Nuclear Donor Research Articles

67 PRODUCTION OF CLONED TRANSGENIC RABBITS FROM MESENCHYMAL STEM CELLS

Mesenchymal stem cells (MSC) are adult stem cells with fibroblast-like morphology, which can be easily isolated from bone marrow and expanded in culture. Mesenchymal stem cells are able to grow from a single cell into a cell clone, which makes them potentially useful for gene targeting. In our recent study we investigated the dynamics of epigenetic reprogramming following nuclear transfer (NT) with MSC and found that these cells can support development of cloned embryos as good as genetically identical fibroblasts (Brero et al. 2009 Cloning Stem Cells 11, 319-329). In the present study we tested whether live cloned rabbits can be produced from MSC. Nuclear donor cells were isolated from a 6-week-old transgenic Ali/Bas rabbit, expanded in culture, and assessed for their differentiation potential. Mesenchymal stem cells were transfected with a green fluorescent protein (GFP) reporter gene construct and stable cell clones were selected (GFP-MSC). The MSC and GFP-MSC were used for NT at passage 3 to 7 after serum starvation for 2 to 4 days. Nuclear transfer was performed essentially as described previously (Yang et al. 2007 Reproduction 133, 219-320). To assess the development to blastocyst, reconstructed embryos were cultured in B2 medium for 5 to 6 days, whereas for in vivo development embryos were cultured only overnight and then transferred into recipients at the 4- to 8-cell stage. In the MSC group, 844 oocytes were used, 793 (94%) of them fused, 698/786 (89%) cleaved, and 48/128 (38%) developed to blastocyst. After transfer of 483 cloned embryos into 13 recipients, 2 from 8 pregnant recipients gave birth to 10 (2.4%) rabbits, from which 2 and 1 survived for more than 7 days and 3 months, respectively. In the GFP-MSC group, 444 oocytes were used, 412 (93%) of them fused, 377/409 (92%) cleaved, and 97/178 (55%) developed to blastocyst. Transfer of 216 cloned embryos into 8 recipients resulted in 4 pregnancies. One recipient gave birth to 6 (3.7%) live and 2 stillborn rabbits, from which 2 and 1 rabbits survived for more than 3 days and 2 weeks, respectively. All cloned rabbits carried a GFP gene, and green fluorescence could be detected in the follicles of the skin under a fluorescence microscope (Zeiss Axiovert200, Carl Zeiss, Germany). Our study demonstrates that live cloned rabbits can be produced from genetically modified MSC, thus paving the way to generate gene targeted animals. This work is supported by Roche Diagnostic GmbH.

76 DEVELOPMENTAL CAPABILITY OF TRANSGENIC NUCLEAR-TRANSFERRED PIG EMBRYOS PRODUCED USING THE NOVEL METHOD OF PSEUDOPHYSIOLOGICAL ACTIVATION

The physicochemical stimuli, which are commonly used for artificial activation of porcine nuclear-transferred (NT) oocytes, can affect detrimentally or cytotoxically the clonal cybrids and thereby inhibit the development or decrease the quality of cloned embryos. Therefore, we have recently developed a novel method of pseudophysiological transcomplementary (transcytoplasmic) activation to stimulate the developmental program of porcine oocytes reconstructed by somatic cell nuclear transfer. The mechanism underlying this original technique of activation is transcytoplasmic influx of sperm-derived proteins triggering intracellular calcium oscillations, which is mediated via heterologous (rabbit) zygote-descended cytoplasts. The purpose of our study was to estimate the in vitro developmental competences of porcine transgenic cloned embryos following pseudophysiological activation of oocytes receiving pWAPhGH-GFPBsd gene construct-nucleofected fetal fibroblast cell nuclei. In the cloning procedure, IVM pig oocytes were used as recipient cells for cell nuclei of positively selected transgenic fibroblast cells. The reconstruction of enucleated oocytes was performed by intracytoplasmic injection of either the somatic cell-derived karyoplast or whole tiny nuclear donor cell. The activation of porcine NT oocytes was achieved by electrofusion of them with the xenogeneic cytoplasts isolated from in vivo-derived rabbit zygotes (i.e. with the so-called zygoplasts), which led to the formation of triple xenocytoplasmic hybrids (xenocybrids). The rabbit zygotes had been flushed postmortem from the separated oviducts of superovulated postpubertal female donors 18 to 20 hafter administration of hCG and copulation. Single rabbit zygote-descended cytoplasts were inserted into the perivitelline space of previously reconstructed pig oocytes. The resulting zygoplast-NT oocyte couplets underwent fusion, which was induced by generation of 2 successive DC pulses of 1.2 kV cm-1 for 60 μs. The electrofusion medium consisted of 0.3 M Ca2+-deprived mannitol supplemented with 0.1 mM MgSO4 and 0.2 mg mL-1 fatty-acid-free BSA. The transcytoplasmically activated xenocybrids were cultured in vitro for 6 to 7 days up to morula/blastocyst stages. A total of 183/207 (88.4%) oocytes reconstructed with nucleofected fibroblast cell nuclei were successfully fused with zygoplasts. Out of 183 cultured NT embryos, 138 (75.4%) were cleaved. The rates of transgenic NT embryos that reached the morula and blastocyst stages yielded 106/183 (57.9%) and 65/183 (35.5%), respectively. In conclusion, the original method of pseudophysiological activation of porcine NT oocytes turned out to be relatively efficient, which has been confirmed by the high percentages of pWAPhGH-GFPBsd transgenic embryos developing in vitro to morula and blastocyst stages.

66 ISOLATION, CHARACTERIZATION, AND NUCLEAR REPROGRAMMING OF STEM CELLS DERIVED FROM ADULT PORCINE LIVER AND FAT

Genetic manipulation of the porcine genome to produce genetically modified pigs for various biomedical and agriculture applications has been hampered by the lack of an ideal cell type that can grow in culture for a long period of time and is amenable to various genetic manipulations with high efficiency. The cell type currently used for various genetic manipulations is fetal fibroblast. These cells have very limited life span in culture and the efficiency of gene targeting is very low. There is no report of isolation of functional embryonic stem cells (ESC) from pig that would have been used to produce transgenic pigs with high efficiency as has been possible in mice. Recently, porcine induced pluripotent stem cells (iPSC) have been reported by 3 groups. However, they have yet to be tested for genetic manipulations and production of transgenic pigs. In this study, we developed a simple but novel strategy to recover stem cells from adult porcine liver and adipose tissues. Small colonies with few cells became visible as early as 2 to 3 days under reduced oxygen conditions on collagen-coated plates, and a full-grown colony with a fibroblast-like morphology took 10 to 14 days to form. Ten single colonies per tissue were isolated, subcultured, and monitored for growth and gene expression. Both of these cell types maintained steady growth through 70 population doublings (at the time of writing) and are still growing without any change in their morphology. Reverse transcription PCR was used to monitor gene expression. Both cell types show strong expression of c-Myc and KLF4. Moreover, low expression of Oct-4 and Lin28, 2 important genes related to pluripotency, was also detected in cells derived from adipose tissues, indicating that these cells could be easily reprogrammed to pluripotent state. These cells maintained a normal karyotype after long-term culture. Cell lines with stable genetic modifications and extended expression of transgene were obtained when these cells were transfected with a plasmid containing the neomycin resistance gene and selected under G418. Further, these 2 cell types, liver stem cells (LSC) and fat stem cells (FSC), and fetal fibroblasts (FF) as a control were used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos. The average fusion rates were 87, 81, and 89% for LSC, FSC, and FF, respectively. Of 2 recipients receiving nuclear transfer embryos produced with each cell type, one established pregnancy at Day 30 (50%). Efficiencies were 5% (11 fetuses/223 embryos transferred), 1.8% (4 fetuses/228 embryos transferred),and 5% (11 fetuses/219 embryos transferred) for LSC, FSC, and FF, respectively. Thus, these adult liver and fat stem cells are attractive cell types for cloning valuable adult animals with high efficiency and for SCNT transgenesis.

399 ISOLATION AND CHARACTERIZATION OF BOVINE MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE

Stem cells have been widely used because of their multi-differentiation ability. Compared with embryonic stem cells, mesenchymal stem cells (MSC) are more easily sourced and cultured, besides being easily obtained from adult individuals. In this regard, bovine MSC is of great interest because of its wide application in basic and applied research, e.g. in somatic cell nuclear transfer (SCNT). Success rates of SCNT are expected to be enhanced with the use of MSC as donor cells because they exhibit a more undifferentiated condition when compared with the most widely used cell type, fibroblast. There are few reports on MSC referring to this species. Therefore, our aim was to isolate and culture adipose MSC from cattle. Mesenchymal stem cells were induced to differentiate into adipocytes and osteocytes to prove their multi-differentiation ability. A small piece of adipose tissue was sourced from the base of the tail of a cow and extensively washed in cold saline solution containing 5% antibiotic. Recovered tissue was minced and digested in 0.001% collagenase 1 and incubated for 3 h. Collagenase was inactivated in alpha minimal essential medium (MEM) supplemented with 15% FCS. The treated tissue was centrifuged and the pellet plated on plastic dishes in alpha MEM supplemented with 15% FCS and 1% antibiotic. Culture medium was replaced every 2 days, and cells were plated on new dishes before reaching 75% confluence. To test their resistance to grow after thawing, cells were frozen after each passage, thawed, and cultured. Cell growth was accompanied through several weeks, and cells were tested for differentiation ability after 3 passages, when adipocyte and osteocyte differentiation was accomplished using a standard protocol. Briefly, adipocyte differentiation was inducted in DMEM containing 2% FCS, isobutyl-methylxanthine, dexamethasone, insulin, and indomethacin. Osteocyte differentiation was carried out in alpha MEM without FCS containing ascorbate-2-phosphate, dexamethasone, and beta-glycerophosphate.To confirm differentiation, a tissue-specific staining was carried out using Oil Red for adipocyte staining and Alizarin Red for osteocyte staining. The cells adhered to plastic dishes shortly after plating, presented fibroblast-like morphology, and showed an exponential growth curve during the first 6 passages. The cells were capable of growing after thawing similarly to unthawed cells. After 1 week under differentiation protocols, standard morphological changes were observed in the cells. Mesenchymal stem cells subjected to differentiation into adipocytes showed an increase in their size, developed lipid-like vesicles, and stained positive for Oil Red. An increase in the cell size was also observed in the cells subjected to differentiation into osteocyte. Moreover, these cells stained positive for Alizarin Red. Altogether these results provide evidence that these cells are multi-potent MSC. Therefore, we conclude that the current methodology was efficient in isolating bovine MSC from adipose tissue, and the cells resemble those isolated from other mammals. Bovine MSC constitute a significant source of nuclear donor cells for future experiments in SCNT. Financial support was provided by FAPESPA and Cnpq.

71 DEVELOPMENT OF CLONED DOG EMBRYOS RECONSTRUCTED WITH PRE-ACTIVATED IN VIVO OOCYTES AND DONOR FIBROBLASTS ARRESTED AT G2/M PHASE USING ROSCOVITINE

The coordination between the cell cycle stages of nuclear donor cells and host oocytes has a critical effect on the development of embryos produced by somatic cell nuclear transfer (SCNT). Here, we investigated (1) whether roscovitine, an inhibitor of cyclin-dependent kinases (CDK) could arrest canine somatic cells at S/G2 phase of the cell cycle; (2) whether IVM metaphase II (MII) oocyte could be induced to telophase II (TII) after activation. Last, we investigated embryo development ability of nonactivated oocytes (MII) or activated oocytes (TII) fused with somatic cells at different stages of the cell cycle. Dog fetal fibroblasts were treated with roscovitine (30 or 60 μg mL-1 at 24, 48, or 72 h) and a control group of donor cells was cultured to reach confluency. The cells were then fixed and stained with 1 mg mL-1 propidium iodide for flow cytometric analysis. For SCNT, IVM dog oocytes were obtained by flushing (approximately 72 h after ovulation) from the oviducts of oocyte donor dog (Canis familiaris) and divided into 2 groups; nonactivated oocytes (MII) and activated oocytes (TII) by 10 μg mL-1 calcium ionophore for 4 min. Following preparation of each donor cell arrested in G0 and G2/M phase, cells of G0 stage were placed into enucleated MII oocytes (MII-G0) and cells of G2/M-phase were placed into enucleated TII oocytes (TII-G2/M). After fusion by electric stimulation, the MII-G0 group was chemically activated and cultured in modified SOF medium (mSOF), and the TII-G2/M group was cultured in mSOF without activation. The embryo developmental competence was estimated by assessing in vitro development under the microscope. Data were analyzed using a statistical analysis system program. Based on flow cytometry, the frequency of cells arrested at G2/M-phase in the 30 and 60 μg mL-1 roscovitine groups was significantly higher than that in control (31.95 and 25.99% v. 19.79%, respectively), but differences were not observed between the 30 and 60 μg mL-1 roscovitine groups (P > 0.05). Also, a significant increase in the proportion of cells at G2/M-phase was observed at 48 and 72 h in both roscovitine groups compared with the group not treated with roscovitine. The proportion of cells at G2/M-phase in the 60 μg mL-1 group at 48 h and the 30 μg mL-1 group at 72 h was the highest among all treatments. For the TII-G2/M group, we injected into enucleated TII oocyte and selected a large cell that arrested at G2/M-phase in cells cultured with 60 μg mL-1 roscovitine for 48 h. For the result of in vitro development of cloned embryo from MII-G0 and TII-G2/M, TII-G2/M group (39.4 and 7.8%) showed an increased cleavage rate and development to 8 cells compared with MII-G0 (23.5 and 2.9%). In the present study, we demonstrated that, in combination with nuclear donor cells at specific cell cycle stages, MII and TII dog oocytes are similarly effective in supporting the reprogramming of somatic cell nuclei. This study was supported by Korean MEST through KOSEF (grant # M10625030005-09N250300510) and BK21 program, RNL BIO, and Natural Balance Korea.

Fates of Donor and Recipient Mitochondrial DNA during Generation of Interspecies SCNT-Derived Human ES-Like Cells

To investigate nuclear donor and cytoplast recipient mitochondria fate and their effects on generation of interspecies somatic cell nuclear transfer (iSCNT)-derived human embryonic stem (ES)-like cells, iSCNT embryos were reconstructed between enucleated goat oocytes and human neural stem cells (hNSCs). A total of 10.74% cleaved embryos (13/121) developed to blastocyst stage. One typical primary ES-like (tpES-like) colony and two nontypical primary ES-like (non-tpES-like) colonies designated as non-tpES-like cell-1 and non-tpES-like cell-2, respectively, were obtained from the inner cell masses of iSCNT blastocysts. The tpES-like cells expressed ESC markers. Both human and goat mtDNA could be detected in the embryos at 2-8-, 16-32-cell, and blastocyst stages, and in tpES-like colony and two non-tpES-like colonies. Human mtDNA copies per cell from embryos at two- to eight-cell stage to the three colonies maintain almost its original level, whereas 2.88 x 10(5) goat mtDNA copies per oocyte decreased to 10.8 copies per tpES-like cell, 493 copies per non-tpES-like cell-1, and 77.6 copies per non-tpES-like cell-2, resulting in 43.75% (8.4/19.2), 1.24% (6.2/499), and 14.63% (13.3/90.9) mtDNA content in tpES-like cell, non-tpES-like cell-1, and non-tpES-like cell-2 was that of nuclear donor, respectively. Human-specific Tfam and Polg mRNA could be detected in cells of the three colonies. However, tpES-like colony failed to be passaged. The mRNA level of CoxIV encoded by nuclear donor in tpES-like cell was higher than that in non-tpES-like cell, but significantly lower than that of human ESC, suggesting proper nuclear-cytoplasmic communication would not be established in tpES-like cells. Thus, the data suggest that (1) goat oocytes could reprogram human neural stem cells (hNSCs) into embryonic state and further support the inner cell mass (ICM) of iSCNT blastocyst to form tpES-like colony; (2) nuclear donor mtDNA could be replicated and maintain its original level during the reduction of recipient mitochondrial DNA copies, (3) nuclear-cytoplasmic communication and recipient mtDNA copies might affect the derivation of iSCNT-derived ES-like cells.

Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Close Relatedness between Exotic Nucleus and Cytoplast Can Improve the Postimplantation Development Rate of Cloned Intersubspecies Embryos

To improve intersubspecies cloning efficiency, this paper provides six kinds of SCNT embryos with different nuclear-cytoplasmic relatedness to compare the relatedness on the cloning efficiency. Three kinds of SCNT embryos with different relatedness are produced by using Boer goat fibroblast cells as nuclear donors and oocytes of Sannen goat, crossbred F1 (Sannen goat x Boer goat) and Boer goat as cytoplast recipients. Four kinds of SCNT embryos with different relatedness are produced by using Sannen goat oocytes as recipients and fibroblast cells of Boer goat, crossbred F2 (crossbred F1 x Boer goat), crossbred F1, and Sannen goat as nuclear donors. Results show that no obvious differences were observed for preimplantation development of these SCNT embryos. However, different nuclear-cytoplasmic relatedness resulted in obvious differences for postimplantation development of these SCNT embryos. The relatedness is complementary: improving either cytoplasmic compatibility relatedness to nucleus or nuclear relatedness to cytoplast could reduce the gestation loss rate, and increase the birth rate of cloned intersubspecies embryos significantly. But a further amelioration of the relatedness did not improve the postimplantation development in direct proportion. These results suggested that close nuclear-cytoplasmic relatedness can improve the postimplantation development rate of cloned intersubspecies embryos.

Efficient Genetic Reprogramming of Unmodified Somatic Neural Progenitors Uncovers the Essential Requirement of Oct4 and Klf4

Significant breakthroughs have been recently achieved in reprogramming somatic cells to a pluripotent embryonic state by the ectopic expression of specific transcription factors. One of the major drawbacks of reprogramming strategies lays in the low efficiency of the process. It is likely that the required complex epigenetic-remodeling events could be cell-type specific and more rational approaches to cell source selection might help to improve the outcome of the procedure. Because the use of somatic stem cells, and specifically neural stem cells (NSCs), as nuclear donors significantly increased the efficiency of somatic cell nuclear transfer, we aimed to determine whether genetically unmodified somatic NSCs could be more easily reprogrammed to pluripotency than unmodified mouse embryonic fibroblasts. Retroviral transduction of the factors Oct4, Sox2, Klf4, and c-Myc successfully reverted NSCs to a pluripotent embryonic stem cell-like state with a 2-fold efficiency increase, faster kinetic, and with a lower number of viral integrations. Quantification analysis of reprogramming-associated genes revealed that NSCs endogenously expressed high levels of Sox2 and c-Myc. Accordingly, NSCs could be successfully induced to pluripotency through the ectopic viral expression of the other two factors (Oct4 and Klf4). These findings suggest that endogenous expression of reprogramming genes could help the reprogramming process and somatic stem cells might be more prone to reprogramming due to their specific genetic background. Genetic-based somatic cell screening might provide essential information for the selection of alternative cell sources more suitable to direct reprogramming.

Epigenetic Reprogramming by Somatic Cell Nuclear Transfer in Primates

We recently demonstrated that somatic cells from adult primates could be reprogrammed into a pluripotent state by somatic cell nuclear transfer. However, the low efficiency with donor cells from one monkey necessitated the need for large oocyte numbers. Here, we demonstrate nearly threefold higher blastocyst development and embryonic stem (ES) cell derivation rates with different nuclear donor cells. Two ES cell lines were isolated using adult female rhesus macaque skin fibroblasts as nuclear donors and oocytes retrieved from one female, following a single controlled ovarian stimulation. In addition to routine pluripotency tests involving in vitro and in vivo differentiation into various somatic cell types, primate ES cells derived from reprogrammed somatic cells were also capable of contributing to cells expressing markers of germ cells. Moreover, imprinted gene expression, methylation, telomere length, and X-inactivation analyses were consistent with accurate and extensive epigenetic reprogramming of somatic cells by oocyte-specific factors.

Production of human lysozyme-transgenic cloned porcine embryos by somatic nuclear transfer

Due to their physiology and organ size, pigs have significant potential as human disease models and as organ transplantation donors. Genetic modification of pigs could provide benefits for both agriculture and human medicine. In this study, five fetal pig fibroblast cell lines from two species (Wuzhishan and Landrace pigs) were transfected using double-marked human lysozyme (HLY) plasmids (pBC1-HLY-GFP-NEO) by a liposome-mediated method. The ratio of green fluorescent protein (GFP)-expressing cells was >95% in sw7, sw8, slw3 and slw6 cell lines, but only 49.3% in slw9 cells. Cells from the four highly transgenic lines were used as nuclear donors to construct embryos, which were then cultured after fusion and activation by electric stimulation. The rate of cleavage was 76.7%, 48 h after activation. After 7 days, 18.5% of cleaved eggs had developed to the blastocyst stage and 93.3% of blastocysts were GFP-positive. These results indicate that transgenic fetal pig fibroblast cell lines could be obtained by a liposome-mediated method, though the transfection efficiency varied between cell lines. Reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive.

Resurrection of a Bull by Cloning from Organs Frozen without Cryoprotectant in a −80°C Freezer for a Decade

Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a −80°C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.

Les applications des animaux génétiquement modifiés (AGM)

The first transgenic animals, mice, were obtained in 1980. The techniques of gene transfer had to be adapted to obtain transgenic animals with an acceptable yield in about fifteen species. When the yield is low (low rate of random integration and targeted integration via homologous recombination), genetic modifications must be achieved in intermediate cells able to participate to the development of chimeric transgenic animals (ES cells, EG cells, iPS obtained by the dedifferentiation of somatic cells) or in somatic cells used as nuclear donor to generate transgenic clones. Various tools make possible a marked increase of homologous recombination efficiency (meganucleases and ZFN), or a gene inactivation at the genome level (direct or conditional knock out) or at the mRNA level (interfering RNAs). Vectors allow a more reliable transgene expression. Genetically modified animals are used mainly to obtain information on biological functions and human diseases. Transgenic animals produce recombinant pharmaceutical proteins in milk and soon in egg white. Pig organs adapted to be tolerated by patients might be tested in humans in five years. The projects based on the use of transgenesis to improve animal production are presently few. Transgenic salmon with accelerated growth might be on the market when their possible escape in oceans will be controlled.

Porcine mesenchymal stem cells - Current technological status and future perspective

Similarities of porcine mesenchymal stem/progenitor cells (MSCs) with human counterpart allow them to be considered as a valuable model system for in vitro studies and preclinical assessments. Effective isolation and expansion of porcine MSCs from different origins, namely bone marrow, umbilical cord Wharton's jelly, amniotic fluid, umbilical cord blood and peripheral blood has been reported. The differentiation of porcine MSCs into mesenchymal lineages under in vitro conditions is consistent and growing evidence has also suggested their transdifferentiation abilities. Results of preclinical studies unveil a time dependent retention, engraftment, migration, ex vivo and in vivo differentiation characteristics and possibility for genetic modification of MSCs. Findings on immunogenicity and the immunomodulatory capacity of porcine MSCs are encouraging and valuable to understand the host compatibility following transplantation. Furthermore, suitability of porcine MSCs as donors in nuclear transfer offers a greater potential to medicine and biopharming. Here, we highlight recent findings in the areas of porcine MSC sources, differentiation ability, transplantation applications and their potential as nuclear donors for somatic cell nuclear transfer.

Porcine nuclear transfer using somatic donor cells altered to express male germ cell function

Recent studies reported that the direct transformation of one differentiated somatic cell type into another is possible. In the present study, we were able to modulate the cell fate of somatic cells to take on male germ cell function by introducing cell extracts derived from porcine testis tissue. Fibroblasts were treated with streptolysin O, which reversibly permeabilises the plasma membrane, and incubated with testis extracts. Our results showed that the testis extracts (TE) could activate expression of male germ cell-specific genes, implying that TE can provide regulatory components required for altering the cell fate of fibroblasts. Male germ cell function was sustained for more than 10 days after the introduction of TE. In addition, a single TE-treated cell was injected directly into the cytoplasm of in vitro-matured porcine oocytes. The rate of blastocyst formation was significantly higher in the TE-treated nuclear donor cell group than in the control cell group. The expression level of Nanog, Sox9 and Eomes was drastically increased when altered cells were used as donor nuclei. Our results suggest that TE can be used to alter the cell fate of fibroblasts to express male germ cell function and improve the developmental efficiency of the nuclear transfer porcine embryos.

Production of Cloned Pigs by Nuclear Transfer of Preadipocytes Following Cell Cycle Synchronization by Differentiation Induction

Four methods of cell cycle synchronization of porcine preadipocytes for use as nuclear donors in somatic cell cloning were compared: serum starvation, differentiation induction, contact inhibition and roscovitine treatment. After three days of differentiation induction, the percentage of nuclear donor cells synchronized at the G0/G1 phase reached a peak value of 91.8%, which was significantly higher (P<0.05) than the percentage attained by serum starvation (84.9-89.8%), contact inhibition (78.3-83.7%) or roscovitine treatment (67.8-80.3%). Cell cycle synchronization by serum starvation, contact inhibition and roscovitine treatment all increased the percentage of apoptotic cells, while no increase was observed when the donor-cell cycle was synchronized by differentiation induction (Annexin V-positive: 15.7% to 19.3% vs. 7.7%, P<0.05; TUNEL-positive: 12.8% to 14.0% vs. 8.3%, P<0.05). Additionally, comparison of the in vitro development of nuclear transfer (NT) embryos formed from the nuclei of differentiation-induced or serum-starved preadipocytes revealed that, in both cases, a high proportion of embryos developed to the blastocyst stage (39.0 and 33.7%, respectively). In this study, NT embryos reconstructed with preadipocytes synchronized by differentiation induction were transferred to four recipient pigs, three of which gave birth to a total of 17 piglets (4.2%, 17/403). These results demonstrate that donor-cell cycle synchronization by differentiation induction enables effective production of cloned pigs. The findings also indicate that differentiation induction of multipotent cells is an excellent method of cell cycle synchronization that permits highly efficient synchronization of cells at the G0/G1 phase.

Advances in the development of a noninvasive embryo model for the evaluation of the quality of cloned embryos subjected to different treatments

Total number of cells in cloned embryos is generally lower than that of in vivo derived embryos and in bovines cell allocation at the blastocyst stage, has been observed to be affected in a large proportion of cloned embryos. The current embryo staining procedures are toxic for mammalian cells and thus can not be used to determine the developmental potential of a stained embryo. Therefore, in the present study we sought to assess the feasibility to develop a noninvasive embryo model that would be suitable for the evaluation of cloned embryos subjected to different nuclear transfer and embryo culture procedures. For doing this, we stably transfected a bovine embryonic fibroblast cell line and generated a number of clones that constitutively expressed a red fluorescent protein (HcRed) in the nuclear compartment of the cell. Those clones with normal chromosomal content were further used as nuclear donor in nuclear transfer procedures (SCNT) to generate transgenic cloned embryos. These embryos expressed the red fluorescent protein in each blastomere, allowing their in vivo evaluation during development, thus demonstrating the potential of this model as a noninvasive tool for the assessment of the quality of cloned embryos.

55 PRELIMINARY STUDIES ON THE DEVELOPMENT OF RECONSTRUCTED EMBRYOS AFTER NUCLEAR TRANSFER IN DROMEDARY CAMEL (CAMELUS DROMEDARIUS)

Experiments were conducted to study the in vitro development of reconstructed dromedary camel embryos after nuclear transfer by a modified zona-free method. Cumulus oocyte complexes, collected from slaughterhouse ovaries were cultured in TCM199 at 38.5°C in an atmosphere of 5% CO2 in air for 32 to 36 h. Matured oocytes were denuded of cumulus cells by repeated pipetting and the zona pellucida was removed by brief incubation in 5 mg mL–1 pronase dissolved in Ca- and Mg-free PBS. Zona-free oocytes were stained with 5 mg mL–1 Hoechst 33342 in H199 supplemented with 7.5 μg mL–1 cytochalasin B and 10% FCS. They were enucleated under constant UV-light exposure in H199 supplemented with cytochalasin B and 10% FCS. The granulosa cells at passage numbers 4 to 15 were used as nuclear donors. The zona-free cytoplasts were individually washed for a few seconds in 300 μg mL–1 of Phytohemagglutinin in H199, then quickly dropped on a single donor cell settled to the bottom of a drop of H199 with 0.5% FCS and pushed together with the mouth pipette. Couplets were electrically fused, at room temperature, with two DC pulses of 100 V cm–1 for 15 μs. Reconstructs were activated 2 h post-fusion, with 5 μm ionomycin for 3 min followed by culture in 6-diethylaminopurine for 4 h. The reconstructs were then cultured individually in either 5 μL drops under oil, in agar wells or in wells of wells (WOW) in a well of 4-well culture plate. Embryo culture medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 50 μg mL–1 gentamycine, 1% insulin-transferrin-selenium (ITS), and 15% estrous dromedary serum. The number of oocytes that had cleaved was recorded on day 2, whilst those developing to morulae and blastocysts were recorded on day 7 of culture. For cell count, the blastocysts were stained with Hoechst and cells counted under a fluorescent microscope at ×400. Data obtained was analysed by chi-square test. About 92% (349/380) of the oocytes were successfully enucleated and 76% (259/340) fused with the attached cells. The cleavage rate was significantly lower (P &lt; 0.05) in reconstructed embryos cultured in droplets (10/72, 14%) as compared with those cultured in agar wells (37/87, 42%) or WOW system (42/96, 44%). The proportions of cleaved embryos reaching morula stage were 0, 83, and 89% in droplets, agar wells, and WOW, respectively. However, only 8% and 5% of the cleaved embryos developed to the blastocyst stage in the agar well and WOW culture systems, respectively. No difference was observed in the cell number of blastocysts produced in agar wells (77.3 ± 8.02) or WOW (78.0 ± 4.2) culture system. To the best of our knowledge, this is the first report of embryo production up to the blastocyst stage after NT in camelids and it shows that NT can be successfully applied for embryo production in camelids. Further studies are needed to optimize the parameters and to improve the efficiency for production of transferable blastocysts in this species. This study was kindly sponsored by H.H. General Sheikh Mohammed bin Rashid Al Maktoum, Ruler of Dubai.

41 PRODUCTION OF CLONED MINIATURE PIGS USING BONE MARROW MESENCHYMAL STEM CELLS

The cloning of miniature pigs (Mini-pig) are considered to advance in genetic engineering technology and xenotransplantation. A few researches have recently been reported successfully produce cloned Mini-pigs using somatic cells, however its efficiency is still low. The present study was aimed to successfully produce cloned Mini-pigs derived from bone marrow mesenchymal stem cells (MSCs) by NT, and compared the developmental ability of cloned Mini-pigs between fetal fibroblast (FF) and differentiated MSCs. For the production of the cloned Mini-pig derived from MSCs, MSCs were isolated from a 1 month old of female Mini-pigs (T-type, PWG Micro-pig®, PWG Genetics Korea, Ltd.). MSCs were differentiated into osteocytes, adipocytes, and chondrocytes under controlled conditions and characterized by cell surface antigen profile using specific markers. These differentiated or undifferentiated MSCs, FFs of Mini-pig were transferred into enucleated oocytes of domestic pigs, and 2-cell stage of 100 NT embryos were surgical transferred to the synchronized recipients. Statistical analysis was performed using one-way ANOVA, t-test, Duncan’s and Tukey’s multiple comparisons test by SPSS (SPSS, Inc., Chicago, IL, USA). The developmental potential of NT embryos derived from MSCs (differentiated and undifferentiated), the rates of blastocyst formation was significantly (P &lt; 0.05) higher than NT embryos derived from FFs, however the NT embryos derived from three different types of differentiated MSCs were significantly (P &lt; 0.05) lower than undifferentiated MSCs. In addition, total cell numbers in NT blastocysts derived from MSCs were significantly (P &lt; 0.05) higher than NT blastocysts derived from FFs, but it did not significantly (P &lt; 0.05) differ between differentiated or undifferentiated MSCs. NT embryos derived from MSCs were transferred to 5 domestic pig recipients, and 5 cloned Mini-pigs were obtained from 2 recipients (one stillbirth and 4 viable offspring). All of them were confirmed by the microsatellite analysis (8 markers) of the genomes of cloned offspring, donor MSCs and recipients. Physical and histological studies are in the process for the characterization of a cloned Mini-pig derived from MSCs as animal model. The results demonstrated that, in vitro developmental ability of NT embryos derived from undifferentiated MSCs were a higher than those from differentiated MSCs or FFs. Moreover, multipotent MSC might have a potential for the production of viable cloned Mini-pigs. Therefore, MSCs as a nuclear donor might a key to improving the production of cloned Mini-pig as animal model for xenotransplantation. This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.

27 HIGHLY VARIABLE EPIGENOMES IN HEALTHY ADULT BOVINE CLONES

Somatic cell nuclear transfer (SCNT) cloning requires epigenetic reprogramming of a differentiated donor cell nucleus. Incorrect reprogramming of epigenetic marks such as methylation of cytosine (5 mC) is associated with compromised development. Clones that survive into adulthood, in contrast, are assumed to be normal and are expected to be used in global food production. However, the epigenetic status of such healthy adult clones has never been investigated. We used a capillary electrophoresis technique to measure 5 mC content in leukocyte DNA from 38 healthy female clones at 1 to 8 years of age. Fibroblasts from 9 adults were used to generate 2 to 9 clones per donor. Four of the studied clone genotypes were from the Holstein breed, and 5 were from the Simmental breed. Individual 5 mC levels ranged from 4.4 to 6.9% with significant differences in mean 5 mC levels of Holstein and Simmental clones (6.50 ± 0.01% and 5.09 ± 0.02%, P &lt; 0.001). The observed variances in 5 mC between clone genotypes of both breeds were similar (0.0204 and 0.0164), and we computed exact restricted likelihood ratio tests on the basis of linear mixed effects models to test for the presence of variability between genotypes. We found that the estimated variance in 5 mC level within clone genotypes from both breeds (0.104) was higher than the estimated variance between clone genotypes (&lt;10–10). Three Holstein donors were analyzed for the 5 mC level of their genome together with their clones. Two donors exhibited the lowest 5 mC levels of the respective genotypes. The 5 mC level of the third nuclear donor was identical to 1 of its 8 clones, higher than 5 of them, and lower than the 2 remaining ones. This clearly demonstrates that the functional reprogramming of a given donor genotype is compatible with a highly flexible methylation status of its DNA and that genomic copies of adult animals have to be considered as epigenome variants. To quantify the contribution of SCNT to the observed variability between clones, we compared Simmental clones with female Simmental monozygotic twins of similar age that were generated by bisection of fertilized embryos. The estimated variability of 5 mC levels within 5 genotypes of clones (0.0636, n = 19) was clearly higher than in 12 twin pairs (0.0091). In clones, the estimated variability within genotypes (0.0636) was thus higher than between (&lt;10–10) genotypes. In contrast, the variability within twin genotypes (0.0091) was lower than between twin genotypes (0.0136). Absolute deviations of 5 mC values of SCNT clones from their genotype means were 5-fold increased as compared with twins, whereas deviations of genotype means from group means were similar for clones and twins. The comparison with twins confirmed a SCNT cloning effect illustrated by a clone-specific variability of DNA methylation and provided new evidence of DNA hypermethylation by SCNT. Our data show that SCNT cloning can no longer be considered (epi)genetically neutral in apparently healthy adult animals. This raises new scientific and regulatory questions about the risks of disseminating cloned (epi)genetics into animal populations. Supported by EU (SABRE), DFG, INRA, and MRES.