Donors For Nuclear Transfer Research Articles

P-405 Nuclear transfer overcomes poor embryo development of in vitro grown mouse oocytes

Abstract Study question Is the nuclear transfer technology able to overcome the compromised embryonic development of in vitro cultured secondary follicles from young B6D2 mice? Summary answer Nuclear transfer technology was able to restore embryonic development to levels similar of in vivo grown oocytes. What is known already In vitro follicle culture has been proposed as a strategy for fertility preservation in cancer patients. Studies in human follicle culture remain scarce, due to the low availability of human tissue. Mouse models have been extensively studied to improve follicle maturation and investigate the potential of in vitro grown (IVG) gametes. Despite significant improvements reported over the years, including increased maturation rates, advanced embryonic development and generation of fertile offspring, the quality of IVG oocytes remains inferior compared to their in vivo grown counterparts. Study design, size, duration The experimental study was conducted between October 2020 and January 2023, after approval by the Animal Ethics Committee of Ghent University Hospital (ECD no 19/60). In total, 108x 16-days-old B6D2 females were used for secondary follicle isolation and in vitro culture, 81x 8-12-week-old females for collection of in vivo grown oocytes, 4x 9-14-week-old B6D2 males for sperm freezing, 5x 10-24-week-old CD1 vasectomised males and 10x 8-12-weeks-old CD1 females as surrogate mothers for embryo transfer. Participants/materials, setting, methods Follicles isolated from 16-days-old B6D2 mice were cultured for 9 days, followed by maturation for 16-18hrs. Mature oocytes were assessed on diameter, spindle morphology, calcium releasing ability, mitochondrial membrane and embryonic developmental potential. In vivo grown oocytes from stimulated mice were used as controls and cytoplasmic donors for nuclear transfer. Spindle (ST) and Pronuclear transfer (PNT) were applied to overcome poor development of IVG embryos, by transferring the spindle/pronuclei of IVG oocytes/zygotes to enucleated controls. Main results and the role of chance In total, 1509 secondary follicles were cultured and 73.6% survived to Day 9. From the 719 cumulus oocyte complexes, 364 (50.6%) oocytes matured. Oocyte diameters were significantly smaller between IVG (67.4μm) and controls (73.1μm) (p < 0.001). Spindle staining revealed a similar number of normal spindles between the two groups (71.4% and 82.6% respectively, p = 0.138). Calcium release was significantly lower in IVG oocytes (1.6) vs controls (5.7) (p < 0.001), as well as mitochondrial membrane potential ((0.9) vs (2.2), p < 0.001). These data implicate a cytoplasmic inferiority in IVG oocytes. Following parthenogenetic activation, 2-cell (59.4%) and blastocyst rate (36.3%) in IVG group was significantly lower than control (89.4% and 88.2% respectively, p < 0.001). For this reason, we performed ST, using controls as cytoplasmic recipients for IVG spindles. The 2-cell rate significantly increased to 100% (p < 0.001) in ST oocytes, reaching 100% blastocyst formation. Next, to create bi-paternal embryos, we performed IVF. From the IVG group, 2-cell (40%) and blastocyst (0%) rates were significantly lower than control (94.4% and 83.3%, p < 0.001). Embryo development was restored following PNT (98% 2-cell and 86% blastocyst rate, p < 0.001). Genetic analysis of PNT embryos revealed that 5/6 were chromosomally normal, similar to controls (8/10). Embryo transfer of PNT and control embryos is ongoing. Limitations, reasons for caution Both ST and PNT are promising technologies to overcome poor embryonic development of IVG oocytes, but the benefit for human IVG oocytes remains to be investigated, along with the safety of the technique. Wider implications of the findings Our results demonstrate that ST and PNT have the potential to restore embryonic developmental competence of IVG oocytes, without increasing chromosomal abnormalities. This technology could be investigated in the future, as a mean to overcome poor quality of IVG oocytes in human. Trial registration number Not applicable

Editorial and News

Editorial and News

Effects of Refrigeration at 5°C for Long Periods of Time on Bovine Ear Skin as a Strategy to Transport Biological Material and Isolate Fibroblasts to Use in the Nuclear Transfer.

Animal cloning is an important technique used to produce clones from valuable farm animals, to rescue animals in risk of extinction, and for producing transgenic animals. The objective of this work was to evaluate the effects of refrigeration on bovine ear skin as a strategy to transport biological material for long periods of time to isolate viable fibroblasts. Ears from eight cows were collected after death and stored for 30 days at 5°C. On days 0, 2, 4, 7, 14, 21, and 30, skin biopsies were cultured in vitro for fibroblast isolation. The time for first fibroblast outgrowth, time to reach 100% confluence. and cell concentration before freezing were observed for each period. In addition, plasma membrane integrity, cell apoptosis, and necrosis in cells were evaluated through fluorescent colorant combination in a flow cytometer from all periods after thawing. Fibroblasts obtained after 30 days of storage, considered a critical period, were tested for embryo production using nuclear transfer (NT) with micromanipulators. All time points allowed for cell culture. The time of cell growth onset was longer in samples refrigerated for 14, 21, and 30 days. The time to reach confluence also increased with longer refrigeration periods. Cells from day 0 reached confluence in 24 ± 2 days, while day 30 cells took 31 ± 0 days. Cell concentration and viability dropped with increased storage time and freezing/thawing, respectively. It was found that a long period of sample storage results in cell damage, making cultivation more difficult and decreasing cell viability post-thawing and cell concentration. However, when cells from day 30 were used as nuclei donors in NT, a 26.05% blastocyst rate after 7 days in culture was obtained. In conclusion, refrigeration at 5°C was shown to be efficient in maintaining viable tissue for up to 30 days, and fibroblasts isolated can be used for cloned embryo production.

Codon optimized membrane cofactor protein expression in α 1, 3 galactosyltransferase knockout pig cells improve protection against cytotoxicity of monkey serum.

In this study, we attempted to upgrade GT -MCP/-MCP pig genetically to express MCP at a higher level and additionally thrombomodulin (TBM), which have respective roles as a complement regulatory protein and a coagulation inhibitor. We constructed a dicistronic cassette consisting of codon-optimized MCP (mMCP) and TBM (m-pI2), designed for ubiquitous expression of MCP and endothelium specific expression of TBM. The cassette was confirmed to allow extremely increased MCP expression compared with non-modified MCP, and an endothelial-specific TBM expression. We thus transfected m-pI2 into ear-skin fibroblasts isolated from a GT -MCP/-MCP pig. By twice selection using magnetically activated cell sorting (MACS), and single-cell culture, we were able to obtain clones over 90% expressing MCP. The cells of a clone were provided as a donor for nuclear transfer resulting in the generation of a GT -MCP/-MCP /mMCP/TBM pig, which was confirmed to be carrying cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Collectively, these results demonstrated an effective approach for upgrading transgenic pig, and we assumed that upgraded pig would increase graft survival.

Generation of two-cell cloned embryos from mouse faecal cell

Cloning animals using nuclear transfer (NT) provides the opportunity to preserve endangered species. However, there are risks associated with the collection of donor cells from a body, which may cause accidental death of the animal. Here, we tried to collect faeces-derived cells and examined the usability of those nuclei as a donor for NT. A relatively large number of cells could be collected from GFP-Tg mouse faeces by this method. After NT, only 4.2% of the reconstructed oocytes formed pseudo-pronucleus. This rate increased up to 25% when GFP and Hoechst were used as a marker to select better cells. However, the reconstructed oocytes/embryos showed several abnormalities, such as shrunken nuclear membranes and abnormal distribution of tubulin, and none of them developed beyond one-cell stage embryos. These developmental failures were caused by not only toxic substances derived from faeces but also intrinsic DNA damage of donor cell nuclei. However, when the serial NT was performed, some of the cloned embryos could develop to the two-cell stage. This method may remove toxic substances and enhance DNA repair in the oocyte cytoplasm. Thus, these results indicate that faeces cells might be useful for the conservation of endangered species when technical improvements are achieved.

Porcine somatic cell nuclear transfer donor sources affect transplant success: A meta-analysis

Herd boars, male domestic pigs used for stud, are economically important, and somatic cell nuclear transfer (SCNT) is a promising technology to expand herd boar yields. However, live births are dictated by donor cell source, and fetal donors may offer more advantages than adult donors. A meta-analysis was conducted to better understand how donor sources affect SCNT outcomes. Of the 1,431 records viewed, 10 were selected for review. Blastocyst formation rates, successful pregnancies, and live births were assessed to measure efficacy. SCNT blastocyst formation differed between adult and fetal donors among the studies. SCNT pigs had more malformed fetuses as well, which negatively affected the post-birth mortality. Organs of porcine fetuses are limited by deficiencies of maternal nutrient and growth hormones, which compromise post-birth adaptations. SCNT pregnancy success is neither determined by donor source nor by live births. Live births are also tied to donor age. Embryos from fetal donors are more frequently healthy likely due to less differentiation and less reprogramming of reconstructed embryos. Adult donors in contrast have more cell differentiation and as such accumulate more mutations and damage. This may reduce reconstructed embryo viability. Finally, SCNT efficiency may be improved with more in vitro passages, but more work is required to validate this concept.

Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.

Epigenetic states of donor cells significantly affect the development of somatic cell nuclear transfer (SCNT) embryos in pigs.

The type and pattern of epigenetic modification in donor cells can significantly affect the developmental competency of somatic cell nuclear transfer (SCNT) embryos. Here, we investigated the developmental capacity, gene expression, and epigenetic modifications of SCNT embryos derived from porcine bone marrow-derived mesenchymal stem cells (BMSCs) and fetal fibroblasts (FFs) donor cells compared to embryos obtained from in vitro fertilization (IVF). Compared to FFs, the donor BMSCs had more active epigenetic markers (Histone H3 modifications: H3K9Ac, H3K4me3, and H3K4me2) and fewer repressive epigenetic markers (H3K9me3, H3K9me2, and DNA methyltransferase 1). Embryos derived from BMSC nuclear-transfer (BMSC-NT embryos) and IVF embryos had significantly higher cleavage and blastocyst rates (BMSC-NT: 71.3 ± 3.4%, 29.1 ± 2.3%; IVF: 69.2 ± 2.2%, 30.2 ± 3.3%; respectively) than FF-NT embryos (58.1 ± 3.4%, 15.1 ± 1.5%, respectively). Bisulfite sequencing revealed that DNA methylation at the promoter regions of NANOG and POU5F1 was lower in BMSC-NT embryos (30.0%, 9.8%, respectively) than those in FF-NT embryos (34.2%, 28.0%, respectively). We also found that BMSC-NT embryos had more H3K9Ac and less H3K9me3 and 5-methylcytosine than FF-NT embryos. In conclusion, our finding comparing BMSCs versus FFs as donors for nuclear transfer revealed that differences in the initial epigenetic state of donor cells have a remarkable effect on overall nuclear reprogramming of SCNT embryos, wherein donor cells possessing a more open chromatin state are more conducive to nuclear reprogramming.

Combined positive effect of oocyte extracts and brilliant cresyl blue stained recipient cytoplasts on epigenetic reprogramming and gene expression in buffalo nuclear transfer embryos.

This study examined the effects of buffalo oocyte extracts (BOE) on donor cells reprogramming and molecular characterisation of oocytes screened via brilliant cresyl blue (BCB) staining and comparison of gene expression profiles of developmentally important genes in blastocysts from IVF and cloned derived from BOE treated donor cells with BCB selected recipient cytoplasts. Relative abundance (RA) of OCT4 and NANOG was increased (P<0.05) and HDAC-1, DNMT-1, and DNMT-3A decreased (P<0.05) in extract treated cells (ETCs). This ETCs dedifferentiated into neuron-like lineage under appropriate induction condition. The RA of NASP, EEF1A1, DNMT1, ODC1 and RPS27A was increased (P<0.05) in BCB+ oocytes, whereas ATP5A1 and S100A10 increased (P<0.05) in BCB- oocytes. Total cell number and RA of OCT4, NANOG, SOX2, DNMT1, IGF2, IGF2R, MNSOD, GLUT1, BAX and BCL2 in cloned blastocysts derived from BCB+ oocytes with ETC more closely followed that of IVF counterparts compared to BCB+ oocytes with extract untreated cell and BCB- oocytes with ETC derived blastocysts. In conclusion, BOE influenced epigenetic reprogramming of buffalo fibroblasts making them suitable donors for nuclear transfer (NT). BCB staining can be effectively used for selection of developmentally competent oocytes for NT. The combined effects of epigenetic reprogramming of donor nuclei by BOE and higher nuclear reprogramming capacity of BCB+ oocytes improve developmentally important gene expression in cloned blastocysts. Whether these improvements have long-term effects on buffalo calves born following embryo transfer remains unknown.

Isolation of Bovine Skin-Derived Precursor Cells and Their Developmental Potential After Nuclear Transfer.

Nuclei from less differentiated stem cells yield high cloning efficiency. However, pluripotent stem cells are rather difficult to obtain from bovines. Skin-derived precursor (SKPs) cells exhibit a certain degree of pluripotency, which has been shown to enhance the efficiency of nuclear transfer (NT) in pigs. In this study, bovine SKPs were isolated and characterized. Results showed that bovine SKPs expressed nestin, fibronectin, vimentin, pluripotency-related genes, and characteristic neural crest markers, such as NGFR, PAX3, SOX9, SNAI2, and OCT4. Bovine SKPs and fibroblasts were used as NT donor cells to examine and compare the preimplantation developmental potential of reconstructed embryos after somatic cell nuclear transfer (SCNT). Bovine SKP-cloned embryos displayed higher developmental competence in terms of blastocyst formation rate and total cell number in blastocysts compared with the bovine embryonic fibroblast-cloned embryos. This study revealed that bovine SKPs may be considered excellent candidate nuclear donors for SCNT and may provide a promising platform for transgenic cattle generation.

Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency.

Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and reacquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesized that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts versus nonstarved mitotically selected (G1) controls. We show that G0 chromatin contains reduced levels of Polycomb group proteins EED, SUZ12, PHC1, and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts that retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, serum starvation induced epigenetic changes, specifically hypotrimethylation, that provide a mechanistic correlate for increased somatic cell reprogrammability.

Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells.

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38–77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.

42 CONVERSION OF THE CHROMATIN OF SOMATIC CELLS INTO SPERMATID-LIKE STRUCTURES

The post-meiotic phase of spermatogenesis is characterised by a radical reorganization of the chromatin, leading to its nucleosomal to toroid transition. The replacement of histones with protamine is a gradual process regulated to the hierarchical translation of repressed mRNAs leading to the following events: incorporation of testis-specific histone variants and general histone hyperacetylation, bromodomain proteins, transition proteins, concluded by protamine incorporation on DNA. In this work, we tested whether the induced expression of human protamine 1 (PR1) in sheep somatic cells could induce a protamine/toroid conformation of interphase nuclei. Sheep adult fibroblasts (SAF) were cultured in DMEM with 10% fetal bovine serum from second to eighth passage. Then, SAF at 80% confluence were transfected with 3 μg of pPR1-red fluorescent protein (RFP) and pRFP (CTR) with lipofectamine. At 4 h post-transfection, cells were treated with 5 nM trichostatin A (TSA; histone deacethylase inhibitor) for an additional 16 h. Transfected cells (visualised through the RFP tag) were analysed for nuclear morphology (transmission electron microscopy), PR1-RFP expression (confocal microscope, RT-PCR, Western blot), cytofluorimeter, DNA damage (comet assay, pH2A.X immune-detection), and chromatin immune-precipitation assays (ChIP). Moreover, to visualise the histone/protamine exchange, we transfected with PR1 mouse GFP-H2B fibroblasts. Protaminized cells were used as donors for nuclear transfer (NT) and TH2B (testis/oocyte-specific histone normally present in male pronucleus, a marker of reprogramming) was detected in pronuclear stage of NT zygotes. The χ2 test was used for statistical analyses. We demonstrated that PR1 translocates into the nuclei and gradually compacts them into elongating spermatid-like structures in 48 h; TSA treatment facilitates the process [TSA 83.3% (50/60); without TSA 55.2%; 58/105; P &lt; 0.0003]. A complete histone-to-protamine exchange was also visualised in GFP-H2B nuclei (mouse fibroblasts) 40h after PR1 transfection. Cytofluorimetric analysis demonstrated that protamine incorporation occurs in any cell cycle stage and without DNA breaks. Next, protamine-protamine binding was excluded by ChIP analysis, which confirmed protamine-DNA binding. Finally, protaminized nuclei transplanted into enucleated oocytes incorporated maternal histone TH2B (3/8), whereas no signal was detected in control cells (0/9), suggesting that protaminized nuclei are better remodelled. We conclude that the induced expression of PR1 forces the somatic chromatin to acquire a structure overlapping elongated spermatid/spermatozoa, a conformation that perfectly matches the nuclear reprogramming machinery of the oocyte. Further work will determine whether protaminized cells are better reprogrammed upon nuclear transfer.

Development and gene expression of porcine cloned embryos derived from bone marrow stem cells with overexpressing Oct4 and Sox2.

The present study compared the potential of porcine bone marrow mesenchymal stem cells (pBMSCs) at different passages as nuclear transfer (NT) donors and the developmental efficiency of NT embryos from donor cells transfected with/without Oct4 and Sox2. Early-passage pBMSCs showed higher proliferation and expression of Oct4 and Sox2 and differentiation potential into mesenchymal lineages than middle- and late-passage pBMSCs. Cleavage rate did not differ among pBMSCs at different passages, but NT embryos with early-passage pBMSCs and middle-passage pBMSCs transfected with Oct4 (Oct4-pBMSCs) had significantly (p<0.05) higher blastocyst development than those with middle-passage pBMSCs. The incidence of apoptotic bodies in NT blastocysts from late-passage pBMSCs and Sox2-transfected middle-passage pBMSCs (Sox2-pBMSCs) was significantly (p<0.05) higher than others. The transcriptional levels of Oct4, Sox2, Nanog, Cdx2, Dnmt3a, and Igf2r genes were significantly (p<0.05) higher in Oct4- and Sox2-pBMSCs NT embryos. Middle-passage pBMSCs NT embryos revealed lower transcriptional levels of Bcl2 than others, except Sox2-pBMSCs NT embryos. The transcriptional level of Bax increased gradually in NT embryos derived from pBMSCs following extended passages and was significantly (p<0.05) higher in Sox2-pBMSCs NT embryos. Our results demonstrated that early-passage pBMSCs are more potent in expressing transcription factors and displayed higher differentiation ability, and middle-passage pBMSCs transfected with Oct4 improved the developmental efficiency of NT embryos, suggesting that high Oct4 expression cells are more efficient as NT donors.

Production GH transgenic goat improving mammogenesis by somatic cell nuclear transfer

Growth hormone is a positive regulator of mammary gland development. Dairy animals that are administered growth hormone display enhanced lactation performance, a desirable agricultural trait. The objective of the current research was to generate an improved milk production phenotype in a large animal model using over-expressed GH in the mammary gland to promote mammogenesis. To this end, we constructed a mammary gland-specific expression vector, pcGH, and demonstrated effective GH expression in goat mammary epithelial cells in vitro by ELISA. Then, to produce transgenic offspring that were capable of stable GH expression in vivo, the linearized pcGH vector was electroporated into goat fetal fibroblasts. Cell colonies that were positive for GH were used as donors for nuclear transfer to enucleated oocytes. A total of 253 morulae or blastocytes developed from the reconstructed embryos were transferred to 56 recipients, resulting in 24 pregnancies at day 35. Finally, six transgenic goats were born. PCR detection confirmed the success of the cloning procedure. To observe the mammogenesis of dairy goats, the GH transgenic goats were mated with a completely healthy buck. In the later pregnancy period, the mammary gland of the GH transgenic goats were extensive than non-transgenic goats. These experiments indicated that the pcGH vector was incorporated into the transgenic goats and affected mammogenesis, which laid a solid foundation for elucidating the impact of GH on mammogenesis and lactation performance.

Cell cycle synchronization of leukemia inhibitory factor (LIF)-dependent porcine-induced pluripotent stem cells and the generation of cloned embryos

Nuclear transfer (NT) from porcine iPSC to create cloned piglets is unusually inefficient. Here we examined whether such failure might be related to the cell cycle stage of donor nuclei. Porcine iPSC, derived here from the inner cell mass of blastocysts, have a prolonged S phase and are highly sensitive to drugs normally used for synchronization. However, a double-blocking procedure with 0.3 μM aphidicolin for 10 h followed by 20 ng/ml nocodazole for 4 h arrested 94.3% of the cells at G2/M and, after release from the block, provided 70.1% cells in the subsequent G1 phase without causing any significant loss of cell viability or pluripotent phenotype. Nuclei from different cell cycle stages were used as donors for NT to in vitro-matured metaphase II oocytes. G2/M nuclei were more efficient than either G1 and S stage nuclei in undergoing first cleavage and in producing blastocysts, but all groups had a high incidence of chromosomal/nuclear abnormalities at 2 h and 6 h compared with non-synchronized NT controls from fetal fibroblasts. Many G2 embryos extruded a pseudo-second polar body soon after NT and, at blastocyst, tended to be either polyploid or diploid. By contrast, the few G1 blastocysts that developed were usually mosaic or aneuploid. The poor developmental potential of G1 nuclei may relate to lack of a G1/S check point, as the cells become active in DNA synthesis shortly after exit from mitosis. Together, these data provide at least a partial explanation for the almost complete failure to produce cloned piglets from piPSC.

Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells

There are growing numbers of recombinant proteins that have been expressed in milk. Thus one can consider the placement of any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Among the transgene introducing techniques, only nuclear transfer (NT) allows 100% efficiency and bypasses the mosaicism associated with counterpart techniques. In this study, in an attempt to produce a transgenic goat carrying the human coagulation factor IX (hFIX) transgene, goat fetal fibroblasts were electroporated with a linearized marker-free construct in which the transgene was juxtaposed to β-casein promoter designed to secret the recombinant protein in goat milk. Two different lines of transfected cells were used as donors for NT to enucleated oocytes. Two transgenic goats were liveborn. DNA sequencing of the corresponding transgene locus confirmed authenticity of the cloning procedure and the complementary experiments on the whey demonstrated expression of human factor IX in the milk of transgenic goats. In conclusion, our study has provided the groundwork for a prosperous and promising approach for large-scale production and therapeutic application of hFIX expressed in transgenic goats.

Induced Pluripotent Stem–Induced Cells Show Better Constitutive Heterochromatin Remodeling and Developmental Potential After Nuclear Transfer Than Their Parental Cells

Recently, reprogramming of somatic cells from a differentiated to pluripotent state by overexpression of specific external transcription factors has been accomplished. It has been widely speculated that an undifferentiated state may make donor cells more efficient for nuclear transfer. To test this hypothesis, we derived induced pluripotent stem cells (iPS cells) from several somatic cell lines: mouse embryonic fibroblast (MEF), adult tail tip fibroblast (TTF), and brain neural stem cells (NSCs). Three dimensional (3D)-fluorescent in situ hybridization (FISH) and quantitative-FISH (Q-FISH) were then used to evaluate constitutive (pericentric and telomeric) heterochromatin organization in these iPS cells and in their parental differentiated cells. Here, we show that important nuclear remodeling and telomeres rejuvenation occur in these iPS cells regardless of their parental origin. When we used these cells as donors for nuclear transfer, we produced live-born cloned mice at much higher rates with the iPS-induced cells than with the parental cell lines. Interestingly, we noticed that developmental potential after nuclear transfer could be correlated with telomere length of the donor cells. Altogether, our findings suggest that constitutive heterochromatin organization from differentiated somatic cells can be reprogrammed to the pluripotent state by induction of iPS cells, which in turn support nuclear transfer procedure quite efficiently.

Production of transgenic embryos through nuclear transfer using ovine fetal fibroblasts transferred with foreign genes

Human ALR gene sequence was amplified by PCR from human total DNA and inserted into pIRES(2)-EGFP vector. The bicistronic eukaryotic expression vector, pIRES-EGFP/ALR, expressing EGFP, Neo(r) and ALR genes was constructed. Sheep fetal fibroblast cells (sEFCs) were transfected with pIRES-EGFP/ALR by the induction of lipofectAMINE(TM). The positive cell clones were selected with medium containing G418 (800 μg/mL). The fluorescence of transgenic cells was examined with a confocal laser scanning microscope. The expression of ALR gene was tested by PCR, RT-PCR and immuno-histochemical staining. The transgenic cells were used as donors for nuclear transfer to enucleated ovine oocytes. Transgenic embryos were tested by confocal laser scanning microscope and immuno-histochemical staining. Results showed that the EGFP and ALR genes linked with IRES were coexpressed simultaneously in sFFCs; the blastocysts formed by nuclear transfer using tranfected donor cells are all transgenic blastocysts. EGFP, ALR and Neo(r) gene were all expressed in the transgenic embryos. In conclusion that a method to construct the positive embryos before pre-implantation which stably express ALR gene by the indication of EGFP expression has been successfully established. The application of this method can simplify the procedure of testing the targets and contribute to the efficiency increasing of transgenic domestic animal production.

425 HIGH-LEVEL EXPRESSION OF LEA29Y IN PANCREATIC ISLETS OF TRANSGENIC PIGS

Among the candidate organs or tissues for pig-to-primate xenotransplantation, pancreatic islets are probably closest to clinical application. Rejection of islet xenografts occurs mainly by cellular mechanisms; that is, T cells. A candidate molecule to protect porcine islets against the attack by human T cells is CTLA-4Ig, which represents the T-cell-inactivating extracellular domain of the human CTLA-4 protein linked to a region of the human immunoglobulin (Ig). This recombinant soluble fusion protein binds to CD80 and CD86, blocking their interaction with CD28 and thereby inhibiting T-cell proliferation and T-cell-dependent antibody production. The survival of human, rabbit, and porcine islets after transplantation into streptozotocin-treated diabetic mice was found to be prolonged after treatment with CTLA-4Ig. In order to facilitate local protection of pig-to-primate islet xenografts, we generated transgenic pigs expressing LEA29Y, a modification of the original CTLA-4Ig with higher potency, specifically in the pancreatic islets. In LEA29Y, 2 amino acids in the binding region of CTLA-4 are altered. The LEA29Y coding sequence was placed under the control of the 1.3-kb core promoter from the porcine insulin gene (INS), and the polyadenylation signal from the bovine growth hormone gene (GH) was added. The construct was linked with a floxed neomycin resistance cassette and transfected into porcine fetal fibroblasts. The cells were selected and stable clones were pooled and used as donors for nuclear transfer. After electrofusion and activation, embryos were transferred to 2 synchronized gilts; 8 piglets survived to term with 7 of them carrying the transgene. Southern blot analysis suggested that the founder animals contain 1 or 2 independent integration sites. Four founders were autopsied at the age of 3 months to evaluate expression of LEA29Y in the pancreatic islets by immunohistochemistry. The ratio of immunohistochemically stained islet cell profiles to all islet cell profiles in the islet profiles visible in the sections was estimated. The staining intensity was also estimated qualitatively, by grading from weak to strong immunoreactivity (brown color, using DAB as chromogen). Although 2 founders exhibited single LEA29Y-positive islet cells in some pancreatic islet profiles, the other 2 founders showed a high percentage of strongly positive cells in all islet profiles examined, suggesting beta-cell specific expression. Fibroblasts from the latter 2 founders are currently being used for recloning to generate multiple pigs with constitutive expression of LEA29Y in the pancreatic islets. The protective effect of this strategy will be tested by transplanting LEA29Y-expressing porcine islets in diabetic mouse models with a humanized immune system and in diabetic nonhuman primate models. Supported by the Deutsche Forschungsgemeinschaft (FOR 535).