Petra Hassel

Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli.

Oropeza M, Petersen B, Carnwath JW, Lucas‐Hahn A, Lemme E, Hassel P, Herrmann D, Barg‐Kues B, Holler S, Queisser A‐L, Schwinzer R, Hinkel R, Kupatt C, Niemann H. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli.
Xenotransplantation 2009; 16: 522–534.

Development and Validation of a Highly Efficient Protocol of Porcine Somatic Cloning Using Preovulatory Embryo Transfer in Peripubertal Gilts

The efficiency of porcine somatic nuclear transfer (born piglets/transferred embryos) is low. Here, we report a highly efficient protocol using peripubertal gilts as recipients synchronized to ovulate approximately 24 h after transfer of cloned embryos. Retrospectively, we compared the efficiency of two different synchronization protocols: In group 1, recipient animals were synchronized to ovulate approximately 6 h prior to surgical embryo transfer while in group 2 the animals were treated to ovulate 24 h after embryo transfer. In total, 1562 cloned embryos were transferred to 12 recipients in group 1; two of them became pregnant (16.7%). One pregnancy was lost on day 32, the second pregnancy went to term, and led to the birth of one healthy piglet after Cesarean section. In group 2, 1531 cloned embryos were transferred to 12 recipients. Nine recipients (75.0%) became pregnant as determined by ultrasound scanning on day 25. All pregnancies went to term and delivered a total of 47 live-born piglets. The cloning efficiency of both groups differed significantly (group 1: 0.1%, group 2: 3.1%, p < 0.05). This modified protocol was then applied in subsequent experiments using different types of transgenic and nontransgenic donor cells with similar success rates. Results show that this protocol is robust and highly reproducible, and can thus be employed for routine production of cloned pigs.

Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys

Petersen B, Ramackers W, Lucas‐Hahn A, Lemme E, Hassel P, Queißer A‐L, Herrmann D, Barg‐Kues B, Carnwath JW, Klose J, Tiede A, Friedrich L, Baars W, Schwinzer R, Winkler M, Niemann H. Transgenic expression of human heme oxygenase‐1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. Xenotransplantation 2011; 18: 355–368.

Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes.

Xenotransplantation is considered to be a promising solution to the growing demand for suitable donor organs for transplantation. Despite tremendous progress in the generation of pigs with multiple genetic modifications thought to be necessary to overcoming the severe rejection responses after pig‐to‐non‐human primate xenotransplantation, the production of knockout pigs by somatic cell nuclear transfer (SCNT) is still an inefficient process. Producing genetically modified pigs by intracytoplasmic microinjection of porcine zygotes is an alluring alternative. The porcine GGTA1 gene encodes for the α1,3‐galactosyltransferase that synthesizes the Gal epitopes on porcine cells which constitute the major antigen in a xenotransplantation setting. GGTA1‐KO pigs have successfully been produced by transfecting somatic cells with zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs), or CRISPR/Cas targeting GGTA1, followed by SCNT.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig‐to‐primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA‐mediated gene silencing. Porcine fibroblasts were transfected with TF‐targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild‐type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF‐α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi‐transgenic pigs for xenotransplantation.

Kidneys From α1,3-Galactosyltransferase Knockout/Human Heme Oxygenase-1/Human A20 Transgenic Pigs Are Protected From Rejection During Ex Vivo Perfusion With Human Blood.

Background Multiple modifications of the porcine genome are required to prevent rejection after pig-to-primate xenotransplantation. Here, we produced pigs with a knockout of the &agr;1,3-galactosyltransferase gene (GGTA1-KO) combined with transgenic expression of the human anti-apoptotic/anti-inflammatory molecules heme oxygenase-1 and A20, and investigated their xenoprotective properties. Methods The GGTA1-KO/human heme oxygenase-1 (hHO-1)/human A20 (hA20) transgenic pigs were produced in a stepwise approach using zinc finger nuclease vectors targeting the GGTA1 gene and a Sleeping Beauty vector coding for hA20. Two piglets were analyzed by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and sequencing. The biological function of the genetic modifications was tested in a 51Chromium release assay and by ex vivo kidney perfusions with human blood. Results Disruption of the GGTA1 gene by deletion of few basepairs was demonstrated in GGTA1-KO/hHO-1/hA20 transgenic pigs. The hHO-1 and hA20 mRNA expression was confirmed by quantitative reverse-transcription polymerase chain reaction. Ex vivo perfusion of 2 transgenic kidneys was feasible for the maximum experimental time of 240 minutes without symptoms of rejection. Conclusions Results indicate that GGTA1-KO/hHO-1/hA20 transgenic pigs are a promising model to alleviate rejection and ischemia-reperfusion damage in porcine xenografts and could serve as a background for further genetic modifications toward the production of a donor pig that is clinically relevant for xenotransplantation.

Generation of HHO-1/HA20/GGTA1-ko pigs by using sleeping beauty transposon and zinc finger nucleases

Program and Abstracts of the 12th Transgenic Technology Meeting (TT2014) The Assembly Rooms, Edinburgh, Scotland, United Kingdom, 6–8 October 2014 The TT2014 Meeting is hosted by: The Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland, United Kingdom. The Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, Scotland United Kingdom. The Institute of Genetics and Molecular Medicine, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU United Kingdom. 123 Transgenic Res (2014) 23:827–909 DOI 10.1007/s11248-014-9820-1 Springer International Publishing Switzerland 2014

Functional enucleation of porcine oocytes for somatic cell nuclear transfer using femtosecond laser pulses

Cloning of several mammalian species has been achieved by somatic cell nuclear transfer over the last decade. However, this method still results in very low efficiencies originating from biological and technical aspects. The highly-invasive mechanical enucleation belongs to the technical aspects and requires considerable micromanipulation skill. In this paper, we present a novel non-invasive method for combined oocyte imaging and automated functional enucleation using femtosecond (fs) laser pulses. After three-dimensional imaging of Hoechst-labeled porcine oocytes by multiphoton microscopy, our self-developed software automatically determined the metaphase plate position and shape. Subsequent irradiation of this volume with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation. We show that functional fs laser-based enucleation of porcine oocytes completely inhibited further embryonic development while maintaining intact oocyte morphology. In contrast, non-irradiated oocytes were able to develop to the blastocyst stage without significant differences to control oocytes. Our results indicate that fs laser systems offer great potential for oocyte imaging and enucleation as a fast, easy to use and reliable tool which may improve the efficiency of somatic cell clone production.

Femtosecond laser based enucleation of porcine oocytes for somatic cell nuclear transfer

Cloning of several mammalian species has been achieved by somatic cell nuclear transfer (SCNT) in recent years. However, this method still results in very low efficiencies around 1% which originate from suboptimal culture conditions and highly invasive techniques for oocyte enucleation and injection of the donor cell using micromanipulators. In this paper, we present a new minimal invasive method for oocyte imaging and enucleation based on the application of femtosecond (fs) laser pulses. After imaging of the oocyte with multiphoton microscopy, ultrashort pulses are focused onto the metaphase plate of MII-oocytes in order to ablate the DNA molecules. We show that fs laser based enucleation of porcine oocytes completely inhibits the first mitotic cleavage after parthenogenetic activation while maintaining intact oocyte morphology in most cases. In contrast, control groups without previous irradiation of the metaphase plate are able to develop to the blastocyst stage. Further experiments have to clarify the suitability of fs laser based enucleated oocytes for SCNT.