Barbara Kessler

Efficient transgenesis in farm animals by lentiviral vectors.

Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV‐PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV‐PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ‐line. Tissue‐specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV‐K14). LV‐K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV‐PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.

Transgenic pigs as models for translational biomedical research.

The translation of novel discoveries from basic research to clinical application is a long, often inefficient, and thus costly process. Accordingly, the process of drug development requires optimization both for economic and for ethical reasons, in order to provide patients with appropriate treatments in a reasonable time frame. Consequently, “Translational Medicine” became a top priority in national and international roadmaps of human health research. Appropriate animal models for the evaluation of efficacy and safety of new drugs or therapeutic concepts are critical for the success of translational research. In this context rodent models are most widely used. At present, transgenic pigs are increasingly being established as large animal models for selected human diseases. The first pig whole genome sequence and many other genomic resources will be available in the near future. Importantly, efficient and precise techniques for the genetic modification of pigs have been established, facilitating the generation of tailored disease models. This article provides an overview of the current techniques for genetic modification of pigs and the transgenic pig models established for neurodegenerative diseases, cardiovascular diseases, cystic fibrosis, and diabetes mellitus.

Glucose Intolerance and Reduced Proliferation of Pancreatic β-Cells in Transgenic Pigs With Impaired Glucose-Dependent Insulinotropic Polypeptide Function

OBJECTIVE The insulinotropic action of the incretin glucose-dependent insulinotropic polypeptide (GIP) is impaired in type 2 diabetes, while the effect of glucagon-like peptide-1 (GLP-1) is preserved. To evaluate the role of impaired GIP function in glucose homeostasis and development of the endocrine pancreas in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPRdn) in pancreatic islets. RESEARCH DESIGN AND METHODS GIPRdn transgenic pigs were generated using lentiviral transgenesis. Metabolic tests and quantitative stereological analyses of the different endocrine islet cell populations were performed, and β-cell proliferation and apoptosis were quantified to characterize this novel animal model. RESULTS Eleven-week-old GIPRdn transgenic pigs exhibited significantly reduced oral glucose tolerance due to delayed insulin secretion, whereas intravenous glucose tolerance and pancreatic β-cell mass were not different from controls. The insulinotropic effect of GIP was significantly reduced, whereas insulin secretion in response to the GLP-1 receptor agonist exendin-4 was enhanced in GIPRdn transgenic versus control pigs. With increasing age, glucose control deteriorated in GIPRdn transgenic pigs, as shown by reduced oral and intravenous glucose tolerance due to impaired insulin secretion. Importantly, β-cell proliferation was reduced by 60% in 11-week-old GIPRdn transgenic pigs, leading to a reduction of β-cell mass by 35% and 58% in 5-month-old and 1- to 1.4-year-old transgenic pigs compared with age-matched controls, respectively. CONCLUSIONS The first large animal model with impaired incretin function demonstrates an essential role of GIP for insulin secretion, proliferation of β-cells, and physiological expansion of β-cell mass.

HLA-E/human beta2-microglobulin transgenic pigs: protection against xenogeneic human anti-pig natural killer cell cytotoxicity

Background. Natural killer (NK) cells participate in pig-to-primate xenograft rejection both by antibody-dependent and -independent mechanisms. A majority of human NK cells express the inhibitory receptor CD94/NKG2A, which binds specifically to human leukocyte antigen (HLA)-E, a trimeric complex consisting of the HLA-E heavy chain, &bgr;2-microglobulin (&bgr;2m), and a peptide derived from the leader sequence of some major histocompatibility complex class I molecules. Methods. To use this mechanism for protection of pig tissues against human NK cell-mediated cytotoxicity, we generated transgenic pigs by pronuclear microinjection of genomic fragments of HLA-E with an HLA-B7 signal sequence and of human &bgr;2-microglobulin (hu&bgr;2m) into zygotes. Results. Three transgenic founder pigs were generated. Northern blot analysis of RNA from peripheral blood mononuclear cells revealed the presence of the expected transcript sizes for both transgenes in two of the three founders. The founder with the highest expression and his offspring were characterized in detail. Fluorescence-activated cell sorting (FACS) and Western blot analyses demonstrated consistent expression of HLA-E and hu&bgr;2m in peripheral blood mononuclear cells. Immunohistochemistry revealed the presence of HLA-E and hu&bgr;2m on endothelial cells of many organs, including heart and kidney. In vitro studies showed that lymphoblasts and endothelial cells derived from HLA-E/hu&bgr;2m transgenic pigs are effectively protected against human NK cell-mediated cytotoxicity, depending on the level of CD94/NKG2A expression on the NK cells. Further, HLA-E/hu&bgr;2m expression on porcine endothelial cells inhibited the secretion of interferon (IFN)-&ggr; by co-cultured human NK cells. Conclusions. This novel approach against cell-mediated xenogeneic responses has important implications for the generation of multitransgenic pigs as organ donors for clinical xenotransplantation.

Xenografted Islet Cell Clusters From INSLEA29Y Transgenic Pigs Rescue Diabetes and Prevent Immune Rejection in Humanized Mice

Islet transplantation is a potential treatment for type 1 diabetes, but the shortage of donor organs limits its routine application. As potential donor animals, we generated transgenic pigs expressing LEA29Y, a high-affinity variant of the T-cell costimulation inhibitor CTLA-4Ig, under the control of the porcine insulin gene promoter. Neonatal islet cell clusters (ICCs) from INSLEA29Y transgenic (LEA-tg) pigs and wild-type controls were transplanted into streptozotocin-induced hyperglycemic NOD-scid IL2Rγnull mice. Cloned LEA-tg pigs are healthy and exhibit a strong β-cell–specific transgene expression. LEA-tg ICCs displayed the same potential to normalize glucose homeostasis as wild-type ICCs after transplantation. After adoptive transfer of human peripheral blood mononuclear cells, transplanted LEA-tg ICCs were completely protected from rejection, whereas reoccurrence of hyperglycemia was observed in 80% of mice transplanted with wild-type ICCs. In the current study, we provide the first proof-of-principle report on transgenic pigs with β-cell–specific expression of LEA29Y and their successful application as donors in a xenotransplantation model. This approach may represent a major step toward the development of a novel strategy for pig-to-human islet transplantation without side effects of systemic immunosuppression.

Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation.

Background:  Multi‐transgenic pigs produced for use in xenotransplantation have to be screened for the presence and expression of porcine endogenous retroviruses (PERV) to select animals with low PERV load. The production of transgenic pigs may also be associated with the integration of the transgene adjacent to or into the locus of a PERV provirus, potentially leading to an enhanced virus expression.

A porcine model of familial adenomatous polyposis.

We created gene-targeted pigs with mutations in the adenomatous polyposis coli (APC) gene (APC) that are orthologous to those responsible for human familial adenomatous polyposis (FAP). One-year-old pigs with the APC(1311) mutation (orthologous to human APC(1309)) have aberrant crypt foci and low- and high-grade dysplastic adenomas in the large intestine, similar to the precancerous lesions that develop in patients with FAP. Dysplastic adenomas accumulate β-catenin and lose heterozygosity of APC. This large-animal, genetic model of FAP will be useful in the development of diagnostics and therapeutics for colorectal cancer. DNA sequence data: NCBI accession number GU951771.

Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle

Duchenne muscular dystrophy (DMD) is caused by mutations in the X-linked dystrophin (DMD) gene. The absence of dystrophin protein leads to progressive muscle weakness and wasting, disability and death. To establish a tailored large animal model of DMD, we deleted DMD exon 52 in male pig cells by gene targeting and generated offspring by nuclear transfer. DMD pigs exhibit absence of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive dystrophic changes of skeletal muscles, impaired mobility, muscle weakness and a maximum life span of 3 months due to respiratory impairment. Unlike human DMD patients, some DMD pigs die shortly after birth. To address the accelerated development of muscular dystrophy in DMD pigs when compared with human patients, we performed a genome-wide transcriptome study of biceps femoris muscle specimens from 2-day-old and 3-month-old DMD and age-matched wild-type pigs. The transcriptome changes in 3-month-old DMD pigs were in good concordance with gene expression profiles in human DMD, reflecting the processes of degeneration, regeneration, inflammation, fibrosis and impaired metabolic activity. In contrast, the transcriptome profile of 2-day-old DMD pigs showed similarities with transcriptome changes induced by acute exercise muscle injury. Our studies provide new insights into early changes associated with dystrophin deficiency in a clinically severe animal model of DMD.

Permanent Neonatal Diabetes in INSC94Y Transgenic Pigs

Mutations in the insulin (INS) gene may cause permanent neonatal diabetes mellitus (PNDM). Ins2 mutant mouse models provided important insights into the disease mechanisms of PNDM but have limitations for translational research. To establish a large animal model of PNDM, we generated INSC94Y transgenic pigs. A line expressing high levels of INSC94Y mRNA (70–86% of wild-type INS transcripts) exhibited elevated blood glucose soon after birth but unaltered β-cell mass at the age of 8 days. At 4.5 months, INSC94Y transgenic pigs exhibited 41% reduced body weight, 72% decreased β-cell mass (−53% relative to body weight), and 60% lower fasting insulin levels compared with littermate controls. β-cells of INSC94Y transgenic pigs showed a marked reduction of insulin secretory granules and severe dilation of the endoplasmic reticulum. Cataract development was already visible in 8-day-old INSC94Y transgenic pigs and became more severe with increasing age. Diabetes-associated pathological alterations of kidney and nervous tissue were not detected during the observation period of 1 year. The stable diabetic phenotype and its rescue by insulin treatment make the INSC94Y transgenic pig an attractive model for insulin supplementation and islet transplantation trials, and for studying developmental consequences of maternal diabetes mellitus.

Transcriptome Changes in the Porcine Endometrium During the Preattachment Phase

ABSTRACT The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Days 11 and 12 of gestation. Concomitant with trophoblast elongation, production of conceptus estrogen, the porcine embryonic pregnancy recognition signal, increases. Conceptus attachment to the uterine surface epithelium starts after Day 13, initiating epitheliochorial placentation. To analyze the transcriptome changes in the endometrium in the course of maternal recognition of pregnancy, deep sequencing of endometrial RNA samples of Day 12 pregnant animals (n = 4) and corresponding nonpregnant controls (n = 4) was performed using RNA sequencing (RNA-Seq). Between 30 000 000 and 35 000 000 sequence reads per sample were produced and mapped to the porcine genome (Sscrofa10.2). Analysis of read counts revealed 2593 differentially expressed genes (DEGs). Expression of selected genes was validated by the use of quantitative real-time RT-PCR. Bioinformatics analysis identified several functional terms specifically overrepresented for up-regulated or down-regulated genes. Comparison of the RNA-Seq data from Days 12 and 14 of pregnancy was performed at the level of all expressed genes, the level of the DEG, and the level of functional categories. This revealed specific gene expression patterns reflecting the different functions of the endometrium during these stages (i.e., recognition of pregnancy and preparation for conceptus attachment). Genes related to mitosis, immune response, epithelial cell differentiation and development, proteolysis, and prostaglandin signaling and metabolism are discussed in detail. This study identified comprehensive transcriptome changes in porcine endometrium associated with establishment of pregnancy and could be a resource for targeted studies of genes and pathways potentially involved in regulation of this process.