Donor Cell Lines Research Articles

30 IN VITRO EXPRESSION OF BAX AND BCL2 GENES IN NUCLEAR DONOR SKIN FIBROBLAST, CUMULUS, AND GRANULOSA CULTURED CELLS DURING CULTURE AND THEIR SOMATIC CELL NUCLEAR TRANSFER-CLONED EMBRYOS IN INDIAN BUFFALOES (BUBALUS BUBALIS)

Somatic cell nuclear transfer (SCNT) involves functional changes in the genome which result in low efficiency for the production of viable and cloned embryos. It is primarily due to incomplete reprogramming of genome of donor cell nuclei in the reconstructed embryos (Vassena et al. 2007 Dev. Biol. 304, 75–89). Expression of BCL2 and Bax can be correlated with apoptosis. BCL2 inhibits apoptosis by regulating the release of cytochrome-c and other proteins from mitochondria (Keep et al. 2007 EMBO J. 26, 825–834). Antiapoptotic BCL2 is antiproliferative by facilitating G0. Bax is proapoptotic and accelerates S-phase progression. The dual functions in apoptosis and cell cycle are coordinately regulated by the BCL2 family and suggest that survival is maintained at the expense of proliferation (Zinkel et al. 2006 Cell Death Differ. 13, 1351–1359). The aim of this study was to estimate the relative expression of BCL2 oncogene and Bax gene in regulating apoptosis, in skin fibroblast, cumulus, and granulosa cells in culture, so that ideal-type donor cell lines are developed for higher success rates in SCNT-derived buffalo cloning. The cell lines up to 25th passage were from all the 3 tissue types by previous method (Gupta et al. 2007 Cell Biol. Int. 31, 1257–1264). The cells between passages 5th to 15th were selected as competent donor cells and transferred into enucleated in vitro-matured oocytes from slaughter ovaries. The couplets were activated electrically (1.5 kV cm–2, 15 μs) and chemically (ionomycin, 6-DMAP, CHX, and Cyto-B) and were cultured up to blastocyst. The cDNA were prepared from the growing cells in culture at 5, 10, and 15 passages from all cell lines and SCNT-cloned blastocysts from these cell lines at respective passages for Bax and BCL2 gene expression analysis. Relative expression of these candidate genes was quantified using real-time PCR. The data was analyzed for 1-way ANOVA and post-hoc Duncan multiple range test at P ≤ 0.05 level of significance. The cell proliferation rate in cultured cells at fifth passage was higher in all the 3 cell lines and declined in subsequent passages (range from 1.06 to 0.67). The relative abundance of Bax mRNA in granulosa cell was comparable with skin fibroblasts but significanly higher than cumulus cells at respective passages. BCL2 mRNA expression was significantly upregulated in cumulus cells as compared to granulosa cells but not with skin fibroblasts. The SCNT blastocyst production rates from granulosa were highest (24.28%) as compared to fibroblast (22.6%) and cumulus (21.4%) at passage 10. Level of Bax and BCL2 mRNA in granulosa and fibroblast SCNT blastocysts was not significantly different from IVF (control), whereas cumulus-derived blastocyst showed abnormal patterns with downregulated expression of Bax mRNA and upregulated expression of BCl2 mRNA. Identification of expressed genes in cells and cloned embryos will help to investigate the causes of developmental abnormality due to deregulation of expression of important gene associated with ART.

Inhibition of Cell-to-Cell Transmission of Human T-Cell Lymphotropic Virus Type 1 In Vitro by Carbohydrate-Binding Agents

Peripheral blood mononuclear cells (PBMCs) from healthy individuals can be infected by human T-lymphotropic virus type 1 (HTLV-1) upon cocultivation of the PBMCs with irradiated HTLV-1-transformed human MT-2 cells. This model system closely mimics HTLV-1 transmission through cell-to-cell contact. Carbohydrate-binding agents (CBAs) such as the alpha(1,3)/alpha(1,6)mannose-specific Hippeastrum hybrid agglutinin and the GlcNAc-specific Urtica dioica agglutinin, and also the small, nonpeptidic alpha(1,2)-mannose-specific antibiotic pradimicin A, were able to efficiently prevent cell-to-cell HTLV-1 transmission at nontoxic concentrations, as evidenced by the lack of appearance of virus-specific mRNA and of the viral protein Tax in the acceptor cells. Consistently, antivirally active doses of CBAs fully prevented HTLV-1-induced stimulation of PBMC growth. The inhibitory effects of CBAs on HTLV-1 transmission were also evident when HTLV-1-infected C5MJ cells were used in place of MT-2 cells as a virus donor cell line. The anti-HTLV-1 properties of the CBAs highlight the importance of the envelope glycans in events underlying HTLV-1 passage from cell to cell and indicate that CBAs should be further investigated for their potential to prevent HTLV-1 infection, including mother-to-child virus transmission by cell-to-cell contact through breast milk feeding.

Mitochondrial DNA Transmission and Transcription After Somatic Cell Fusion to One or More Cytoplasts

Following fertilization, mitochondrial DNA is inherited from the oocyte and transmitted homoplasmically. However, following nuclear transfer, mitochondrial DNA can be transmitted from both the donor cell and recipient oocyte, resulting in a state of heteroplasmy. To determine whether the genetic diversity between donor cell and recipient cytoplast mitochondrial DNA influences development, we generated bovine embryos by fusing a donor cell to one or more enucleated cytoplasts. Analysis of mitochondrial DNA from embryos, fetal tissues, and blood samples from offspring revealed that early preimplantation embryos from two or three cytoplasts had significantly more mitochondrial DNA variants than fetal tissues. Phylogenic analysis of embryos generated using single cytoplasts divided the mitochondrial DNA sequence variants into three separate groups with various amounts of genetic divergence from the donor cell line. In heteroplasmic tissue and blood samples, the predominant mitochondrial DNA population was significantly more divergent from the donor cell than the less frequent allele. Furthermore, analysis of the mitochondrially encoded cytochrome B gene showed that two heteroplasmic alleles encoded for different amino acids, and the ratios of mitochondrial DNA/mRNA for each allele differed significantly between tissues. The degree of evolutionary distance between the donor cell and the cytoplast and the variability in heteroplasmy between tissues may have an impact on more divergent intergeneric nuclear transfer and the use of this approach for the generation of embryonic stem cells.

Messenger RNA expression patterns of histone‐associated genes in bovine preimplantation embryos derived from different origins

Histone modification genes in bovine embryos: The mRNA expression pattern of histone-related genes was determined in bovine oocytes and embryos. We compared immature and in vitro-matured oocytes, either before or after enucleation and activation, in vitro produced embryos (zygotes, 8-16 cell stages, blastocysts), embryos cloned with female or male donor cells; parthenogenetic embryos, and in vivo-derived blastocysts to detect deviations from the normal expression pattern. A sensitive semi-quantitative endpoint RT-PCR assay was used to reveal differences in histone deacetylation [histone deacetylase 2 (HDAC2)]; histone acetylation [histone acetyltransferase 1 (HAT1)]; histone methylation [histone methyltransferases (SUV39H1, G9A)]; heterochromatin formation [heterochromatin protein 1 (HP1)]; and chromatin-mediated transcription regulation [zygote arrest 1 (ZAR1)]. With the exception of ZAR1, these mRNAs were present throughout preimplantation development. The relative abundance of mRNAs for histone methyltransferases (SUV39H1 and G9A) and for heterochromatin-associated protein (HP1) differed significantly before and after activation of the bovine embryonic genome. The similarity of HAT1 gene expression in 8-16 cell embryos and blastocysts suggests that histone acetylation is primarily affected by in vitro culture only prior to embryonic genome activation. HDAC2 gene mRNA expression was not affected by in vitro culture and/or cloning before and after activation of the embryonic genome. The donor cell line affected mRNA expression patterns of genes involved in reprogramming cloned embryos suggesting epigenetic dysregulation. Results show that both in vitro production and somatic cloning alter the mRNA expression of histone modifying genes in bovine embryos.

Identification of tumor suppressive activity by irradiation microcell‐mediated chromosome transfer and involvement of alpha B‐crystallin in nasopharyngeal carcinoma

In previous studies, we successfully refined nasopharyngeal carcinoma (NPC) critical regions (CRs) mapping to chromosome 11q13 and 11q22-23. The chromosome 11 fragment containing the 1.8 Mb NPC CR at 11q13 (CR1), the CR at 11q22.3 mapped near D11S2000 (CR2), part of the CR at 11q23.1-11q23.2 overlapping with D11S1300 and D11S1391 (CR3), and the CR at cell adhesion molecule 1 (CADM1) locus (CR4), was chosen as the chromosome 11 donor cell line for the present study. Gamma irradiation was applied to cleave this truncated chromosome into smaller fragments and a new panel of donor cells containing further deleted fragments was produced. Subclones XMCH3.2 and XMCH3.4 were chosen for subsequent transfer to HONE1 cells; each contains a single copy of deleted chromosome 11 fragment with or without CR2 and the THY1 locus, previously shown to be involved in NPC. Both resultant chromosome 11 fragments in XMCH3.2 and XMCH3.4 caused tumor suppression. The association of alpha B-crystallin (CRYAB), a gene identified as being differentially expressed by gene profiling of NPC and an immortalized nasopharyngeal epithelial cell line, and which is located near CR3, was found to be associated with tumor suppression in all the tumor-suppressive hybrids. In addition, the expression level of this gene was down-regulated in the 7 NPC cell lines and in 5 out of 14 normal/tumor tissue pairs in the present study. Both promoter hypermethylation and allelic loss may be involved in the inactivation of this gene, suggesting its possible role in NPC development.

Effect of Phosphonated Carbocyclic 2′-Oxa-3′-Aza-Nucleoside on Human T-Cell Leukemia Virus Type 1 Infection In Vitro

There is currently little research and development of new compounds with specific anti-human T-cell leukemia virus type 1 (HTLV-1) activity. The few antiretrovirals that have been tested against HTLV-1 in vitro have already been developed into anti-human immunodeficiency virus (HIV) drugs. Here, we show the effects of a newly synthesized family of phosphonated nucleoside compounds, phosphonated carbocyclic 2'-oxa-3'-aza-nucleosides (PCOANs), on HTLV-1 infection in vitro. To ascertain the anti-HTLV-1 activity of PCOANs, peripheral blood mononuclear cells from healthy donors were infected in vitro by coculture with an HTLV-1 donor cell line in the presence of three prototype PCOAN compounds. PCOANs were able to completely inhibit HTLV-1 infection in vitro at a concentration of 1 microM, similar to what has been observed for tenofovir and azidothymidine. Treatment with PCOANs was associated with inhibited growth of HTLV-1-infected cells, and their effects were 100 to 200 times more potent than that of tenofovir. The mechanisms involved in the anti-HTLV-1 effects of PCOANs can mainly be ascribed to their capacity to inhibit HTLV-1 reverse transcriptase activity, as ascertained by means of a cell-free assay. PCOANs caused little reduction in proliferation or induction of apoptotic cell death of uninfected cells, showing toxicity levels similar to tenofovir and lower than azidothymidine. Overall, these results indicate that the family of PCOANs includes potential candidate compounds for long-lasting control of HTLV-1 infection.

Bovine Oocytes with the Potential to Reprogram Somatic Cell Nuclei Have a Unique 23-kDa Protein, Phosphorylated Transcriptionally Controlled Tumor Protein (TCTP)

Despite the long-held assumption that reprogramming factors are present in mammalian oocytes at the second metaphase stage, the molecular nature of these factors is not known. Here, we demonstrated that oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP). Injection of TCTP double-stranded RNA into germinal vesicle oocytes decreased the potential of nuclear-transferred (NT) oocytes, but not in vitro fertilized oocytes, to develop into blastocysts. Phosphorylated TCTP is considered to facilitate the first step of somatic cell reprogramming. After transfer of blastocysts that developed from NT oocytes fused with cumulus cells in which phosphorylated TCTP peptide was previously incorporated, the recipient pregnancy rate (47%) increased and the abortion rate (13%) decreased. Moreover, all seven cloned calves survived for at least 1 month after parturition, and had no morphologic abnormalities. The present study demonstrated that pretreatment of donor cells with phosphorylated TCTP peptide has a beneficial effect on the potential of bovine somatic cell nuclei to develop into normal cloned calves. Before widespread application of TCTP for bovine cloning, however, a large-scale embryo transfer study using different donor cell lines of various origins is necessary.

Red Deer Cloned from Antler Stem Cells and Their Differentiated Progeny1

The significance of donor cell differentiation status for successful cloning by somatic cell nuclear transfer (SCNT) is unclear. Here, we cloned a new species, red deer (Cervus elaphus), from multipotent antler stem cells and their differentiated progeny. Cultured donor cell lines from male antlerogenic periosteum (AP) were left undifferentiated or chemically induced to initiate osteogenesis or adipogenesis. Based on their morphology and marker gene expression profile, donor cells were classified as undifferentiated AP cells, presumptive osteoblasts, or adipocytes. Adipocytes upregulated adipogenic markers procollagen type I alpha 2 (COL1A2), peroxisome proliferator-activated receptor gamma 2 (PPARG), and gylceraldehyde-3-phosphate dehydrogenase (GAPDH), and downregulated antlerogenic transcripts POU-domain class 5 transcription factor (POU5F1) and parathyroid hormone (PTH)-like hormone (PTHLH). Despite differences prior to NT, transcript abundance of donor-specific markers COL1A2, PPARG, GAPDH, and POU5F1 did not differ significantly in cloned blastocysts (P = 0.10, 0.50, 0.61, and 0.16, respectively). However, donor cell and blastocyst expression levels were completely different for most genes analyzed, indicating their successful reprogramming. The type of donor cell used for NT (AP, bone, and fat cells), had no effect on in vitro development to blastocysts (93 [38%] of 248 vs. 32 [44%] of 73 vs. 59 [32%] of 183, respectively). Likewise, development to weaning was not significantly different between the three cell types (2 [4%] of 46 vs. 2 [29%] of 7 vs. 4 [13%] of 31, for AP vs. bone vs. fat, respectively). Microsatellite DNA analysis confirmed that the eight cloned red deer calves were genetically identical to the cells used for NT.

Transcriptional reprogramming of somatic cell nuclei during preimplantation development of cloned bovine embryos

While somatic cell nuclear transfer (SCNT) techniques have been successfully implemented in several species to produce cloned embryos and offspring, the efficiencies of the procedures are extremely low, possibly due to insufficient reprogramming of somatic nuclei. Employing GeneChip microarrays, we describe global gene expression analysis of bovine in vitro fertilized (IVF) and SCNT blastocysts as well as respective donor cell lines to characterize differences in their transcription profiles. Gene expression profiles of our donor cell lines were significantly different from each other; however, the SCNT and IVF blastocysts displayed surprisingly similar gene expression profiles, suggesting that a major reprogramming activity had been exerted on the somatic nuclei. Despite this remarkable phenomenon, a small set of genes appears to be aberrantly expressed and may affect critical developmental processes responsible for the failures observed in SCNT embryos. Our data provide the most comprehensive transcriptome database of bovine IVF and SCNT blastocysts to date.

5 TRANSCRIPTIONAL PROFILING OF PIG IN IN VIVO, IN VITRO-FERTILIZED, AND NUCLEAR TRANSFER-DERIVED BLASTOCYSTS AND THE DONOR SOMATIC CELL LINE

The objective of this study was to perform transcriptional profiling between in vivo (IVV), in vitro-fertilized (IVF), and nuclear transfer (NT) blastocyst stage embryos, along with the donor cell line used for NT, in order to identify candidate genes that may contribute to the suboptimal phenotypes of cloned pigs. IVV samples were collected surgically 8 days post-estrus. IVF and NT embryos were transferred into recipient gilts on Day 0 or 1 of estrus and were subsequently collected 6 days later by uterine flush. NT oocytes were activated using one of three methods:NT-1 (electrical activation/fusion), NT-2 (electrical activation/fusion + treatment with proteasomal inhibitor MG 132), or NT-3 (electrical fusion + thimerosal/dithiothreitol (DTT) activation). NT was performed by using pCAG-EGFP positive fetal fibroblast cells to avoid collection of parthenogenetic blastocysts. Donor cells were collected post-NT in pools of 100. Three pools of 10–15 embryos were collected for each treatment. Each pool was analyzed twice, resulting in three biological and two technical replicates. A reference design was used and the reference RNA represented a pool of both reproductive and non-reproductive tissues. Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA) and amplified by using an Ovation Ribo-SPIA linear amplification kit (NuGEN Technologies, Inc., San Carlos, CA, USA). Amplified cDNA from blastocysts or cells was labeled with Cy5 and compared to cDNA from the reference sample labeled with Cy3. The cDNAs were hybridized to an in-house printed pig reproductive tissue-specific 19 968 spot cDNA microarray. Microarray images were acquired using a GenePix� 4000B scanner. Spot quality was assessed and results files were constructed using GenePix Pro 4.0. Lowess normalization and analysis was performed in Genespring 7.3.1 (Agilent Technologies, Inc., Palo Alto, CA, USA). Two comparisons were made: IVF versus IVV, and a comparison of all treatments IVV, IVF, NT-1, NT-2, NT-3, and donor cell line. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg False Discovery Rate multiple correction test. The IVF and IVV comparison resulted in 0 differentially detected cDNAs. The IVV, IVF, NT-1, NT-2, NT-3, and donor cell line comparison detected 1477 differentially detected cDNAs, including heat shock proteins (HSPD1 and HSPE1), which are lowly expressed in the donor cell line, and X inactive-specific transcript (XIST), which has higher expression in IVV and IVF compared to that in NT blastocysts. A standard correlation was performed on both comparisons. The R2 value for the IVV and IVF comparison was 0.892, while the R2 value for all samples was 0.716. These results illustrate that IVV and IVF blastocysts, developed within the uterus, are nearly identical. However, a comparison of blastocysts in all treatments including NT and the donor cell line revealed many differentially expressed genes that can be further evaluated for biological function and usefulness as potential markers of quality embryo development after NT.

Donor Cell Lines Considerably Affect the Outcome of Somatic Nuclear Transfer in the Case of Bovines

Since the first successful nuclear transfer (NT) experiments were carried out, various somatic cell types have been used as donor cells for production of cloned animals. In most experiments, fibroblasts are used since they only need to be isolated and cultivated. Recently, some researchers have shown that different cell cultures from different sources possess different capacities to support preimplantation development of NT embryos. The blastocyst rates obtained in our previous studies varied and were as high as 45% in relation to the number of reconstructed embryos. This led us to question whether the origin and culture conditions of the defined male and female fibroblast lines could be responsible for the differences in developmental potency. Taking all our results into consideration, we conclude that different fibroblast lines recovered from the same tissue and cultivated under equal culture conditions could produce dramatically different blastocyst rates. The influence of cell line itself is higher than the influence of passage number. The observed effects of cell cycle stage, chromosomal aberrations, and diminished vitality are important but not sufficient to discriminate well-qualified nuclear donor cells. We speculate that some epigenetically regulated deviations in the gene expression program are responsible for these phenomena. Explanation of the underlying mechanisms should contribute to better understanding of epigenetic reprogramming and may ultimately assist reprogramming in the laboratory.

Banking on Human Embryonic Stem Cells: Estimating the Number of Donor Cell Lines Needed for HLA Matching

Banking on Human Embryonic Stem Cells: Estimating the Number of Donor Cell Lines Needed for HLA Matching

Birth of Cloned Pigs from Zona-Free Nuclear Transfer Blastocysts DevelopedIn Vitrobefore Transfer

The objective of this work was to obtain cloned pig offspring by uterine transfer of blastocysts produced by zona-free manipulation. We started by defining the most suitable culture media for growing pig nuclear transfer embryos produced by zona-free micromanipulation comparing NCSU-23aa with Synthetic Oviduct Fluid (SOFaa) and with in vivo culture in the sheep oviduct. We found that parthenogenetic development to day 7 blastocyst in NCSU-23aa and sheep oviduct was significantly superior as compared to SOFaa (61.8%, 64% and 42.4 respectively) although blastocyst cell number was higher in the latter. Interestingly, when we compared the two media for the culture of nuclear transfer (NT) embryos derived from 3 different donor cell lines, we observed lower rates of development with NCSU-23aa (from 24.5% to 32.4%) while with SOFaa the development was significantly higher for two donor cell lines as compared to the third (44.4%, 48.9% and 20.6% respectively). A total of 244 blastocysts grown in SOFaa were transferred in four synchronized sows on day 5 or 6 of development. Two recipients farrowed 6 and 8 piglets corresponding to an efficiency of development to term of 8% and 16% of the transferred embryos respectively. Eleven pigs are now 10 month of age and those that have reached puberty have been proven to be fertile. Finally, this is the first report on the production of cloned pigs derived from the transfer of NT embryos at the blastocyst stage.

Epigenetic Marks in Cloned Rhesus Monkey Embryos: Comparison with Counterparts Produced In Vitro1

Until now, no primate animals have been successfully cloned to birth with somatic cell nuclear transfer (SCNT) procedures, and little is known about the molecular events that occurred in the reconstructed embryos during preimplantation development. In many SCNT cases, epigenetic reprogramming of the donor nuclei after transfer into enucleated oocytes was hypothesized to be crucial to the reestablishment of embryonic totipotency. In the present study, we focused on two major epigenetic marks, DNA methylation and histone H3 lysine 9 (H3K9) acetylation, which we examined by indirect immunofluorescence and confocal laser scanning microscopy. During preimplantation development, 67% of two-cell- and 50% of eight-cell-cloned embryos showed higher DNA methylation levels than their in vitro fertilization (IVF) counterparts, which undergo gradual demethylation until the early morula stage. Moreover, whereas an asymmetric distribution of DNA methylation was established in an IVF blastocysts with a lower methylation level in the inner cell mass (ICM) than in the trophectoderm, in most cloned blastocysts, ICM cells maintained a high degree of methylation. Finally, two donor cell lines (S11 and S1-04) that showed a higher level of H3K9 acetylation supported more blastocyst formation after nuclear transfer than the other cell line (S1-03), with a relatively low level of acetylation staining. In conclusion, we propose that abnormal DNA methylation patterns contribute to the poor quality of cloned preimplantation embryos and may be one of the obstacles to successful cloning in primates.

Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer

BackgroundSomatic cell nuclear transfer (SCNT) provides an appealing alternative for the preservation of genetic material in non-domestic and endangered species. An important prerequisite for successful SCNT is the availability of good quality donor cells, as normal embryo development is dependent upon proper reprogramming of the donor genome so that embryonic genes can be appropriately expressed. The characteristics of donor cell lines and their ability to produce embryos by SCNT were evaluated by testing the effects of tissue sample collection (DART biopsy, PUNCH biopsy, post-mortem EAR sample) and culture initiation (explant, collagenase digestion) techniques.ResultsDifferences in initial sample size based on sample collection technique had an effect on the amount of time necessary for achieving primary confluence and the number of population doublings (PDL) produced. Thus, DART and PUNCH biopsies resulted in cultures with decreased lifespans (<30 PDL) accompanied by senescence-like morphology and decreased normal chromosome content (<40% normal cells at 20 PDL) compared to the long-lived (>50 PDL) and chromosomally stable (>70% normal cells at 20 PDL) cultures produced by post-mortem EAR samples. Chromosome stability was influenced by sample collection technique and was dependent upon the culture's initial telomere length and its rate of shortening over cell passages. Following SCNT, short-lived cultures resulted in significantly lower blastocyst development (≤ 0.9%) compared to highly proliferative cultures (11.8%). Chromosome stability and sample collection technique were significant factors in determining blastocyst development outcome.ConclusionThese data demonstrate the influence of culture establishment techniques on cell culture characteristics, including the viability, longevity and normality of cells. The identification of a quantifiable marker associated with SCNT embryo developmental potential, chromosome stability, provides a means by which cell culture conditions can be monitored and improved.

Proliferative characteristics and chromosomal stability of bovine donor cells for nuclear transfer

Few studies have characterized donor cell lines in terms of proliferative capacity and chromosomal stability. Abnormal phosphorylation patterns of the histones during metaphase could lead to abnormal chromosome segregation and extensive chromosome loss during mitosis. Suboptimal culture conditions may lead to abnormal histone H3 phosphorylation patterns, ultimately inducing missegregation and loss of chromosomes. The objective of the present study was to determine proliferative characteristics, chromosomal stability, and level of histone phosphorylation in cell lines established by explants and enzymatic dissociation. Proliferative characteristics, percentage of aneuploid cells, and relative levels of phosphorylated histone H3 (ser10) were determined at different population doublings (PD) by cell counting, karyotyping, and flow cytometry, respectively. The level of aneuploidies was high and remained elevated throughout the study independent of the technique used to establish the primary culture. Some cell lines had up to 50% of aneuploid cells during early passages. Multinucleated cells and abnormal spindle configurations were observed after prolonged time in culture (60 and 41%, respectively). An increase in the relative level of phosphorylated histone occurred after extended time in culture (55.7 during early passages vs. 102.6 at late passages). These data demonstrate the importance of determining chromosome content and the selection of healthy cell lines to decrease the percentage of aneuploid reconstructed embryos and increase the efficiency of nuclear transfer (NT).

38 RABBIT NUCLEAR TRANSFER AND IN VIVO-FERTILIZED EMBRYOS FAIL TO EXPRESS A MOUSE Oct-4 PROMOTER-DRIVEN EGFP REPORTER GENE

Reprogramming of a donor cell genome during somatic cell nuclear transfer (SCNT) is largely dependent on appropriate expression of 'pluripotency'? genes, such as Oct-4 (POU5F1). Recently, we transfected bovine fetal fibroblasts with GOF18-ΔPE-EGFP, a reporter gene construct for the Oct-4 promoter and assessed the expression of Oct-4 after SCNT (Wuensch et al. 2006 Reprod. Fertil. Dev. 18, 144). Our previous study on DNA methylation reprogramming revealed that rabbit in vivo-fertilized and cloned embryos differ from bovine embryos in respect to this epigenetic modification (Shi et al. 2004 Biol. Reprod. 71, 340–347), suggesting differences in the mechanism of epigenetic reprogramming between these two species. In this study, we tested whether GOF18-ΔPE-EGFP could be used to monitor Oct-4 expression in rabbit cloned embryos. The reporter gene construct included the EGFP gene flanked by a 9-kb fragment of the murine Oct-4 upstream region with a deletion in the proximal enhancer (PE) and a 9-kb fragment containing the nontranscribed murine structural Oct-4 gene. The 21.2-kb fragment GOF18-DPE-EGFP was released from the vector backbone by NotI digestion and purified with QIAquickGel Extraction Kit (Qiagen, Hilden, Germany) after gel electrophoresis. Four stable transfected colonies of rabbit fetal fibroblasts (RFF), none of which exhibited green fluorescence, were used for SCNT. The resulting embryos were examined on Days 2–5 by fluorescence microscopy. To detect endogenous Oct-4 expression, in vivo-fertilized embryos were stained with anti-mouse Oct-3 antibody and then incubated with secondary Alexa 488-conjugated goat anti-mouse antibody. The most prominent endogenous Oct-4 expression was detected in in vivo-fertilized embryos at the morula and blastocyst stages. Depending on the donor cell line used for nuclear transfer, cleavage and blastocyst rates ranged from 56 to 97% and from 33 to 49%, respectively. When a total of 230 cloned embryos at the 2-to 16-cell stages and 93 cloned morulae and blastocycts were examined by fluorescence microscopy, none of the examined embryos exhibited fluorescence signals indicating the lack of Oct-4 promoter activity. Taking into account the fact that both cloned and in vivo-fertilized rabbit embryos have specific patterns of DNA methylation reprogramming, which are different from that of bovine embryos, we injected GOF18-ΔPE-EGFP gene constructs into pronuclei of in vivo-fertilized zygotes. None of the 74 injected embryos, which were examined at the 2-cell to blastocyst stages, showed fluorescence signals. Our results demonstrate that rabbit nuclear transfer and in vivo-fertilized embryos are unable to activate a mouse Oct-4 promoter-reporter construct. Potential reasons include incompatibilities between mouse Oct-4 promoter sequences and rabbit transcription factors as well as specific mechanisms of epigenetic reprogramming in the rabbit. This work was supported by the Bayerische Forschungsstiftung.

Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching

Human embryonic stem (hES) cells are a promising source for transplantation to replace diseased or damaged tissue, but their differentiated progeny express human leucocyte antigens (HLAs) that will probably cause graft rejection. The creation of a bank of HLA-typed hES cells, from which a best match could be selected, would help reduce the likelihood of graft rejection. We investigated how many hES cell lines would be needed to make matching possible in most cases. The number of hES cell lines needed to achieve varying degrees of HLA match was estimated by use of, as a surrogate for hES-cell donor embryos, blood group and HLA types on a series of 10,000 consecutive UK cadaveric organ donors. The degree of blood group compatibility and HLA matching for a recipient population consisting of 6577 patients registered on the UK kidney transplant waiting list was determined, assuming all donor hES cell lines could provide a transplant for an unlimited number of recipients. A bank of 150 consecutive donors provided a full match at HLA-A, HLA-B, and HLA-DR for a minority of recipients (<20%); a beneficial match (defined as one HLA-A or one HLA-B mismatch only) or better for 37.9% (range 27.9-47.5); and an HLA-DR match or better for 84.9% (77.5-90.0). Extending the number of donors beyond 150 conferred only a very gradual incremental benefit with respect to HLA matching. A panel of only ten donors homozygous for common HLA types selected from 10,000 donors provided a complete HLA-A, HLA-B and HLA-DR match for 37.7% of recipients, and a beneficial match for 67.4%. Approximately 150 consecutive blood group compatible donors, 100 consecutive blood group O donors, or ten highly selected homozygous donors could provide the maximum practical benefit for HLA matching. The findings from these simulations have practical, political, and ethical implications for the establishment of hES-cell banks.

A region on human chromosome 4 (q35.1→qter) induces senescence in cell hybrids and is involved in cervical carcinogenesis

Human papillomavirus (HPV) types 16 and 18 are known to play a major role in cervical carcinogenesis. Additional genetic alterations are required for the development and progression of cervical cancer. Previously, we showed that the introduction of an entire human chromosome 4 into HPV-immortalized cells by microcell-mediated chromosome transfer (MMCT) can induce senescence in cell hybrids. In the present study, we established eight new murine donor cell lines harboring different fragments of the human chromosome 4. These were tested for their ability to induce senescence by MMCT into HPV16-immortalized keratinocytes (HPK II) and cervical carcinoma cells (HeLa). By exclusion, we could identify a region for a putative senescence gene or genes at 4q35.1-->qter. Further evidence that this locus may be involved in cervical carcinogenesis was obtained by studying sections of high-grade cervical intraepithelial neoplasias (CIN2/3) and cervical cancers from 87 women using a combination of interphase fluorescence in situ hybridization (I-FISH) and microsatellite PCR. I-FISH indicated copy number loss at 4q34-->qter. Microsatellite analysis showed that loss of one or more alleles at chromosome 4 was more frequent in the cervical carcinomas than in the CINs. Loss of heterozygosity (LOH) affected four areas, D4S412 (4p16.3), D4S2394 (4q28.2), D4S3041 (4q32.3), and D4S408 (4q35.1), and was highest at D4S408. LOH at terminal 4q has been reported previously for cervical carcinomas and other human malignancies. This is the first report associating allelic loss at 4q34-->qter with high-grade intraepithelial neoplasia and cervical carcinoma, and the first experimental evidence that this locus or these loci can induce senescence in cervical carcinoma cells and HPV16-immortalized cells.

Cats cloned from fetal and adult somatic cells by nuclear transfer

This work was undertaken in order to study the developmental competence of nuclear transfer (NT) into cat embryos using fetal fibroblast and adult skin fibroblast cells as donor nuclei. Oocytes were recovered by mincing the ovaries in Hepes-buffered TCM199 and selecting the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark color. Homogenous ooplasm was cultured for maturation in TCM199+10% fetal bovine serum (FBS) for 12 h and used as a source of recipient cytoplast for exogenous somatic nuclei. In experiment 1, we evaluated the effect of donor cell type on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate were not different between fetal fibroblasts and adult skin cells (71.2 vs 66.8; 71.0 vs 57.6; 4.0 vs 6.1% respectively; P < 0.05). In experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of the seven recipient queens was delivered naturally of 2 healthy cloned cats and 1 stillborn from fetal fibroblast cells of male origin 65 days after embryo transfer. One of three recipient queens was delivered naturally of 1 healthy cloned cat from adult skin cells of female origin 65 days after embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning.