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Genetic manipulation in pigsDavid H. and Cesare Galli aMassachusetts General Hospital, Transplantation Biology Research Center, Boston, Massachusetts, USA Recent developments in the field of genetic engineering have made it possible to add, and bLaboratorio di Tecnologie della Riproduzione,Avantea srl, Cremona and Clinical Veterinary delete or exchange genes from one species to another. This technology has special Department, University of Bologna, Bologna, Italy relevance to the field of xenotransplantation, in which the elimination of a species- specific disparity could make the difference between success and failure of an organ transplant. This review focuses on developments in both the techniques and Transplantation Biology Research Center, 13th Street,Building 149-9019, Boston, MA 02129, USA applications of genetically modified animals.
Tel: +1 617 726 4065; fax: +1 617 726 4067; Advances have been made using existing techniques for genetic modifications of swine Current Opinion in Organ Transplantation 2009, and in the development of new, emerging technologies, including enzymatic engineering and the use of small interfering RNA. Applications of the modified animalshave provided evidence that genetically modified swine have the potential to overcomeboth physiologic and immunologic barriers that have previously impeded this field. Theuse of alpha-1,3-galactosyltransferase gene-knockout animals as donors have shownmarked improvements in xenograft survivals.
SummaryTechniques for genetic engineering of swine have been directed toward avoidingnaturally existing cellular and antibody responses to species-specific antigens. Organsfrom genetically engineered animals have enjoyed markedly improved survivals innonhuman primates, especially in protocols directed toward the induction of tolerance,presumably by avoiding immunization to new antigens.
Keywordsgenetic engineering, knockout, transplantation, xenograft, xenotransplantation Curr Opin Organ Transplant 14:148–153ß 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins Techniques for genetic modifications of swine One of the major advantages of the pig as a potential Techniques for genetic modification are crucial for xeno- xenograft donor is the ability of the genome of these transplantation research. Major advances have been animals to be modified by modern techniques of genetic made since the first transgenic large animals, including engineering. The breeding characteristics of these transgenic pigs, were obtained through pronuclear micro- animals, in particular their relatively short gestation time injection mainly by the use of assisted reproductive and large litter size, combined with the availability of technologies These advances made possible the first techniques for oocyte manipulation and artificial inse- inactivation (knockout) of a gene in a large animal model mination, have made it possible to carry out both by combining genetic engineering on cultured somatic addition of genes (i.e. transgenic animals) and elimin- cells with somatic cell nuclear transfer (SCNT) ation of gene expression (i.e. knockout animals) forselected genes involved in xenograft rejection. Some of the first transgenic animals were produced for this Pronuclear microinjection is now seldom used, having purpose just over a decade ago whereas knockout been largely replaced by more efficient and less expens- animals for alpha-1,3-galactosyltransferase (Gal) ive techniques. Klose et al. used microinjection to have become available over the past 5 years. We intend to insert the tumor necrosis factor-alpha-related apoptosis- review here both the techniques available for producing inducing ligand into pigs; ubiquitous expression levels genetic modifications of swine and some of the most were detected in three lines without side effects. Martin recent data using such modified animals in the field of et al. developed transgenic pigs that express the human T cell inhibitory molecule, human cytotoxic T 1087-2418 ß 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Genetic manipulation in pigs Sachs and Galli lymphocyte antigen 4-immunoglobulin, under the con- genesis and silencing of lentiviral sequences by trol of the neuron-specific enolase promoter. Expression methylation and high frequency of mosaicism in founder was found in various areas of the brain in these transgenic pigs, including the mesencephalon, hippocampus andcortex. Kues et al. also generated transgenic Another class of viruses used was the adeno-associated pigs by microinjecting an autoregulatory, tetracycline- virus (AAV). Recently, Rogers et al. produced a responsive, bicistronic expression cassette (NTA). The cystic fibrosis transmembrane conductance regulator expression pattern correlated inversely with the methyl- (CFTR)-null pig using AAV-mediated gene targeting ation status of the NTA transcription start sites, indicat- and SCNT. These authors generated a pig with both ing epigenetic silencing of one of the NTA cassettes.
null (knockout) and DF508 (knock-in) modifications.
Gene targeting using the AAV approach has resulted in a very efficient strategy for obtaining knockout of the Recently, a technique to produce multitransgenic pigs CFTR gene that is not expressed in fibroblasts.
has been optimized, incubating genes for three fluor-escent proteins with spermatozoa, which were sub- sequently used for insemination. Seven of the 18 piglets SCNT has become the leading tool for generating born were reported to be transgenic by PCR analysis animals from genetically engineered somatic cells.
The benefits of this technique, compared with pronuclear SCNT works better in pigs than in other large animals microinjection, are efficiency, low cost and ease of use.
A recent innovation to make the technique Nevertheless, the insertion is random and the transgene more user-friendly is the zona-free system. Zona removal can be rearranged, thus affecting the expression levels.
may be performed after enucleation for zona-free fusion, The expression of the transgene in the long term remains activation and culture or before enucleation controversial Sperm-mediated gene transfer for handmade cloning Major limitations are (SMGT) has also been used with a nonviral episomal represented by the lack of embryonic stem cell technol- vector that can offer several advantages, including ogies. Somatic cells are being used, but they have a the prevention of insertional mutagenesis and position limited lifespan, thus restricting the time the cells can effects on expression. Twelve of 18 fetuses that were be cultured in vitro for genetic manipulation. For xeno- recovered retained the episomal reporter gene, and nine transplantation purposes, the use of miniature pigs is expressed it. Intracytoplasmic sperm injection-mediated more attractive for a number of reasons. However, these gene transfer, a variant of SMGT, in combination with breeds do not carry the same reproductive efficiency as SCNT produced transgenic pigs by coincubating commercial breeds, and they are not as readily available.
sperm with the bicistronic vector, human albumin green SCNT efficiency in miniature pigs, using miniature sow fluorescent protein (GFP), and microinjecting the sper- recipients, is very low. When miniature pig embryos are matozoa directly into the ooplasm. After the transfer transferred to commercial sows, the outcome is improved of 702 embryos into five gilts, two out of 35 fetuses but not to the level of the commercial breeds recovered were transgenic. Using somatic cells from Using a combination of SCNT with heterozygous cells the transgenic pigs for nuclear transfer, 767 embryos were for alpha-Gal and crossbreeding of the resulting animals, retransferred to five recipients, resulting in six live trans- a commercial line of pig homozygous for the knockout genic piglets. This is not more efficient than the two of the Gal epitope has been generated with a low level techniques used separately; however, it can be suitable of inbreeding that is beneficial for reproductive when a large construct (ranging from À100 to more than À1000 kb, i.e. YAC, BAC, microchromosome) has to betransfected Several new technologies are becoming available that Lentiviral gene transfer is extremely efficient, with 80– may be of great benefit in the future to the construction of 100% of the animals being born transgenic after oocyte or embryo infection or somatic cell culture infection.
Lentiviruses have been used in a variety of experiments to transduce cells with various transgenes. These exper- Transposons, also called ‘jumping genes’, are class II iments include small interfering RNA (siRNA) knock- mobile genetic elements; they are small segments of down for stem cells and somatic cells, and nuclear transfer DNA able to move from one DNA to another using (see below) for generating desired modifications. Lenti- transposition mediated by enzymes (transposases).
viruses are unlikely to be used in clinical applications DNA ‘cut and paste’ transposons have been used for because of the risks associated with multiple integration.
precise and efficient delivery of DNA expression cas- Such risks include oncogene activation, insertional muta- settes in vertebrate cells. In a recent study it was Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
shown that cotransfection of PEGE cells with Sleeping (PERV); for example ‘knockdown’ of PERV expression Beauty, Passport Tol2 and piggyBac, with their corre- has been shown in transgenic pigs expressing siRNA sponding transposase expression constructs, resulted corresponding to the viral pol2 sequence On the respectively in 13.5, five, 21 and 28-fold increases over contrary, recently, an alternative strategy to reduce PERV transfection without transposase. In addition to increas- transmission was proposed using APOBEC3G, which ing the efficiency of integration, transposase-mediated is a single-stranded DNA cytosine deaminase that can transgenesis precisely integrates a single copy of the inhibit many retrovirus elements. It engages assembling transposon into one or more locations in the genome, retrovirus particles, accesses the RNA genome-containing avoiding the integration of G/C-rich prokaryotic elements virus core and upon reverse transcription, deaminates of the vector and avoiding transgene concatemerization cDNA cytosines to uracils, inactivating the coding capacity that can cause shutdown of gene expression. Other powerful tools in genome modifications are representedby Cre and FLP recombinases that catalyze a conserva- tive DNA recombination event between two short Most transgenic animals carry transgenes expressed in all recombinase recognition sites (RRSs), loxP and FRT.
tissues. Under specific circumstances, and to avoid This can permit deletion or inversion of the DNA possible side effects due to undesired transgene expres- between two RRSs, depending on their orientation sion in some tissues, it may be necessary to have a promoter . Also, in lentiviral-mediated transgenesis, the use that is inducible or tissue-specific. Although this technol- of some drugs such as cytokines or proteasome inhibitors ogy has been well established in the mouse, information is can increase lentiviral gene transfer Santoni de only recently becoming available in the pig Sio et al. has shown that human hematopoietic stemcells (HSCs) can be transduced to high efficiency by ashort exposure to lentivirals in the presence of stem cell factor, thrombopoietin, interleukin-6 and Flt3L. More- over, it was shown that the proteasome restricts lentiviral The use of genetically modified animals in transplant transduction in HSCs, and on using the reversible research has necessarily lagged behind the technological peptide–aldehyde proteasome inhibitor, MG132, and advances that have been made in producing such animals.
the peptide–boronate inhibitor, PS-341, during the However, the effects of these modifications are already lentiviral-GFP transduction period, there is a substantial drug dose-dependent increase in the frequency of trans-gene-expressing cells and in their mean fluorescence intensity. Zinc finger nucleases show promise in improving The first transgenic modifications of swine reportedly the efficiency of gene targeting by introducing DNA involved the introduction of human complement-regulat- double-strand breaks in target genes, which then stimulate ory proteins (CRPs) as transgenes into outbred swine. The the cell’s endogenous homologous recombination machin- three CRPs most extensively studied have been human ery. Many studies have been performed on human and decay-accelerating factor (hDAF) , CD46 and mouse cells A strategy to speed multitransgenic CD59 Although early studies using these transgenic pig production is represented by the recent adaptation of animals showed clear advantages in terms of inhibition of the 2A system from foot and mouth disease virus to complement-mediated graft destruction, none of these mammalian transgenic technology In this system, modifications was capable of preventing eventual humoral the open reading frame consists of multiple individual rejection on its own Subsequent studies have cDNAs separated by sequences encoding 2A and furin attempted to combine the use of these transgenic animals cleavage sites. A single complex mRNA is produced and with additional modifications or treatments designed to translated into a single polypeptide that is cleaved into further reduce antibody or complement-mediated activity.
individual exogenous proteins at the 2A sites.
For example, Lam et al. have reported the effect ofchronic complement inhibition by soluble complement receptor type 1 (SCR1, TP10) to the treatment regimen for In cells transfected with siRNA vectors, targeted mRNAs renal transplantation of hDAF pig kidneys into cyno- are degraded by endonuclease activity and the amount of molgus monkeys but without significant improvement protein translated is reduced by over 95%, thus resulting in xenograft survival. McGregor et al. reported a in a significant knockdown and is an alternative approach median survival of 96 days for pig hearts from CD46 to achieving more complex and difficult knockouts. This transgenic pigs transplanted heterotopically in baboons technique is particularly useful when more than one copy using rituximab and thymoglobulin as induction ther- of the endogenous gene is present and the usual knock- apy along with a standard immunosuppressive regimen out approach is not feasible. This situation is indeed the including tacrolimus, sirolimus steroids and an inhibitor of case of pathogens such as porcine endogenous retrovirus anti-Gal antibodies. Shimizu et al. reported on a series Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Genetic manipulation in pigs Sachs and Galli of pig-to-baboon renal xenografts using kidneys from hDAF pigs and treating with extracorporeal absorption Although the study by Hering et al. has suggested of anti-Gal antibodies, additional inhibition of comple- that Gal is not a problematic antigen on adult porcine ment with cobra venom factor and strong T-cell immuno- islets, non-Gal antigens may indeed still play a role.
suppression. Nevertheless, humoral xenograft rejection Komoda et al. have demonstrated that islets from occurred, characterized by both antibody deposition and transgenic pigs expressing N-acetylglucosaminyltransfer- ase-III enjoy prolonged survival after transplantation intocynomolgus monkeys.
The study by Shimizu et al. points out that eventhough dysregulated coagulation is part of the rejectionpicture, immune-mediated damage probably initiates the rejection process. This hypothesis has been substantiated by studies from Byrne et al. who showed that Studies using the new GalT-KO organs as xenografts to increasing immunosuppression rather than anticoagula- nonhuman primates have been carried out in several tion was capable of extending cardiac xenograft survival laboratories with very divergent results, depending on of CD46 transgenic pig hearts in baboons.
Although until the recent availability of alpha-1,3-galac- Pig-to-primate organ xenotransplantation using chronic tosyltransferase gene-knockout (GalT-KO) pigs, anti-Gal antibodies were considered to be the major cause of Chen et al. found little advantage of the use of GalT- humoral rejection in pig-to-primate xenotransplantation, KO pig kidneys over previous transgenic kidneys when there were already some studies indicating that antinon- transplanting into baboons using two potent immuno- Gal antibodies might also play a role. One such study was suppressive regimens. Because they found a marked reported by Chen et al. who demonstrated that life- evidence for elevated anti-non-Gal antibodies at the time supporting renal xenografts from hDAF transgenic pigs of rejection, they concluded that antibodies to non-Gal into baboons, in which anti-Gal antibodies were chroni- antigens might present a major barrier to xenotransplan- cally neutralized by soluble Gal-containing polymers, anti- tation, even using GalT-KO pigs. Kuwaki et al. non-Gal antibodies still led to acute humoral rejections reported improvement in the survival of heterotopically transplanted pig hearts in baboons when GalT-KO pigswere substituted for hDAF pigs as the donors. However, although one animal in these studies showed a survival of Because of the difficulty of experiments involving organ a GalT-KO heterotopic heart for 179 days even xenografts in vivo, a number of studies have been this survival was not markedly greater than the maximum reported using cellular parameters of xenograft rejection achieved in the same model using hDAF grafts, the to examine the effects of transgenic modifications. Forte longest of which survived 139 days In both these et al. and Lilienfeld et al. have demonstrated studies, the rejection observed showed evidence for that natural killer (NK) cytotoxicity by human NK cells initiation by antibodies, suggesting that even with T-cell toward porcine lymphoblastoid and endothelial cells immunosuppression, there was sufficient T-cell help to could be inhibited markedly by the expression of human produce T-cell-dependent antibody responses to non- leukocyte antigen E (HLA-E) on the cell surface of these Gal antigens. Tseng et al. analyzed the same animals porcine cells. Although it is not clear what role NK cells and suggested that anticoagulation with aspirin may have play in the direct rejection of xenograft organs, they been a major factor in delaying the onset and progression clearly could be important in the rejection of cellular of thrombotic microangiopathy in the longest surviving transplants, such as those involved in the induction of heterotopic hearts. However, Shimizu et al. demon- tolerance through mixed chimerism Also, using strated that these thrombotic lesions were always accom- in-vitro assays, Diaz-Roman et al. have provided panied by evidence for both T-cell and antibody activity, evidence that some of the lack of protection from making it unclear whether the thrombotic complications complement-mediated lysis by hDAF in vivo in non- were primary or secondary to immune reactivity in these human primates could be due to a difference between the complement of humans and that of nonhumanprimates. Manzi et al. have shown that transfection Approaches directed toward tolerance induction of a pig endothelial cell line with a truncated form of In contrast to results obtained using immunosuppressive human SCR1 is capable of protecting these cells from drug regimens, Yamada et al. have demonstrated complement-mediated lysis by human natural anti- prolonged survival of life-supporting vascularized renal bodies. However, transgenic pigs expressing SCR1 have xenografts when vascularized thymic tissue was cotrans- planted at the same time as the kidney. Although hDAF Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
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Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
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51 Komoda H, Miyagawa S, Omori T, et al. Survival of adult islet grafts from This study described the use of zinc finger nucleases to significantly increase the transgenic pigs with N-acetylglucosaminyltransferase-III (GnT-III) in cynomol- efficiency of homologous recombination.
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transcripts, thus reducing the probability of PERV transmission during xenotrans-plantation.
55 Tseng YL, Kuwaki K, Dor FJ, et al. alpha1,3-Galactosyltransferase gene- knockout pig heart transplantation in baboons with survival approaching 37 Jonsson SR, LaRue RS, Stenglein MD, et al. The restriction of zoonotic PERV 6 months. Transplantation 2005; 80:1493–1500.
transmission by human APOBEC3G. PLoS ONE 2007; 2:e893.
38 Diamond LE, Quinn CM, Martin MJ, et al. A human CD46 transgenic pig model Shimizu A, Hisashi Y, Kuwaki K, et al. Thrombotic microangiopathy associated system for the study of discordant xenotransplantation. Transplantation 2001; with humoral rejection of cardiac xenografts from alpha1,3-galactosyltrans- ferase gene-knockout pigs in baboons. Am J Pathol 2008; 172:1471–1481.
39 Byrne GW, McCurry KR, Martin MJ, et al. Transgenic pigs expressing human The results of this study indicate that the characteristic thrombotic microangio- CD59 and decay-accelerating factor produce an intrinsic barrier to comple- pathy seen histologically in porcine heart xenografts transplanted into baboons are ment-mediated damage. Transplantation 1997; 63:149–155.
associated with antibody-mediated rejection, which may be the primary cause of 40 Sachs DH, Fishman JA. Xenotransplantation. In: Brenner BM, editor. Brenner & Rector’s The Kidney. Philadelphia: Saunders Elsevier; 2008. pp. 2183– 57 Yamada K, Yazawa K, Shimizu A, et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransfer- This chapter provides a recent and comprehensive review of the field of xeno- ase gene-knockout donors and the cotransplantation of vascularized thymic transplantation with emphasis on an alternative source of renal transplants.
41 Lam TT, Hausen B, Hook L, et al. The effect of soluble complement receptor This study presents the first results obtained using GalT-KO animals as a source of type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate orthotopic renal xenografts to baboons. Survival of life-supporting kidneys of up to life-supporting kidney xenografts. Xenotransplantation 2005; 12:20–29.
83 days is reported using a treatment regimen directed toward the induction oftolerance through cotransplantation of vascularized thymic tissue.
42 McGregor CG, Davies WR, Oi K, et al. Cardiac xenotransplantation: recent preclinical progress with 3-month median survival. J Thorac Cardiovasc Surg 58 Mezrich JD, Haller GW, Arn JS, et al. Histocompatible miniature swine: an inbred large-animal model. Transplantation 2003; 75:904–907.
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A physician should be consulted if Lyme disease is suspected. Only the rash is distinctive enough for a clinical diagnosis without laboratory confirmation. In the absence of an EM rash, Lyme disease may be difficult to diagnose because its symptoms and signs vary among individuals and can be similar to those of many other diseases. Conversely, other arthritic or neurologic diseases may be mis


Prof. Dr. Bernd Hoppe, Hôpital Universitaire de Cologne, Service de Une augmentation de l’élimination urinaire de l’Oxalate s’appelle « Hyperoxalurie ». Elle est une des causes principales d’une lithiase urinaire récidivante (calculs urinaires) ou d’une néphrocalcinose progressive (= Calcification ou sclérose rénale, c’est-à-dire un dépôt de cristaux d’oxalate de calc

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