bluuebunny/crossref_metadata_2025 huggingface dataset:
340PRODUCTION OF TRANSGENIC PORCINE BLASTOCYSTS BY HANDMADE CLONING
Authors: Kragh P.M. , Vajta G. , Corydon T.J. , Bolund L. , Callesen H.
Published: February 2004

The present study demonstrates the application of the recently developed handmade cloning (HMC) technique in production of transgenic porcine blastocysts. The HMC technique was originally established for bovine nuclear transfer (Vajta et al., 2003, Biol. Reprod. 68, 571–578), and has the advantages of being less demanding and more productive than traditional nuclear transfer techniques. Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries and matured for 41h. Subsequently, the cumulus cells were removed by pipetting in 1mgmL−1 hyaluronidase in HEPES-buffered TCM-199; zonae pellucidae were removed by incubation in 2mgmL−1 pronase in HEPES-buffered TCM-199 supplemented with 2% cattle serum (T2) for 1min. Bisection was performed by hand under a stereomicroscope using a microblade in 5μgmL−1 cytochalasin B in TCM-199 supplemented with 20% cattle serum (T20). Demi-oocytes were incubated in 5μgmL−1 Hoechst 33342 in T20 for 10min, followed by examination under UV light to select the halves containing no chromatin, i.e., the cytoplasts. Porcine fibroblasts harvested from an ear skin biopsy were transfected with pN1-EGFP (Clontech) using Lipofectamine (Gibco, Life Technologies). G418 selection (0.8mgmL−1) was applied 48h after transfection, and well separated G418-resistant cell colonies originating from a single transfected cell were isolated, expanded, and cryopreserved. Days before, nuclear transfer cells were grown to a confluent monolayer in DMEM supplemented with 10% FCS. Fusions were performed 43h after start of maturation. One cytoplast was attached to one fibroblast in 500μgmL−1 phytohemagglutinin dissolved in T2. In the fusion chamber, covered with fusion medium (0.3M mannitol, 0.1mM MgSO4, 0.05mM CaCl2, and 0.01% PVA), one cytoplast-fibroblast pair was fused with one cytoplast in a single step. The fusions were performed with a double DC pulse of 65V, each pulse for 20μs and 0.1s apart from each other. Successfully fused embryos were activated 1h after the end of fusion by incubation in 2μM calcium ionophore A23187 in T20 for 5min followed by 3-h incubation in microdrops of culture medium (NCSU-23 with 4mgmL BSA) containing 2mM 6-dimethylaminopurine. Activated embryos were cultured individually in microdrops of culture medium for 7 days. In four independent experiments, 93% of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, and 37/37, respectively). On Day 7 after activation, the blastocyst rates (per successfully reconstructed embryos) were 6% (2/31), and 7% (1/15), 7% (2/28), and 3% (1/37), respectively. Green Fluorescent Protein was expressed in all cells of the developing blastocysts. The results show that transgenic porcine blastocysts can be produced using HMC, and the technique may also be applied for the production of transgenic pigs.
343NUCLEAR LOCALIZATION SIGNAL AND CELL SYNCHRONY ENHANCES GENE TARGETING
EFFICIENCY IN FETAL BOVINE FIBROBLASTS
Authors: Mir B. , Piedrahita J.A.
Published: February 2004

The use of primary somatic cells for nuclear transfer has facilitated the manipulation of the domestic animal genome via homologous recombination. Yet, the absolute frequency of homologous recombination (HR) in somatic cells is two orders of magnitude lower than in ES cells whereas frequencies of non-homologous end joining are higher. While a few loci have been targeted in somatic cells using enrichment strategies similar to those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells, and difficulties in cloning the cell after targeting. We present evidence that the use of a nuclear localization signal (nls) and S-phase cell cycle synchronization by thymidine block enhances targeting efficiency at the hypoxanthine phosphoribosyl transferase locus in primary fetal bovine fibroblasts. We designed two hypoxanthine phosphoribosyl transferase (HPRT)-targeting constructs, HPRT-DEx6 and HPRT-DEx6-nls. Both constructs have a 31-bp deletion and a PGK-puro insertion in exon 6 to ensure inactivation of the HPRT locus. Additionally, the HPRT-DEx6-nls construct contains a 180-bp cassette comprised of two 72-bp tandem repeats from SV40 enhancer known to act as nuclear localization signal. Diploid male cells that undergo targeted gene disruption at the single copy X-linked HPRT locus can be selected with 8-Azaguanine (8-AG) as HPRT cells are resistant to 8-AG; all transformants, random and targeted, can be selected in puromycin. Male primary bovine fibroblasts were electroporated with linearized targeting constructs, and plated in media containing puromycin or puromycin plus 8AG. All experiments were done in triplicate and data were analyzed by two-way ANOVA with NLS and cell synchrony as the two factors. Significance was set at P<0.01. While the total number of insertions (random plus targeted) with both constructs were equivalent, the HPRT-DEx6-nls construct produced a significantly higher number of targeted colonies (1–2 8AG-resistant colonies per 9.5×106 cells) than HPRT-DEx6 where no targeted events were seen. Cells were synchronized in the S-phase of the cell cycle by a 2mM thymidine treatment for 24 hours and electroporated with the linearized targeting constructs. Compared to non-synchronous cells, the total number of insertions (random plus targeted) was reduced by 59-fold in constructs with or without nls (P<0.01), while targeted insertions increased 6-fold in the HPRT-DEx6-nls construct, from an average of 10 per million cells without nls to 7.6 per 10 million with nls (P<0.01). All 8AG-resistant colonies were verified by Long Range-PCR, and PCR products confirmed by end sequencing. This finding has important implications for targeting in somatic cells, as with a drastic reduction in the number of random insertions, and increased targeting due to the presence of the nls, identification of a targeted colony is greatly facilitated even in cases where no enrichment protocols are available.
35ANALYSIS OF EPIGENETIC MODIFICATIONS AND GENOMIC IMPRINTING IN NUCLEAR
TRANSFER DERIVED BOS GAURUS×B. TAURUS CONCEPTI
Authors: Dindot S.V. , Farin P. , Farin C. , Alexander J. , Crosier E. , Walker S. , Long C. , Piedrahita J.A.
Published: February 2004

Somatic cell nuclear transfer in cattle is an inefficient process hindered by low pregnancy rates and fetal placental abnormalities. Improper or incomplete epigenetic reprogramming of the donor genome has been implicated as a cause for these aberrations and has been investigated extensively in mice. Here we report the use of a bovine interspecies model (Bos gaurus×B. taurus) for the assessment and characterization of epigenetic modifications and genomic imprinting in 40-day-old female nuclear transfer (NT)-derived fetuses and placentas. Previously, we identified genomic imprinting at the IGF2, GTL2 and XIST loci in the Bos gaurus×B. taurus fetuses. These results indicated maternal and paternal imprinting of the IGF2 and GTL2 loci, respectively, in the chorion, allantois, liver, lung and brain, whereas the XIST locus was maternally imprinted solely in the chorion of females. We extended this analysis to 40-day-old NT fetuses derived from a hybrid lung fibroblast cell line (female). Analysis of the donor cell line indicated conservation of imprinting of the IGF2 and GTL2 loci and bialleic expression of the XIST locus, presumably from the random patterns of X-chromosome inactivation. Analysis of three NT and three control pregnancies indicated disruption of genomic imprinting at the XIST locus in the chorions of all three clones compared to controls. In contrast, proper allelic expression of the IGF2 and GTL2 loci was observed in all fetuses and placentas. Quantification of maternal and paternal XIST transcripts in the chorion of clones and controls demonstrated a significant skewing from preferential paternal expression in controls (95.0±0.882, mean±S.E.) to mixed paternal and maternal expression in clones (73.6±5.2), (t-test; P<0.05). In an attempt to determine the cause for the abnormal allelic expression of the XIST locus in the chorion of the clones, methylation analysis of the XIST Differentially Methylated Region (DMR) was performed. Methylation-sensitive restriction digests and subsequent PCR of the XIST DMR indicated patterns were not different between controls and clones. However, when genome-wide and promoter-specific methylation analysis (bisulfite sequencing) was extended to the satellite I repeat element and epidermal cytokeratin promoter, hypermethylation was observed in the chorion of clones. These results demonstrate disruption of genomic imprinting in XIST locus in the placenta of 40-day-old clones independent of DMR methylation. They also indicate that cloning is associated with increased levels of methylation in selected genomic regions in the chorion.
36COMPARISON OF THE DEVELOPMENTAL POTENTIAL OF CAPRINE NUCLEAR TRANSFER
EMBRYOS DERIVED FROM IN VITRO AND IN VIVO MATURED OOCYTES
Authors: Echelard Y. , Memili E. , Ayres S.L. , O'Coin M. , Chen L.H. , Meade H.M. , Behboodi E.
Published: February 2004

The objective of this study was to compare the development to the blastocyst stage of reconstructed caprine nuclear transfer (NT) embryos derived from two sources of ova. In vivo oocytes were flushed from the oviduct of superovulated donors by exposing the reproductive tract via a small ventral laparotomy. In vitro oocytes were collected from ovaries supplied by an abattoir located in Purdue, IN. Oocytes were aspirated, cultured in maturation medium (M199 +10% goat serum, 3μgmL−1 LH, 3μgmL−1 FSH and 0.22mM sodium pyruvate), and shipped overnight (38°C, air). Donor cell preparation and NT procedures were as previously reported (Behboodi et al., 2001 Theriogenology 55, 254 abst). Donor cells were transfected female fetal fibroblasts that were synchronized by 4 days of serum starvation, followed by a 10-hour exposure to medium containing 10% FCS. Oocytes were enucleated, karyoplast-cytoplast couplets were reconstructed, fused and then activated simultaneously by a single electrical pulse. Couplets containing in vitro oocytes were incubated in the presence of 5μgmL−1 ionomycin after fusion. Fused couplets were co-cultured in TCM199 with 10% FCS and oviductal epithelial cells for 8–10 days (38°C, 5% CO2). Embryos that developed in vitro to the blastocyst stage were surgically transferred to recipients. Pregnancies were confirmed by ultrasonography. One live kid was delivered on Day 150 of gestation via elective C-section. Southern blotting analysis confirmed that it was derived from the transgenic donor cell line. These experiments show that in vivo matured oocytes not only better support caprine NT embryo development to the blastocyst stage, but also can result in live birth (table). Although fusion and cleavage rates were similar in the two groups, development to the blastocyst stage was significantly higher (Student’s t-test) in the group utilizing in vivo-matured oocytes. In conclusion, this is the first live goat produced from goat NT blastocysts developed in vitro. This suggests that in vivo matured oocytes may be superior to oocytes developed in vitro for generating live animals from NT blastocysts.

Table 1
37DEVELOPMENTAL POTENTIAL OF CLONE CELLS IN MURINE CLONE-FERTILIZED
AGGREGATION CHIMERAS
Authors: Eckardt S. , Leu N.A. , McLaughlin K.J.
Published: February 2004

In both murine and porcine preimplantation stage clones, mosaicism in gene expression has been observed, indicating variation in transcription of some genes between cells of the individual clone (Boiani M et al., 2002 Genes Dev. 16, 1209–1219; Park KW et al., 2002 Biol. Reprod. 66, 1001–1005). This observation raises the question as to whether all blastomeres within one early-stage clone are equivalent, or whether there are differences in developmental potential. To address this, we aggregated preimplantation-stage clone embryos with fertilized embryos and assessed contribution of Oct4-GFP expressing cells of clone origin in blastocysts and in vitro outgrowths. In normal embryos, the Oct4-GFP transgene is expressed during preimplantation stages and reflects expression of Oct4 protein. Mouse cumulus cell clones were produced from cells transgenic for Oct4-GFP (Szabó PE et al., 2002 Mech. Dev. 115, 157–160) as described (Boiani M et al., 2002 Genes Dev. 16, 1209–1219). Four-cell-stage clones and synchronous fertilized non-transgenic embryos were aggregated in micro-wells after removal of the zona pellucida using acid Tyrode’s solution. Aggregates were cultured to the blastocyst stage in -MEM supplemented with bovine serum albumin (0.4% w/v). All control chimeras produced from four-cell-stage fertilized non-transgenic and Oct4-GFP transgenic embryos formed blastocysts, and 15 of 20 had GFP-expressing cells. The majority of clone-wild-type aggregates developed to the blastocyst stage (35/40); however, contribution of GFP-expressing cells was observed in fewer blastocysts compared to controls (12/35; P<0.05). Contribution of GFP expressing clone cells to the ICM varied between 30% and 100% of cells as determined by subjective evaluation of GFP fluorescence overlaying bright-field images. During in vitro outgrowth formation of synchronous aggregation chimeras of clone and wild-type embryos, maintenance of clone contribution to the ICM mound was observed, but at a lower frequency (12% v. 34% at the blastocyst stage). The results suggest that aggregation with fertilized cells does not provide benefit to clone blastomeres during preimplantation stages. Possibly, clone blastomeres may not be competitive with wild-type blastomeres, or are developmentally asynchronous, which will be tested using asynchronous chimeras.
39MATERNAL ORIGIN OF OOCYTES AFFECTS IN UTERO
DEVELOPMENTAL CAPACITY AND PHENOTYPE OF BOVINE NUCLEAR TRANSFER
FETUSES
Authors: Hiendleder S. , Reichenbach H.-D. , Wenigerkind H. , Bebbere D. , Brem G. , Zakhartchenko V. , Wolf E.
Published: February 2004

Epigenetic phenomena have been recognized as an important issue in somatic cell nuclear transfer (SCNT) and have been linked to SCNT success rate. We have previously reported a significant maternal lineage effect of oocyte donors on the number of transferable embryos obtained after SCNT (Brüggerhoff et al., 2002 Biol. Reprod. 66, 367–373), pointing to ooplasmic maternal genetic/epigenetic factors important for early embryonic development. We have now extended this study and generated a set of cloned embryos using recipient oocyte pools from defined or random maternal sources that were identified by pedigree data and mitochondrial DNA (mtDNA) haplotype, to study effects of nuclear-cytoplasmic interactions on in utero developmental capacity and fetal phenotype. Embryos were reconstructed from granulosa cells of a Bos taurus (Brown Swiss) cow using oocytes with B. taurus A (Simmental), B. taurus B (Simmental), B. indicus (Dwarf Zebu), or random B. taurus (Brown Swiss) cytoplasm. Two to three embryos per recipient heifer (Simmental) were transferred on Day 7 after SCNT, and fetuses were recovered after slaughter of recipients on Day 80. The number of pregnancies obtained per transferred embryo on Day 28 was similar for the B. taurus combinations (0.11–0.15) but much higher for a limited number of embryos reconstructed with B. indicus cytoplasm (0.50). Developmental capacity of embryos derived from the four oocyte pools varied considerably and resulted in 8 (11.1%), 3 (4.6%), 2 (33%) and 10 (15.2%) viable fetuses with B. taurus A, B. taurus B, B. indicus and random B. taurus cytoplasm, respectively. When fetuses in resorption were included, however, the percentages for recovered fetuses on Day 80 were similar (12.5, 13.8 and 16.7%) in all B. taurus combinations. Body weight, crown rump length, thorax circumference (P<0.05), and femur length (P<0.01) of viable fetuses with B. taurus A cytoplasm differed from fetuses with random B. taurus cytoplasm and showed less variation. Furthermore, body weights of fetuses with B. taurus A or B, or B. indicus cytoplasm, were not significantly different from those of B. taurus (Brown Swiss) control fetuses produced by artificial insemination, but fetuses reconstructed with cytoplasts from randomly collected B. taurus oocytes indicated fetal overgrowth (80.6±6.5g v. 107.9±5.8g, P<0.01). Our data point to complex ooplasm-dependent epigenetic modifications and/or nuclear DNA mtDNA interactions that affect developmental capacity and fetal weight and dimensions in SCNT. This might also be relevant for crossbreeding in farm animals and other reproductive technologies such as ooplasmic transfer in human-assisted reproduction
4CHROMOSOMAL STABILITY OF AFRICAN WILD CAT (FELIS SILVESTRIS LIBICA) SOMATIC CELLS AND CLONED EMBRYOS
Authors: Giraldo A.M. , Gomez M.C. , Dresser B.L. , Harris R.F. , King A.L. , Pope C.E.
Published: February 2004

Nuclear transfer (NT) procedures are generally inefficient and chromosomal abnormalities have been suggested as one causative factor. Embryos derived by somatic cell NT show a high incidence of aneuploidy, which seems to be reflective of the donor cell line with high chromosomal abnormalities (Bureau et al., 2003, Theriogenology 59, 239). Also, aneuploidies in some cell lines increase progressively with the number of passages (Denning et al., 2001, Cloning 3, 221–231). Therefore, the aim of the present study was to analyze the chromosomal stability of donor cells at different passages as well as that of reconstructed embryos derived from these cells. A primary culture of African wild cat (AWC) fibroblast cells was established from a skin sample and cultured until cells stopped dividing at passage 9. Chromosome numbers were determined in cells using a karyotyping technique (Iwasaki et al., 1992 J Exp Zool 261, 79–85) at passages 1 and 3 through 9. At passages 1, 3, 4 and 9, NT of AWC fibroblast cells into domestic cat cytoplasts was done (Gomez et al., 2003, Biol Reprod 69, 1032–1041). Reconstructed blastocysts were treated with colcemid (0.28μgmL−1) and fixed for karyotyping to evaluate chromosome numbers in the blastomeres. Blastomere metaphases were often highly contracted or overlapping, so that only 1 to 7 sets of metaphase chromosomes per embryo were spread sufficiently to allow determination of ploidy. No attempt to produce NT embryos using cells at passages 5 through 8 was done because the percentages of aneuploidies of these passages were not significantly different (P>0.05). Data were analyzed by chi-square test (P<0.05). As shown in the Table, the percentage of aneuploidy in the somatic cells increased progressively with duration of culture. Furthermore, the percentage of chromosomal abnormalities in reconstructed embryos was similar to that of the cells from which they were derived. Accordingly, for future NT, we propose to use donor cells at early passages when the percentage of cells with chromosomal abnormalities is still relatively low. Research was funded partially by the John & Shirley Davies Foundation.

Chromosomal abnormalities in AWC cells and cloned embryos
41EFFECTS OF MATURATION PERIOD OF PORCINE OOCYTES ON DEVELOPMENT
FOLLOWING SOMATIC CELL NUCLEAR TRANSFER
Authors: Hoelker M. , Petersen P. , Lemme E. , Lucas-Hahn A. , Niemann H.
Published: February 2004

Despite intensive research, porcine nuclear transfer is still characterized by low success rates. To determine the effect of maturation period of porcine oocytes on subsequent development following nuclear transfer, we investigated fusion rate, induction of activation and development to blastocyst stage of somatic cells. For this we used MII-oocytes after 38, 40, and 42h of maturation culture as recipients. Oocytes surrounded by a compact cumulus mass were selected and placed into North Carolina State University (NCSU) 37 oocyte maturation medium supplemented with 0.1mgmL−1 cysteine, 10ngmL−1 epidermal growth factor, 10% porcine follicular fluid, 50μm 2-mercaptoethanol, 0.5mgmL−1 cAMP, 10 IU each of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) for 22h in humidified air with 5% CO2 at 38.5°C. Subsequently the oocytes were moved to fresh NCSU37 maturation medium without cAMP, eCG and hCG and incubated for an additional 16, 18, and 20h. In the first experiment, a total of 878 MII-arrested oocytes were enucleated, fused with pig fetal fibroblasts in calcium-free medium and activated approximately 3h later with an electrical stimulus. This was followed by incubation in 6-dimethylaminopurine for 3h and subsequent analysis of development in vitro. Maturation period had no effect on the frequencies of fusion (87% v. 75% v. 84%, respectively), and cleavage (82% v. 81% v. 87%, respectively), but when MII-oocytes recovered at 40h of maturation were used as recipients, 41/279 (14,8%) the numbers of cloned embryos developing to the blastocyst stage on Day 7 of culture was significantly (ANOVA followed by multiple pairwise comparisons using Tukey test, 6 replicates, P<0,05) higher than those of embryos reconstituted with oocytes collected at 38h (27/285, 9.6%) and 42h (16/314, 4.9%). In the second experiment, reconstructed embryos derived from oocytes matured for 40h were surgically transferred to the oviducts of synchronized German Landrace gilts. Transfers were made on the first day of standing oestrus within 3h of activation to assess their development in vivo. Synchronization was achieved by injections of 1500IU eCG followed by 500IU hCG 3 days later. Of 4 recipients receiving an average of 150 zygotes (range, 136 to 163), 2 became pregnant as determined by ultrasound between Days 25 and 35 of gestation. Of the two pregnant recipients, one subsequently farrowed 4 piglets on Day 115 of pregnancy. These results indicate that the maturation period is critical and affects development of porcine nuclear transfer embryos. This study was funded by the Deutsche Forschungsgemeinschaft (DFG; SFB265).
43IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS WITH
6-DMAP FOLLOWING FUSION
Authors: IM G.-S. , Lai L. , Liu Z. , Hao Y. , Murphy C.M. , Prather R.S.
Published: February 2004

Although nuclear transfer (NT) has successfully produced cloned piglets, the development to blastocyst and to term is still low. Activation of the NT embryos is one of the key factors to improve the developmental ability of porcine NT embryos. Electric pulses as well as chemicals have been used to activate porcine NT embryos. This study was conducted to investigate the effect of continued activation following fusion pulses on in vitro development of porcine NT Embryos. Oocytes derived from a local abattoir were matured for 42 to 44h and enucleated. Ear skin cells were obtained from a 4-day-old transgenic pig transduced with eGFP recombinant retrovirus. Enucleated oocytes were reconstructed and cultured in PZM-3 in a gas atmosphere of 5% CO2 in air. Cleavage and blastocyst developmental rates were assessed under a stereomicroscope on Day 3 or 6. Blastocysts were stained with 5μg of Hoechst 33342 and total cell number was determined with an epifluorescent microscope. In Experiment 1, oocytes were activated with two 1.2kV/cm for 30μs (E) in 0.3M mannitol supplemented with either 0.1 or 1.0mM Ca2+. In each treatment, activated oocytes were divided into three groups. The first group was control (E). Other two groups were exposed to either ionomycin and 6-DMAP (E+I+D) or 6-DMAP (E+D) immediately after the electric pulses. In Experiment 2, fusion was conducted by using 1.0mM Ca2+ in the fusion medium. Fused NT embryos were divided into three treatments. NT embryos were fused and activated simultaneously with electric pulse as a control (C); the second group was treated with 6-DMAP immediately after fusion treatment (D0); and the third group was treated with 6-DMAP at 20min (D20) after fusion. In experiment 1, for 0.1mM Ca2+, developmental rates to the blastocyst stage for E, E+I+D or E+D were 12.5, 26.7 and 22.5%, respectively. For 1.0mM Ca2+, developmental rates to the blastocyst stage were 11.4, 28.3 and 35.6%, respectively. The activated oocytes treated with 6-DMAP following the electric pulses by using 1.0mM Ca2+ in fusion medium had higher (P<0.05) developmental rates to the blastocyst stage. In Experiment 2, developmental rates to the blastocyst stage for C, D0 or D20 were 10.0, 12.3, and 19.9%, respectively. Developmental rate to the blastocyst stage was higher (P<0.05) in D20. Fragmentation rates were 19.9, 10.8, and 9.0%, respectively. Regardless of Ca2+ concentration in fusion medium, continued treatments with chemicals following electric pulses supported more development of porcine activated oocytes. Treating NT embryos with 6-DMAP alone after fusion was completed by using 1.0mM Ca2+ in fusion medium improved the developmental rates to the blastocyst stage and prevented fragmentation accompanied by electric fusion. This study was supported by NIH NCRR 13438 and Food for the 21st Century.
54THE DEVELOPMENT AND CHARACTERIZATION OF MITOCHONDRIAL DNA
(MTDNA)-DEPLETED CAPRA HIRCUS FETAL FIBROBLASTS: CANDIDATE DONORS FOR SOMATIC
CELL NUCLEAR TRANSFER (SCNT)
Authors: Lloyd R.E. , Alberio R. , Bowles E.J. , Campbell K.H.S. , St. John J.C.
Published: February 2004

Mammalian mtDNA is approximately 16.6kb in size. It has 37 genes, 13 of which encode protein subunits of the oxidative phosphorylation (OXPHOS) system, the major ATP-generating pathway of the cell. Normally, mammals inherit a single mtDNA genome (homoplasmy) from their mother. Somatic cell nuclear transfer (SCNT) violates this strict maternal, homoplasmic inheritance of mtDNA as cytoplasm is transferred along with the nucleus, which often results in an oocyte harboring both donor and recipient mtDNA genomes (heteroplasmy). This been previously reported (reviewed St. John JC 2002 Theriogenology 57, 109–123). To overcome the problem of donor mtDNA transmission, we have developed and characterized mtDNA-depleted C. hircus (goat) cells for use as donors in SCNT. C. hircus primary foetal fibroblast cells were established in culture and depleted of their mtDNA by supplementing their growth medium with a low concentration, 50ngmL−1, of ethidium bromide (EthBr). Conventional PCR, using a series of primers designed specifically for goat mtDNA, was used to screen for the presence of mtDNA during the EthBr treatment. In addition, mitochondrial organization, activity and morphology in the cells were analyzed using the mitochondrial specific fluoroprobe JC1. mtDNA-encoded and mitochondrial transcription factor A (mtTFA) transcript levels were analysed using RTPCR. Furthermore, both mtDNA depleted and non-depleted cells were characterised using immunocytochemistry to detect the expression of specific protein subunits of the OXPHOS system. Progressive mtDNA depletion was observed, using conventional PCR, in cells treated for 3 to 25 days with EthBr, while 42 days of culture resulted in complete depletion. RTPCR showed a progressive reduction followed by complete elimination of the mtDNA-encoded ND1, ND2, ND3, COX I and mtTFA transcripts. In addition, the expression of mtDNA-encoded protein subunits, e.g. COXI, of the OXPHOS system were reduced following mtDNA depletion whereas the expression of nuclear-DNA encoded protein subunits, e.g. COXVic, were unaltered. We hypothesize that the elimination of mtDNA and mtDNA transcripts from the donor cells will facilitate normal mtDNA replication and transcription in SCNT embryos, thus maintaining the strict unimaternal transmission of mtDNA to the offspring. Consequently, genetically identical offspring will be generated which have identical nuclear and mitochondrial DNA content, assuming oocytes from the same ovary are used. This technique is important for the generation of offspring for the livestock industry and animal models for the analysis of single gene disorders as well as the propagation of endangered species.
63INTERFERON-TAU EXPRESSION FROM PRIMARY TROPHECTODERM OUTGROWTHS OF
BOVINE BLASTOCYSTS: COMPARISON BETWEEN IVP, NT, AND PARTHENOGENIC
EMBRYOS
Authors: Ocon O.M. , Talbot N.C. , Powell A.M. , Ealy A.D.
Published: February 2004

Interferon-tau (IFN-tau) is expressed soon after bovine blastocyst formation and might be useful as a marker of appropriate biological function in embryos produced by nuclear transfer. To assess this possibility we have compared IFN-tau levels in the conditioned medium of primary trophectoderm cultures derived from IVP, nuclear transfer (NT), or parthenogenic bovine embryos. Embryos were produced from in vitro-matured cumulus-oocyte complexes processed from local slaughterhouse ovaries or obtained from Bomed, Inc. (Madison, WI, USA). In vitro fertilization, NT, and parthenogensis were as previously described (Talbot et al., 2000 Tissue and Cell, 32, 9–27) except that embryo culture was in G1/G2 medium in 5% oxygen (Lane et al., 2003 Theriogenology, 60, 407–419). Each 8–11-day embryo was cultured individually in a 4-well plate well (Nunc) with STO feeder cells using DMEM medium containing 10% fetal bovine serum as previously described (Talbot et al., 2000 Biol. Reprod. 62, 235–247). Any contaminating epiblast or endoderm was physically dissected and discarded so as to produce pure trophectoderm outgrowths. The success/failure ratio for colony formation was similar for IVP and NT embryos (IVP=155/29; NT=104/25), but was significantly different (P<0.05) for parthenogenic embryos (54/43). Trophectoderm colonies reached diameters of 1 to 1.5cm in 3–4wk, and, at this time, 72-h-conditioned cell culture medium was harvested, frozen, and measured for IFN-tau anti-viral activity as previously described (Talbot et al., 2000 Biol. Reprod. 62, 235–247). From 313 observations, IFN-tau production was analyzed as a two-factor mixed linear model. Differences in IFN-tau production by type of embryo were statistically significant (F=42.61; P<0.0001; df=2). Mean comparisons were done with Sidak adjusted P-values so that the experiment-wise error was 0.05. IFN-tau production means for IVP-, NT-, and parthenogenic-derived trophectoderm were 4311IUmL−1 (n=155), 626IUmL−1 (n=104), and 1595IUmL−1 (n=54), respectively. The results show that mean IFN-tau production from trophectoderm cultures derived from NT embryos is significantly reduced in comparison to IVP- and parthenogenote-derived cultures. Parthenogenote-derived cultures also produced significantly less IFN-tau than IVP embryos on average. IFN-tau production from trophectoderm outgrowths may be a useful measure of NT reprogramming success.
66EFFECTS OF CONTACT INHIBITION INTERVALS V. SERUM DEPRIVATION ON
DEVELOPMENT OF PORCINE NUCLEAR TRANSFER DERIVED EMBRYOS
Authors: Petersen B. , Hoelker M. , Kues W. , Niemann H.
Published: February 2004

Contact inhibition and serum deprivation are commonly used to synchronize donor cells at the G0/G1 state of the cell cycle prior to use in nuclear transfer. Here we compared the effects of serum deprivation (SD) and different intervals of contact inhibition (CI) of the donor cells on the blastocyst rate. One batch from pooled porcine fetal fibroblasts (passage 3) was used in this study. The cells were thawed, seeded to a six-well plate and cultured in Dulbecco‘s Modified Eagles Medium (DMEM) supplemented with 2mM glutamine, 1% non-essential amino acids, 0.1mM mercaptoethanol, 100UmL−1 penicillin, 100mgmL−1 streptomycin containing 10% fetal calf serum (FCS). Serum deprivation was achieved by culturing cells in DMEM containing 0.5% FCS for 48h. Cells of the CI groups were grown to 100% confluency and kept in that state for 24h, 48h and 72h. Immediately after cell cycle synchronization, cells were used in nuclear transfer. Cell cycle state of the cells was evaluated by FACS analysis at 24h after beginning of CI and prior to nuclear transfer. Blastocyst rate was determined 7 days after nuclear transfer. An average of 38-42h in vitro matured oocytes were used in nuclear transfer (NT). NT was performed as described previously (Betthauser J et al., 2000 Nat. Biotechnol. 17, 456–461). There were no differences in the proportion of cells in G0/G1 of the cell cycle in any of the treatment groups (85.0%, 85.8%, 85.5% and 86.3% for SD and CI at either 24h, 48h and 72h, respectively). After nuclear transfer (for each CI group n=336–384 reconstr. embryos; SD n=215) there was a statistically significant difference in the fusion rate between 48h CI and SD cells (74.8% v. 87.5%, t-test P<0.050). Blastocyst rate (blastocysts/fused) differed significantly between SD, 24h CI and 48h CI (17.4%; 9.1%, 9.6%, t-test P<0.050), there was no difference between SD and 72h CI and within the CI groups (72h CI 10.6%). Four transfers of reconstructed embryos (72h CI, n=138–163 embryos/gilt, 1-cell embryos) to prepuberal Landrace gilts led to 2 initial pregnancies determined at Day 25 by ultrasound. One pregnancy was lost at Day 35; the other recipient remained pregnant and farrowed 4 piglets. One piglet was stillborn and one died 7h after birth; the remaining two piglets are healthy and now 4 months old. Four transfers of embryos (n=96–110) reconstructed with SD cells revealed two initial pregnancies determined on Day 25 by ultrasound. Again, one was lost on Day 35, and the other one is now at Day 100. Our results show that, despite similar proportions of cells being in G0/G1 of the cell cycle, cells either contact-inhibited for 72h or serum-deprived both show higher rates of blastocyst development compared to cells contact-inhibited for shorter time periods. Both donor cell preparations can lead to full term development of nuclear transfer-derived embryos. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, SFB 265).
7UTILIZATION OF CELL PROFILING TO EVALUATE BOVINE SPERMATOZOA IN NORMAL AND SIMULATED MICROGRAVITY
Authors: Andrews J.C. , Winters-Hilt S.
Published: February 2004

We developed a method to evaluate bovine sperm membranes in normal (1G) and simulated microgravity (Sim-μG). Bovine spermatozoa are used as a model system because they have cellular membranes analogous to those of other cell types, and yet are much simpler because they have no cytoplasm and do not participate in DNA transcription or mRNA translation. They can be cultured as single cells and are easily evaluated for membrane characteristics using flow cytometry. These features make the mammalian spermatozoon a useful model for exploring the principles of membrane structure/function in the presence of a variety of environmental challenges such as simulated microgravity. Cryopreserved, washed beef bull sperm (4–8×106mL−1)were incubated under non-capacitating conditions (modified glucose-free Tyrode’s medium containing low bicarbonate, HEPES buffer, pyruvate and 3mgmL−1 BSA V; 23°C in air), and these spermatozoa remained alive for 24–48h at 1G. To simulate μG, spermatozoa were incubated under the same conditions, in a HARV 10 rotating wall vessel (RWV, Synthecon, Inc, Houston, TX, USA) at 9rpms. Spermatozoa were incubated in 1G and Sim-μG environments for 2.5–4.5h and subsequently exposed to 0, 60 or 80μgmL−1 LC for 0, 4, 8, 12, 16 and 20min. Three fluorochrome combinations were used as probes at each [LC]/time point: (1) propidium Iodide (dead status)+SYBR 14 (live status); (2) PI+FITC-PSA (acrosome reactions [ARs]); (3) PI+MitoTracker Deep Red (mitochondrial activity). Approximately 1million spermatozoa from 3 bulls were evaluated over 4 days. Data were acquired on a FACSVantage SE flow cytometer, and initially analyzed (quality control) using the bundled FACSVantage SE software package (Cell Quest, BD BioSciences, San Jose, CA, USA). This provided graphics of simple cell relations (fluorescence v. LC exposure time). For further statistical analysis, and incorporation of non-parametric statistical tools (including pattern recognition using Support Vector Machines), the data were processed using a collection of Perl scripts and C programs. Results: Live/dead status: When Sim-μG+60μgmL−1 LC sperm were compared to 1G+60μgmL−1LC, and 80μgmL−1 LC sperm, their profiles were more similar to the 1G 80μgmL−1 LC profiles. AR status: the Sim-μG+60μgmL−1 LC profiles were similar to the 1G+60μgmL−1 LC profiles. Mitochondrial Status: the Sim-μG+60μgmL−1LC profiles were more similar to 1G+80μgmL−1 LC profiles. Summary: although Sim-μG sperm lost their motility within 3h, they were alive. Cell profiles indicate that Sim-μG sperm nuclear membranes are less stable and their mitochondria are less functional than the 1G controls, but their acrosomes are intact indicating that fertilizing potential may remain. Additional experiments are needed to determine the time course for Sim-μG, induced changes, and whether Sim-μG sperm can penetrate eggs. Funding: NASA (2002)-Stennis-24 and The University of New Orleans.
71EFFECT OF CALCIUM IONOPHORE AND CYTOCHALASIN D ON ACTIVATION AND IN
VITRO DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS
Authors: Rzucidlo S.J. , Arat S. , Stice S.L.
Published: February 2004

The activation of oocytes is one of the most important steps for a successful cloning, and chemicals used for activation can affect the viability of cloned offspring. Therefore, some of them may be omitted for activation to eliminate their possible detrimental effect on nuclear transfer (NT) embryos. The objective of this study was to examine the effect of calcium ionophore (CaI, A23187, Sigma, St. Louis, MO, USA) and cytochalasin D (CD) on activation and in vitro development of nuclear transfer units derived from bovine granulosa cells (GCs) treated with the cell cycle inhibitor, roscovitine. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin, rIGF-1, bFSH, and bLH. GCs were isolated from ovarian follicles and cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Prior to NT, donor cells were exposed to 15mM roscovitine for 24 hours and small cells were used for NT. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused using a 20μs DC pulse of 40V/150μm. Two hours after fusion, NT units were assigned into four groups and activated by CaI (5μM for 10min.), and then incubated with cycloheximide (CHX, 10μgmL−1)+CD (2.5μgmL−1) for 1h, followed by CHX alone (without CD) for 5h (Group I) or CaI (for 10min.), followed by CHX alone for 6h (Group II). In the Group III, NT units were activated by CHX (10μgmL−1)+CD (2.5μgmL−1) for 1h, followed by CHX for 5h, and Group IV, CHX alone for 6h. The base activation medium was TCM199 with 1% FBS for CaI and 10% FBS for CHX and CD. After activation, NT units were cultured for 7 days in BARC medium. Differences in activation, cleavage and blastocyst formation rates among treatments were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. Our data showed that CaI and CD did not affect the activation and in vitro development of NT embryos derived from roscovitine-treated GCs. It suggests that both chemicals may be redundant during cloning procedure. This study was supported by a grant from ProLinia, Inc and TUBITAK, Turkey (VHAG-1908-102V048).

Table 1
In vitro development of NT embryos in different activation treatments
72EFFECTS OF GENE EXPRESSION IN BOVINE EMBRYOS RECONSTRUCTED WITH
FIBROBLASTS TRANSFECTED WITH LUCIFERASE GENE ON THE SUBSEQUENT
DEVELOPMENT
Authors: Saeki K. , Tamari T. , Kasamatsu A. , Shirouzu K. , Taniguchi S. , Matsumoto K. , Hosoi Y. , Iritani A.
Published: February 2004

During embryo development, embryonic gene activation (EGA) is one of the first critical events. Inappropriate EGA results in failure of further development. We have reported that gene expression in bovine embryos reconstructed with fibroblasts begins at 48 hours postfusion (hpf) and reaches a maximum level at 60hpf as detected by their bioluminescence following injection of chicken β-actin/firefly luciferase fusion gene (β-act/luc+) into their nuclei (Saeki et al., 2001 Theriogenology 55, 289). In the present study, effects of gene expression in embryos reconstructed with bovine fibroblasts transfected with luciferase gene on their subsequent development to the blastocyst stage were examined. Cultured bovine fibroblasts taken from an ear of a female calf were transfected with plasmid containing β-act/luc+/IRES/EGFP and neor using GeneJammer (StrataGene, La Jolla, CA, USA). Neomycin-resistant cells were selected by culturing with G418. Then, EGFP-positive colonies were further selected under fluorescence microscopy to obtain stably transfected cells. The transfected cells were cultured for several passages. Growing (50 to 60% confluence, GCs) and serum-starved cells (SCs) were used as donor cells. In vitro-matured bovine oocytes derived from slaughterhouse ovaries were enucleated at 20h post maturation. Enucleated oocytes were electrofused with the cells, and activated with calcium ionophore and cycloheximide. Luminescence in the embryos was detected with an imaging photon counter at 0 and 60hpf. Luminescence-positive (P) and -negative (N) embryos were cultured separately at each detection time. Embryos were cultured until 168hpf, and examined for cleavage and blastocyst development. Experiments were repeated 3 times, and totals of 91 and 123 embryos were reconstructed with GCs and SCs, respectively. Data were analyzed with Fisher’s PLSD test following ANOVA by Stat View software (Ver. 5.0). At 0hpf, luminescence was detected in 55 and 4% of embryos reconstructed with GCs and SCs, respectively. At 60hpf, luminescence was detected in 47 and 28% of P and N embryos with GCs, and 17 and 40% of P and N embryos with SCs at 0hpf, respectively. Cleavage rates were not different among groups (P>0.05). Blastocysts were obtained only from the groups of embryos that were N at 0hpf and P at 60hpf (8% with GCs and 17% with SCs). No embryos in the other groups developed to the blastocyst stage. These results suggest that appropriate gene expression in embryos reconstructed with somatic cells is important for their subsequent development and that detecting the reporter gene expression can be used for selection of viable cloned embryos.
75HIGHLY EFFICIENT AND RELIABLE CHEMICALLY ASSISTED ENUCLEATION METHOD
FOR HANDMADE CLONING IN CATTLE AND SWINE
Authors: Vajta G. , Peura T.T. , Lai L. , Murphy C.N. , Prather R.S. , Kragh P.M. , Holm P. , Callesen H.
Published: February 2004

In bovine and porcine nuclear transfer, most traditional enucleation procedures require potentially harmful chromatin staining and UV illumination. The purpose of our work was to find an efficient and reliable chemically-assisted procedure for enucleation connected to the handmade cloning (HMC) technique without chromatin staining. Slaughterhouse-derived oocytes were collected and matured in vitro. At 21 (bovine) or 43 (porcine) h after the start of maturation, cumulus cells were removed with vortexing and oocytes were further incubated in the maturation medium supplemented with 0.5μgmL−1 demecolcine for 2h. Subsequently, zonae pellucidae were digested with 2mgmL−1 pronase in the presence of 10% cattle serum (CS) for 6 to 8min and washed in HEPES-buffered TCM-199 medium and 20% CS. Bisection was performed in the same medium by hand under a stereomicroscope by using a microblade. A small membrane protrusion observable on the surface of oocytes was used as an orientation point. One-third of the cytoplasm connected to this protrusion was removed, and the cytoplasts and karyoplasts were collected separately. Bovine cytoplasts were used as recipients for HMC experiments (Vajta et al., 2003, Biol. Reprod. 68, 571–578) with fetal fibroblasts as donors, and reconstructed embryos were cultured for 7 days. In Experiment 1 (3 replicates), the possibility of oriented bisection at different time points was determined on a total of 225 bovine oocytes. At 5, 15, 25, 35 and 55min after the end of pronase digestion 64, 91, 93, 72 and 59% of oocytes had membrane protrusions (P<0.05 between all groups, SAS Genmod) illustrating the time-dependent manner of the protrusion. In Experiment 2, the efficiency and reliability of enucleation was measured. Bisection was performed between 5 and 35min after pronase digestion. Subsequently both supposed cytoplasts and karyoplasts were stained with Hoechst and investigated under UV light. In cattle (9 replicates), bisection was successfully performed in 94% (519/552) of oocytes, and 98% (507/517) of those bisected were enucleated, i.e. the chromatin was entirely in the presumptive karyoplast. In swine (3 replicates), 91% (302/331) of oocytes were successfully bisected and 95% (280/296) were enucleated. In Experiment 3 (cattle; 4 replicates), blastocyst per reconstructed embryo rates were 47% (139/293), illustrating the high developmental ability in vitro. Considering that no oocyte selection based on the presence of polar body was performed, the above system seems to be more efficient and reliable than other enucleation methods. Moreover, expensive equipment (inverted fluorescent microscope) and a potentially harmful step (staining and UV illumination) can be eliminated from the HMC without compromising the high in vitro efficiency.
77CLONED EMBRYO DEVELOPMENT IN VITRO: COMPARISON OF CONVENTIONAL AND
INDUCED ENUCLEATION PROCEDURES FOR BOVINE CYTOPLAST PREPARATION
Authors: Wang M.-K. , Overstrom E.W.
Published: February 2004

Induced enucleation (IE) of oocytes with demecolcine produces competent ooplasts for SCNT as demonstrated previously in mouse, goat, cow and pig. Whether bovine IE cytoplasts are more or less competent than conventionally enucleated MII oocytes to support nuclear reprogramming of somatic chromatin and embryo development in vitro is not known. This study compared in vitro development of cloned bovine embryos produced by conventional and IE enucleation methods. Three experimental groups were: (1) Parthenogenetic controls. In vitro-matured, MII-arrested bovine oocytes were activated by a single (1×Act, 10μM ionomycin in Tyrodes-HEPES, 5min) or double activation (2×Act; 1×Act, wash 5min, 10μgmL−1 cycloheximide [CHX] 20min, repeat 1×Act) followed by incubation in CHX and 5μgmL−1 cytochalasin B (CB) for 6h, and then culture (BARC medium) for 7 days. (2) Conventional SCNT. MII oocytes were enucleated by micromanipulation in HEPES-buffered enucleation medium (BARC containing 7.5μgmL−1 CB, 5μgmL−1 Hoechst 33342, 10% FBS) under UV illumination (3–5s). Donor cells (fibroblasts, passage 7–9) were inserted into the perivitelline space, and the reconstructed couplets activated (1×Act). Reconstructed couplets were then electrofused, placed in BARC medium containing 10μgmL−1 CHX and 5μgmL−1 CB (6h), and then cultured for 7 days. (3) IE SCNT. MII oocytes were activated (1×Act), placed into BARC-5% FBS containing 0.4μgmL−1 demecolcine (DEME), 10μgmL−1 CHX, 2μgmL−1 cytochalasin D for 20min, then 20min without DEME, then returned to DEME. At 1–1.5h post-activation, the extruding second polar body (PB2) containing nuclear chromatin was removed by micromanipulation, couplets were reconstructed and fused as above, and additionally activated (two pulses, 20–30V/mm, 20μs). Embryos were cultured in 10μgmL−1 CHX and 5μgmL−1 CB medium for 4–5 hour, then BARC for 7 days. The results (Table 1) reveal that 2×Act increases embryo development at Day 2, but not Day 7. Further, there are no significant differences in embryo development rates between conventional and IE SCNT protocols. Respectively, 46%, 32% and 21% of cleaved control (1×Act), conventional and IE embryos developed to 16 cells on Day 7. In vitro development of cleavage embryos to the blastocyst stage was greater in controls (25–32%) than in conventional (22%) and IE (17%) SCNT groups on Day 7. Further comparisons of in vivo development between conventional and IE SCNT methods following embryo transfer are warranted. Supported by ACT, Cyagra and USDA NRI \#2001-35205-09966.

Table 1
Embryo development: Conventional v. induced enucleation