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Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53R167H mutant allele, orthologous to oncogenic human mutant TP53R175H and mouse Trp53R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.

BackgroundSomatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency.Results109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring.ConclusionAlthough the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.

SummaryDirect transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs) expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

We have examined the use of RNA interference as a means of downregulating gene expression and provide the first comparison of shRNA and artificial miRNA constructs for transgenic livestock. Several in vitro assays were performed to identify the most effective RNAi constructs. shRNA and miRNA constructs achieved significant downregulation of two porcine target genes: the milk whey protein beta-lactoglobulin and the tumour suppressor p53. Results of different assays were, however, sometimes at variance, indicating that no one assay can be relied upon to predict the effectiveness of an RNAi construct. Our findings are that screening of RNAi constructs is most informative if carried out in primary cells that express the target gene and are competent for somatic cell nuclear transfer. Importantly, the use of miRNA constructs makes tissue specific gene knockdown in large animals a realistic possibility.

The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.

Xenotransplantation could alleviate the serious shortage of human donor organs and tissues, but clinically acceptable donor pigs require a complex battery of genetic modifications. The precise requirements continue to be refined as knowledge increases, but a current ‘wish list’ includes removal of Gal and non‐Gal surface antigens, expression of complement regulators, anti‐thrombotic or anticoagulant factors, vascular protection and immunomodulatory genes, and some means of preventing possible zoonotic infection. Several pig lines have already been produced for xenotransplantation. Other than α1,3GT gene‐targeted animals, all contain individual randomly integrated transgenes. Transgenes integrated at different sites can however exhibit wide variations in the level and pattern of expression due to the influence of different chromatin environments and endogenous regulatory elements. The “position variegation effect” has been a long‐standing source of inefficiency and contributes to the inadequate expression observed for example in human complement regulator transgenes CD46, CD55 and CD59. This is exacerbated by the structure of traditional transgene expression vectors. In particular, viral promoters, small promoters, and cDNA or minigene rather than genomic sequences, are not reliable means of obtaining abundant uniform expression in transgenic animals. Novel approaches are therefore required to produce complex multi‐transgenic animals. Fortunately a broad range of new technologies are becoming available that significantly increase the chances of success. For example, construction of large transgenes has been aided by the use of BACs for molecular cloning and in vivo recombination (recombineering) in bacteria. The position effect can be overcome by incorporating elements such as nuclear matrix attachment regions, or employing episomal vectors such as mammalian artificial chromosomes. Placement of transgenes at predetermined favourable sites by a variety of recombination systems has become routine in the mouse and will soon be extended to pigs and other livestock. Viral‐ and transposon based vectors greatly improve the efficiency in obtaining transgenic animals. Although size‐restricted these are ideal for gene knockdown by short hairpin or tissue specific micro RNAs. Gene targeting became a reality in livestock species more than a decade ago with the advent of somatic cell nuclear transfer, but remains more difficult than in mouse ES cells. Promoter‐trap or large BAC based vectors offer incremental improvements, but new tools, particularly meganucleases and zinc finger nucleases (ZFNs) are set to revolutionise the field. ZFNs allow gene knockout directly in a fertilised oocyte, circumventing the need for cultured cells and nuclear transfer. More interestingly it has recently been shown that ZFNs can facilitate other forms of gene targeting such as gene replacement in the oocyte. Approaches that still rely on cell‐mediated transgenesis are also set to gain new possibilities with the announcement of pigs derived from ...