically significant non-human carbohydrate antigens, including Gala1,3Gal (Gal) antigens, which are responsible for hyperacute immunologic rejection in pig-to-human xenotransplantation. They further discussed the importance of studying human, pig, and a1,3-galactosyltransferase gene-knockout 308
(GT-KO) pig glycoprofiles and of developing adequate pig-to-human glycan databases. Islet and cellular transplantation
Transportation of pig pancreases from distant breeding centers to experienced islet production facilities is associated with impaired islet quality and outcome. Brandhorst et al. [3] compared the protective effect of perfluorohexyloctane (F6H8) with that of perfluorodecalin (PFD) on long-term stored pig pancreases, using the split lobe model to minimize donor variability. Pancreases were dissected into the connecting and splenic lobe, intraductally flushed with University of Wisconsin solution and immersed for 8 to 10 h in either pre-oxygenated F6H8 or PFD. Pancreatic lobes stored in pre-oxygenated F6H8 had a significantly higher intrapancreatic pO2 value compared with pancreata in oxygen pre-charged PFD, which correlated with a higher ATP-to-inorganic
Xenotransplantation literature update phosphate ratio. Compared with PFD, treatment with F6H8 improved the glucose-stimulated insulin response, the viability, and the post-culture survival, but did not influence islet yield or purity. In summary, this study suggests that F6H8 improves the quality of pig islets isolated after prolonged cold ischemia. Mettler et al. [4] evaluated a new method to reduce the transplantation volume of microencapsulated islets by magnetic separation of encapsulated islets from empty capsules. Rat islets were labeled with three different superparamagnetic iron oxide nanoparticles (SPION): dextran-coated SPION, siloxane-coated SPION, and heparin-coated SPION. Labeling with dextran-coated SPION reduced the graft volume by 70%, with a 33% loss of islet-containing capsules. Heparin-coated SPION labeling led to a 46% reduction in graft volume and a 4.5% loss of islet-containing capsules. No purification was achieved with siloxane-coated SPION due to its toxicity to the primary islets. Furthermore, injection of heparin-coated SPIONlabeled encapsulated rat islets into the hamstrings of mice allowed for in vivo imaging after transplantation. In conclusion, magnetic purification of encapsulated islets can reduce the graft volume without significantly impairing their function. Human hepatocyte chimeric mice are useful for research on drug metabolism and toxicity and on intrahepatic pathogens. However, the small body size of these mice restricts the availability of biologic samples for biochemical analyses and makes surgical manipulation difficult. To achieve larger chimeric animals for such studies, Tachibana et al. [5] generated human hepatocyte chimeric rats. Rats at 2 weeks of age were treated with hepatotoxin retrorsine to inhibit hepatocyte proliferation and transplanted 3 days later via the portal vein with syngeneic rat or human hepatocytes under immunosuppression. Rat hepatocytes engrafted and repopulated the liver at ratios of 16 and 48% at 3 and 6 weeks after transplantation, respectively. Human hepatocytes engrafted the rat livers at a repopulation ratio of 2.5% at 3 weeks post-transplantation, comparable with the ratio seen in the humanized chimeric mouse models. In conclusion, xenogeneic human hepatocytes were able to engraft and grow in rat livers for at least 3 weeks post-transplantation. Immunobiology
Kumar et al. [6] investigated whether anti-pig antibody levels correlated with an individual’s geographic location during childhood, age, gender, diet, and history of vaccination. Blood samples of 75 human volunteers of all ABO blood groups
who had lived at least the initial 18 yr of their lives in a specific region of the world were analyzed. Antibody binding to GT-KO pig cells was less than to wild-type cells. With age, a reduction in anti-pig IgM and anti-Gal IgM, but a slight increase in anti-non-Gal IgG, was recorded. Women had higher levels of anti-Gal IgM than men, and blood group A subjects had higher levels of anti-pig IgM and IgG than those of group AB. Typhoid or measles-mumps-rubella vaccination was associated with lower anti-non-Gal IgG or anti-Gal IgG, respectively, whereas influenza vaccination was associated with higher anti-non-Gal IgG. Diet had no influence on antibody levels. Finally, the mean anti-non-Gal and anti-Gal IgG levels in subjects from Middle Eastern countries were significantly higher than in subjects from all other geographic regions, except for Japan and South-East Asia, respectively. The authors conclude that clinical trials of xenotransplantation may be influenced by various factors, including the recipient’s geographic location during childhood, possibly associated with exposure to different microorganisms. Extracorporeal perfusion of porcine livers represents a potential therapeutic approach for patients in fulminant hepatic failure. However, when perfused with human blood, porcine livers consume significant amounts of human red blood cells (hRBC) and platelets. Porcine Kupffer cells are involved in the phagocytosis of hRBC and are able to bind to N-acetylneuraminic acid on hRBC via the lectin sialoadhesin. Waldman et al. [7] examined whether blocking of porcine sialoadhesin with specific monoclonal antibodies (mAb) prevents the recognition and destruction of hRBC. Wild-type pig livers were perfused ex vivo with isolated hRBC for 72 h in the presence of antiporcine sialoadhesin or isotype control mAb. Compared to treatment with isotype control mAb, the addition of anti-porcine sialoadhesin mAb reduced the loss of hRBC over a 72-h period and prolonged pig liver metabolic function throughout the perfusion. This study shows the involvement of sialoadhesin in mediating the destruction of hRBC in extracorporeal porcine liver xenoperfusion. Residual Gal epitopes present after glutaraldehyde fixation of clinical bioprosthetic heart valves (BHV) induce an increase of circulating human anti-Gal antibodies early after BHV implantation. However, a quantitative determination of the xenogeneic Gal epitopes in commercially available BHV has never been performed. Therefore, Naso et al. [8] determined the number of Gal epitopes in seven different models of BHV (five of bovine and two of porcine origin). The porcine EpicTM (St. Jude Medical, St. Paul, MN, USA) valve was the 309
Schneider and Seebach only one where the Gal antigen was completely shielded. In the composite TrifectaTM (St. Jude Medical), valve cusps of bovine pericardial tissue were negative for Gal, while the stent cover strip of porcine pericardium still maintained 30% of active Gal antigens originally present in native tissue. All other tested BHV expressed significant amounts of residual Gal antigens. The authors suggest that the quantitative analysis of Gal epitopes in BHV might become a mandatory quality control tool able to correct, modify, and redirect further research toward the production of Gal-free BHV. Donor animals
The Hanganutziu-Deicher antigen, a glycan containing N-glycolylneuraminic acid at its terminal, is a major non-Gal antigen that is implicated in xenograft rejection. To generate pigs devoid of this non-Gal antigen, Kwon et al. [9] produced zinc finger nuclease-mediated monoallelic/biallelic male and female cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs. CMAH KO pigs had decreased transcript levels of the Hanganutziu-Deicher antigen. There was no presence of N-glycolylneuraminic acid in fibroblasts derived from biallelic CMAH KO pigs. Both monoallelic and biallelic CMAH KO pigs were healthy and showed no signs of abnormality and off-target mutations. The authors conclude that CMAH KO pigs generated on the GT-KO background could serve as an important model for xenotransplantation research. Xenozoonosis
Jung et al. [10] analyzed the promoter activity and methylation status of long terminal repeat (LTR) elements of porcine endogenous retrovirus (PERV) in NIH miniature pigs. LTR in PERV elements showed promoter activity that could affect neighboring functional genes. The methylation status and promoter activities of three LTR structures (PERV-LTR1, LTR2, and LTR3) belonging to the PERV-A family were examined. The PERV LTR3 element exhibited hypomethylation and stronger promoter activity than the other LTR elements in human liver cells. Several transcription factors such as Nkx2-2 and Elk-1 positively influenced the high transcriptional activity of the PERV LTR3 element. Ethics
Experts on xenotransplantation, ethics, and the ophthalmologic society in Korea established the consensus about the conditions for undertaking 310
clinical trials of xenocorneal transplantation in Korea (Kim et al. [11]). The authors reviewed the key ethical requirements and progress of a Korean regulatory framework for such clinical trials. Further, recommendations were provided, which are essentially based on the International Xenotransplantation Association (IXA) islet xenotransplantation consensus statement. These include donor pig source, quality control, required preclinical efficacy, prevention of PERV transmission, patient selection, and informed consent in xenocorneal transplantation. This consensus statement will build the basis for the Korean Food and Drug Administration to discuss final regulatory guidelines for clinical trials in Korea and for the International Xenotransplantation Association to develop international consensus standards on xenocorneal transplantation. References 1. COOPER DK, HARA H, EZZELARAB M et al. The potential of genetically-engineered pigs in providing an alternative source of organs and cells for transplantation. J Biomed Res 2013; 27: 249–253. 2. JANG KS, KIM YG, ADHYA M, PARK HM, KIM BG. The sweets standing at the borderline between allo- and xenotransplantation. Xenotransplantation 2013; 20: 199–208. 3. BRANDHORST H, IKEN M, SCOTT WE III et al. Quality of isolated pig islets is improved using perfluorohexyloctane for pancreas storage in a split lobe model. Cell Transplant 2013; 22: 1477–1483. 4. METTLER E, TRENKLER A, FEILEN PJ et al. Magnetic separation of encapsulated islet cells labeled with superparamagnetic iron oxide nano particles. Xenotransplantation 2013; 20: 219–226. 5. TACHIBANA A, TATENO C, YOSHIZATO K. Repopulation of the immunosuppressed retrorsine-treated infant rat liver with human hepatocytes. Xenotransplantation 2013; 20: 227–238. 6. KUMAR G, SATYANANDA V, FANG J et al. Is there a correlation between anti-pig antibody levels in humans and geographic location during childhood? Transplantation 2013; 96: 387–393. 7. WALDMAN JP, VOGEL T, BURLAK C et al. Blocking porcine sialoadhesin improves extracorporeal porcine liver xenoperfusion with human blood. Xenotransplantation 2013; 20: 239–251. 8. NASO F, GANDAGLIA A, BOTTIO T et al. First quantification of alpha-Gal epitope in current glutaraldehyde-fixed heart valve bioprostheses. Xenotransplantation 2013; 20: 252–261. 9. KWON DN, LEE K, KANG MJ et al. Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs. Sci Rep 2013; 3: 1981. 10. JUNG YD, LEE JR, KIM YJ et al. Promoter activity analysis and methylation characterization of LTR elements of PERVs in NIH miniature pig. Genes Genet Syst 2013; 88: 135–142. 11. KIM MK, LEE JJ, CHOI HJ et al. Ethical and regulatory guidelines in clinical trials of xenocorneal transplantation in Korea; the Korean xenocorneal transplantation consensus statement. Xenotransplantation 2013; 20: 209–218.
