Sharon J. Harrison

Production of Cloned Pigs from Cultured Fetal Fibroblast Cells

Abstract Somatic cell nuclear transfer was used to produce live piglets from cultured fetal fibroblast cells. This was achieved by exposing donor cell nuclei to oocyte cytoplasm for approximately 3 h before activation by chemical means. Initially, an experiment was performed to optimize a cell fusion system that prevented concurrent activation in the majority of recipient cytoplasts. Cultured fibroblast cells were fused in medium with or without calcium into enucleated oocytes flushed from superovulated gilts. Cybrids fused in the presence of calcium cleaved at a significantly (P < 0.05) greater rate (69%, 37 out of 54) after 2 days of culture compared with those fused without calcium (10%, 7 out of 73), suggesting that calcium-free conditions are needed to avoid activation in the majority of recipient cytoplasts during fusion. In the second experiment, cybrids fused in calcium-free medium were activated approximately 3 h later with ionomycin, followed by incubation in 6-dimethylaminopurine to determine development in vitro. Following 2 days of culture, cleavage rates of chemically activated and unactivated cybrids (fusion without activation control) were 93% (100 out of 108) and 7% (2 out of 27), respectively. After an additional 5 days of culture, activated cloned embryos formed blastocysts at a rate of 23% (25 out of 108) with an average inner cell mass and trophectoderm cell number of 10 (range, 3 to 38) and 31 (range, 16 to 58), respectively. In the third experiment, activated nuclear transfer embryos were transferred to the uteri of synchronized recipients after 3 days of culture to assess their development in vivo. Of 10 recipients receiving an average of 80 cleaved embryos (range, 40 to 107), 5 became pregnant (50%) as determined by ultrasound between Day 25 and Day 35 of gestation. Of the five pregnant recipients, two subsequently farrowed one piglet per litter originating from two different cell culture lines. In this study, efficient reprogramming of porcine donor nuclei by fusing cells in the absence of calcium followed by chemical activation of recipient cytoplasts was reflected in high rates of development to blastocyst and pregnancy initiation leading to full term development.

Production of homozygous α-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer

Abstract:  We report here our experience regarding the production of double or homozygous Gal knockout (Gal KO) pigs by breeding and somatic cell nuclear transfer (SCNT). Large White × Landrace female heterozygous Gal KO founders produced using SCNT were mated with Hampshire or Duroc males to produce a F1 generation. F1 heterozygous pigs were then bred to half‐sibs to produce a F2 generation which contained Gal KO pigs. To determine the viability of mating Gal KO pigs with each other, one female F2 Gal KO pig was bred to a half‐sib and subsequently a full‐sib Gal KO. F1 and F2 heterozygous females were also mated to F2 Gal KO males. All three types of matings produced Gal KO pigs. To produce Gal KO pigs by SCNT, heterozygous F1s were bred together and F2 fetuses were harvested to establish primary cultures of Gal KO fetal fibroblasts. Gal KO embryos were transferred to five recipients, one of which became pregnant and had a litter of four piglets. Together our results demonstrate that Gal KO pigs can be produced by breeding with each other and by SCNT using Gal KO fetal fibroblasts.

In Vitro and In Vivo Characterization of Putative Porcine Embryonic Stem Cells

We have developed a new method for the isolation of porcine embryonic stem cells (ESCs) from in vivo-derived and in vitro-produced embryos. Here we describe the isolation and characterization of several ESC lines established using this method. Cells from these lines were passaged up to 14 times, during which they were repeatedly cryopreserved. During this time, ESCs maintained their morphology and continued to express Oct 4, Nanog, and SSEA1. These cells formed embryoid bodies in suspension culture, and could be directed to differentiate into various lineages representative of all three germ layers in vitro. When injected into blastocysts these cells localized in the inner cell mass of blastocysts. To examine their pluripotency further, cells were injected into host blastocysts and transferred to recipient animals. Of the six transfers undertaken, one recipient became pregnant and gave birth to a litter of one male and three female piglets. Microsatellite analysis of DNA extracted from the tail tissue of these piglets indicated that two female piglets were chimaeric.

Efficient Generation of α(1,3) Galactosyltransferase Knockout Porcine Fetal Fibroblasts for Nuclear Transfer

Pigs are currently considered the most likely source of organs for human xenotransplantation because of anatomical and physiological similarities to humans, and the relative ease with which they can be bred in large numbers. A severe form of rejection known as hyperacute rejection has been the major barrier to the use of xenografts. Generating transgenic pigs for organ transplantation is likely to involve precise genetic manipulation to ablate the α(1,3) galactosyltransferase (galT) gene. In contrast to the mouse, homologous recombination in livestock species to ablate genes is hampered by the inability to isolate functional embryonic stem cells. However, nuclear transfer using genetically targeted cultured somatic cells provides an alternative means to producing pigs deficient for galT. In this study we successfully produced galT+/− somatic porcine fetal fibroblasts using two approaches; positive negative selection (PNS) using an isogenic targeting construct, and with a promoterless vector using non-isogenic DNA.

Effect of DNA concentration on transgenesis rates in mice and pigs.

A retrospective analysis of transgenesis rates obtained in seven pronuclear microinjection programs was undertaken to determine if a relationship existed between the amount of DNA injected and transgenesis rates in the pig. Logistic regression analysis showed that as the concentration of DNA injected increased from 1 to 10 ng/μl, the number of transgenics when expressed as a proportion of the number liveborn (integration rate) increased from 4% to an average of 26%. A similar relationship was found when the number of molecules of DNA injected per picolitre was analysed. No evidence was obtained to suggest either parameter influenced integration rate in mice when the same constructs were injected. The number of transgenics liveborn when expressed as a proportion of ova injected (efficiency rate), increased as DNA concentration increased up to 7.5 ng/μl and then decreased at 10 ng/μl for both species suggesting that at this concentration DNA (or possible contaminants) may have influenced embryo survival. The relationship between efficiency and the number of molecules injected per picolitre was complex suggesting that the concentration at which DNA was injected was a better determinant of integration and efficiency rates. In conclusion, the present study suggests that transgenes need to be injected at concentrations of between 5 and 10 ng/μl to maximise integration and efficiency rates in pigs.

Isolation and in vitro characterization of putative porcine embryonic stem cells from cloned embryos treated with trichostatin A.

We report here the establishment and characterization of putative porcine embryonic stem cell (ESC) lines derived from somatic cell nuclear transfer embryos (NT-ESCs). These cells had a similar morphology to that described previously by us for ESCs derived from in vitro produced embryos, namely, a polygonal shape, a relatively small (10-15 μm) diameter, a small cytoplasmic/nuclear ratio, a single nucleus with multiple nucleoli and multiple lipid inclusions in the cytoplasm. NT-ESCs could be passaged at least 15 times and vitrified repeatedly without changes in their morphology, karyotype, or Oct-4 and Nanog expression. These cells formed embryoid bodies and could be directed to differentiate in vitro to cell types representative of all three germ layers. Following their injection into blastocysts, these cells preferentially localized in the inner cell mass. In conclusion, we have isolated putative porcine ESCs from cloned embryos that have the potential to be used for a variety of applications including as a model for human therapeutic cloning.

SECRETION OF EUKARYOTIC GROWTH HORMONES IN ESCHERICHIA COLI IS INFLUENCED BY THE SEQUENCE OF THE MATURE PROTEINS

We report the construction of secretion plasmids expressing the fusion proteins, OmpA::pGH (pSpGH.01) and OmpA::hGH (phGH.01), and compare the secretion of mature porcine growth hormone (pGH) and human growth hormone (hGH) employing Escherichia coli. E. coli [phGH.01] secreted 10-15 micrograms hGH/ml/A600 cells into the periplasmic space, representing 30% of total periplasmic proteins. E. coli [pSpGH.01], however, secreted 30-fold less mature pGH. On the basis that both pSpGH.01 and phGH.01 are stably maintained in E. coli and in vitro transcription/translation data showed equivalent expression of OmpA::pGH and OmpA::hGH precursors, we attribute the higher secretion of hGH to the translocation-competent OmpA::hGH protein configuration. Two OmpA::GHF (growth hormone fusion) precursors, OmpA::GHF.02 and OmpA::GHF.03, both with hGH helix 3/helix 4 together instead of the pGH equivalent, secreted mature proteins as efficiently as OmpA::hGH. We propose that hGH helices 3 and 4 in these OmpA::GHF precursors play a major role in the folding of the precursor to a translocation-competent state, mimicking the translocation-competent nature of the OmpA::hGH precursor.

Transgenic perspectives in xenotransplantation, 2001.

1. HAMMER C, THEIN E. Determining significant physiologic incompatibilities. Graft 2001; 4: 108. 2. STANKOVICOVA T, SZILARD M, DE-SCHNEEBERGER I, SIPIO K. M cells and transmural heterogeneity of action potential configuration in myocytes from the left ventricular wall of the pig heart. Cardiovasc Res 2000; 45: 952. 3. KIRKMAN R. Of swine and man: organ physiology in different species. In: HARDY M, ed. Xenograft 25. Amsterdam: Elsevier Sciences, 1989: p. 125. 4. CRICK S, ONG D. Localisation and quantitation of autonomic innervation in the porcine heart. J Anat 1999; 195: 341/359. 5. BRENNER P, SCHMOECKEL M, REICHENSPURNER H et al. IG-Therasorb immunoapheresis in orthotopic xenotransplantation of baboons with landrace pig hearts. Transplantation 2000; 69: 208. 6. KALADY M, LAWSON J, SORREL R, PLATT J. Decrease fibrinolytic activity in porcine-to-primate cardiac xenotransplantation. Mol Med 1998; 4: 629. 7. CHENG D, ONG D. Anaesthesia for non-cardiac surgery in heart transplanted patients. Can J Anaesth 1993; 40: 1981. 8. MOLLEVI D, JAURRIETA E, RIBAS Y et al. Liver xenotransplantation: changes in lipid and lipoprotein concentration after long term graft survival. J Hepatol 2000; 32: 655. 9. MINGUELA A, RAMIREZ P, CARRACOSA C et al. Identification of porcine proteins in baboon sera after pig liver xenotransplantation. Transplant Proc 1999; 31: 2635. 10. MAJADO M, RAMIREZ P, MINGUELA A et al. Evolution of blood coagulation factors and hemotherapeutic support in three pig-to-baboon orthotopic liver xenotransplants. Transplant Proc 1999; 31: 2622. 11. RAMIREZ P, CHAVEZ R, MAJADO M et al. Life-supporting human complement regulator decay accelerating factor transgenic pig liver xenograft maintains the metabolic function and coagulation in the non-human primate for up to 8 days. Transplantation 2000; 70: 989. 12. CRUZADO J, TORRAS J, RIERA M et al. Effect of human natural antibody depletion and complement inactivation on early pig kidney function. Exp Nephrol 1999; 7: 217. 13. MARUYAMA S, CANTU E, DEMARTINO C et al. Membranous glomerulonephritis induced in the pig by antibodies to angiotensin converting enzyme. Am J Soc Nephrol 1999; 10: 2102. 14. COZZI E, BHATTI F, SCHMOECKEL M et al. Long term survival of non-human primates receiving life-supporting transgenic porcine kidney xenografts. Transplantation 2000; 70: 15. 15. PRZEMEK M, VANGEROW B, LOSS M et al. Hemodynamic consequences of porcine kidney xenograft reperfusion in cynomolgus monkeys. Transplantation 2001; 71: 1512. 16. BENNET W, SUNDBERG B, LUNDGREN T et al. Damage of porcine islets of Langerhans after exposure to human blood in vitro, or after intraportal transplantation to cynomolgus monkeys: protective effects of sCR1 and heparin. Transplantation 2000; 69: 711. 17. DAGETT C, YEATMAN M, LODGE A et al. Total respiratory support from swine lungs in primate recipients. J Thorac Cardiovasc Surg 1998; 115: 19. 18. KAPLON R, PLATT J, KWIATKOWSKI P et al. Absence of hyperacute rejection in pig to primate orthotopic xenografts. Transplantation 1995; 59: 410. 19. DAGGETT C, YEATMAN M, LODGE A et al. Swine lungs expressing human complement-regulatory proteins. J Thorac Cardiovasc Surg 1997; 113: 390. 20. YEATMAN M, DAGGETT C, LAU C et al. Human complement regulatory proteins protect swine lungs from xenogeneic Injury. Ann Thorac Surg 1999; 67: 769. 21. SCHELZIG H, SIMON F, KRISCHER C et al. Ex-vivo hemoperfusion (eHPS) of pig lungs with whole human blood: effect of complement inhibitor with a soluble C1-esterase inhibitor. Ann Transplant 2001; 6: 34. 22. HAMMER C. In vivo microscopic assessment of microcirculatory changes in a concordant xenogeneic primate experimental set-up. Ann Transplant 2001; 6: 17. 23. HAMMER C, WAGNER F, THEIN E. Microvasculature after xenografting. Curr Opin Organ Transpl 2001; 6: 47. 24. THEIN E, SEVILMIS G, MüENZING S et al. Evaluation of a system for the perfusion of isolated, rodent organs. Xenotransplantation 2001; 8: 94. 25. ALVAREZ B, DOMENECH N, ALONSO F et al. Molecular and functional characterisation of porcine LFA-1 using monoclonal antibodies to CD 11a and CD 18. Xenotransplantation 2000; 7: 258. 26. TERAJIMA H, THIAENER A, HAMMER C et al. Attenuation of hepatic microcirculatory failure during in situ xenogeneic rat liver perfusion by heat shock preconditioning. Transplant Proc 2000; 32: 1111. 27. ROBSON S, COOPER D, D’APICE A. Disordered regulation and platelet activation in xenotransplantation. Xenotransplantation 2000; 7: 166. 28. ALWAYN I, ROBSON S. Understanding and preventing the coagulation disorders associated with xenograft rejection. Graft 2000; 4: 50.

Transgenic perspectives in livestock science: a review

The limitations of existing transgenic technology, the potential of cloning technology to overcome these, as well as technologies which may be available in the future for inserting new genetic material are discussed. Currently, transgenic livestock are produced by injecting hundreds to thousands of copies of a particular transgene into the pronucleus of a fertilised egg. This method suffers from a number of inherent limitations that prevent the full potential of this technology from being explored. Most of these limitations stem from the fact that it is impossible to control the site at which the transgene becomes inserted. Transgenic technology holds considerable promise for the livestock industries as well as having important biomedical applications. However, before any of these possibilities can be realised, technology is required whereby a single copy of a particular transgene can be inserted or ‘knocked in’ at a site that does not interfere with expression, as well as having the capacity to ‘knockout’ existing genes. This is possible in mice using a combination of homologous recombination and embryonic stem cell technologies. However, despite considerable effort worldwide, embryonic stem cells are yet to be isolated from any of the livestock species. The ability to clone these now means that somatic cells most notably fetal fibroblasts, can used for gene targeting purposes instead of embryonic stem cells. However, this method is not without its limitations and it is possible that more efficient methods will be developed in the future. In particular, the use of mammalian artificial chromosomes will extend this technology to allow combinations of transgenes as well as chromosomal segments to be incorporated, allowing us to explore the full potential of transgenic technology for agricultural as well as biomedical applications.

Multipotent Cell Types in Primary Fibroblast Cell Lines Used to Clone Pigs using Somatic Cell Nuclear Transfer

We have previously demonstrated that the use of porcine mesenchymal stem cells (MSCs) isolated from the bone marrow can increase the proportion of somatic cell nuclear transfer (SCNT) embryos that develop to the blastocyst stage compared with adult fibroblasts obtained from the same animal. The aim of the present study was to determine if MSCs are also present in primary cultures of adult fibroblasts which are commonly used for cloning live animals. To do this we chose a primary culture of adult fibroblasts that we had previously used to clone pigs. Single cell clones were isolated using low-density plating. After seven days of culture 63% of colonies displayed typical fibroblast morphology, while the remainder appeared cobblestone-like in appearance. Two of the 57 clones that displayed fibroblast morphology differentiated into adipocytes but not chondrocytes or osteocytes (uni-potent clones). Three of the 33 cobblestone-like clones differentiated into chondrocytes only, while 3 differentiated into adipocytes and chondrocytes but not osteocytes (bi-potent clones). One of the bi-potent cobblestone-like clones was then used for SCNT and in vitro development compared with a fibroblast-like clone which did not differentiate. Both cell types produced blastocysts at similar rates. In conclusion we have identified uni-potent and bi-potent cell types in primary cultures of adult fibroblasts used previously to clone live piglets.