Donors For Nuclear Transfer Research Articles

Production of Cloned Calves by the Transfer of Somatic Cells Derived from Frozen Tissues Using Simple Portable CO2 Incubator

The ability of frozen-thawed fetal skin was examined to generate viable cell lines for nuclear transfer. Fetal skin frozen at -20°C, -30°C or -80°C in the presence of 5% DMSO used as tissue explants to generate somatic cells. The resultant confluent cells were then used as donors for nuclear transfer (NT). Of the bovine NT embryos reconstracted from the somatic cells, 62.3%, 76.6% to 65% showed cleavage 70.5%, 81.9% to 78.5% reached the stage of morula formation and 39.7%, 43.2% or 47.6% reached the blastocyst stage. There was no significant difference in development when the NT embryos were compared with those reconstracted from fresh somatic cell derieved skin tissues (72%, 75.3%, and 45.2%, for cleavage, and development to morula and blastocyst stage, respectively). NT embryos were then placed in a portable CO2 incubator and carried to China from Japan by air. After reaching to farm, two NT embryos were transferred to each of 5 recipients. We obtained 2 NT calves which birth weights is 30kg and 36kg female, and gestation periods is 281 and 284 days, respectively. There were no observation any abnormality from those calves. The results indicated that cell lines derieved from bovine fetal skin cryopreserved by a simple method could be used as donors in nuclear transfer using the portable CO2 incubator. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 2 : 168-173)

33DEVELOPMENT OF BOVINE EMBRYOS CLONED WITH FETAL FIBROBLASTS ARRESTED AT G0/G1 PHASE BY DIFFERENT TREATMENTS

Numerous factors have an effect on the development of cloned embryos, and one of the most important might be the synchronization between donor nuclei and recipient ooplasts. The objective of this study was to examine the effect of donor cell treatments for G0/G1 synchronization and the donor cell type on development and incidence of apoptosis in cloned cattle embryos. Primary cultures were established from a female fetus on Day 50 of gestation and adult ear skin biopsies. The cells were used for assessements of cell cycle and apoptosis, and for nuclear transfer. Cells were randomly allocated into 3 experimental treatment groups after 6–8 passages: Group 1 (confluent), cells were cultured in DMEM supplemented with 10% FBS until 90% confluent; Group 2 (serum-starvation), cells were cultured in DMEM supplemented with 0.5% FBS for 5 days; Group 3 (Roscovitine), cells were cultured in DMEM supplemented with 10% FBS and 30μM Roscovitine for 12h. Cell cycle and apoptosis were analyzed using flow cytometry after labelling with DAPI and YO-PRO-1, respectively. At 19h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and fused by a single DC pulse (1.6kV/cm, 60μs) delivered by a BTX 200. After activation with the combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1, 5h), the eggs were cultured in CR1aa medium for 3 days and additionally cultured in CR1aa medium supplemented with 30mgmL−1 BSA for 5 days at 39°C in a humidified atmosphere of 5% CO2 in air. Differences between groups were analyzed using one-way ANOVA after arc-sine transformation of the proportional data. There were no significantly differences in the incidence of cells arrested at G0/G1 for fetal fibroblasts cultured in the three treatment groups (87%, 83% and 80%; confluent, serum starvation and Roscovitine, respectively). More cells were apoptotic in Group 2 compared to the cells in Groups 1 and 3 (12% v. 6 and 6%, respectively) (P<0.05). Blastocyst development of cloned embryos was significantly (P<0.05) higher when fetal fibroblasts from Group 1 were used, compared to Groups 2 and 3 (35.1%, v. 31 and 29.7%, respectively). Similar results were observed in the use of ear skin fibroblasts as nuclear transfer donor cells (32.7%, v. 24 and 24%, respectively). These results suggest that fetal fibroblasts can be effectively synchronized at G0/G1 by three different treatments, including growth to confluence, serum-starvation and Roscovitine treatment. However, based on blastocyst development and levels of apoptosis, the use of confluent fetal fibroblasts as donor cells is more effective than using cells synchronized by serum-starvation or Roscovitine treatment in the production of cloned bovine embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 30012-05-3-SB010 and Cho-A Pharm. Co. LTD.]

Reprogramming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning.

Germ cells undergo epigenetic modifications as they develop, which suggests that they may be ideal donors for nuclear transfer (cloning). In this study, nuclei from confirmed embryonic germ cells were used as donors to determine whether they are competent for cloning and at which stage they are most competent. Embryos cloned from migrating 10.5-days-postcoitum (dpc) primordial germ cells (PGCs) showed normal morphological development to midgestation but died shortly thereafter. In contrast, embryos cloned from later-stage germ cells were developmentally delayed at midgestation. Thus, donor germ cell age inversely correlated with the developmental stage attained by cloned embryos. The methylation status of the H19- and Snrpn-imprinting control regions in germ cell clones paralleled that of the donors, and revealed that demethylation, or erasure of imprints, was already initiated in PGCs at 10.5 dpc and was complete by 13.5 dpc. Similarly, clones derived from male 15.5-dpc germ cells showed increased methylation correlating with the initiation of de novo methylation that resets imprints at this stage, and clones from neonatal germ cells showed nearly complete methylation in the H19 imprinting control region. These results indicate that the epigenetic state of the donor nucleus is retained in cloned embryos, and that germ cells are therefore inadequate nuclear donors for cloning because they are either erasing or resetting epigenetic patterns.

Evaluation of gene targeting by homologous recombination in ovine somatic cells.

Mouse models for some human genetic diseases are limited in their applications since they do not accurately reproduce the phenotype of the human disease. It has been suggested that larger animals, for example sheep, might produce more useful models, as some aspects of sheep physiology and anatomy are more similar to those of humans. The development of methods to clone animals from somatic cells provides a potential novel route to generate such large animal models following gene targeting. Here, we assess targeting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in ovine somatic cells using homologous recombination (HR) of targeting constructs with extensive (>11 kb) homology. Electroporation of these constructs into ovine fetal and post-natal fibroblasts generated G418-resistant clones, but none analyzed had undergone HR, suggesting that at least for this locus, it is an extremely inefficient process. Karyotyping of targeted ovine fetal fibroblasts showed them to be less chromosomally stable than post-natal fibroblasts, and, moreover, extended culture periods caused them to senesce, adversely affecting their viability for use as nuclear transfer donor cells. These data stress the importance of donor cell choice in somatic cell cloning and suggest that culture time be kept to a minimum prior to nuclear transfer in order to maximize cell viability.

Gene expression in cloned bovine fetal liver.

Nuclear transfer (NT) is a method of animal reproduction that bypasses fertilization and propagates known combinations of genes. Currently NT is an inefficient process. Attempts have been made to increase the efficiency of this procedure, but most have been deemed unsuccessful. Some problems associated with NT are unusually large birth weights, and physical abnormalities in developing liver, heart, and brain. Despite numerous studies performed on NT animals, the factors behind the anomalies remain unknown. It is possible that nuclear reprogramming is the basis of poor development rates, meaning, when the donor cells are fused with enucleated eggs the nuclei may not regain the full ability to direct cell differentiation in subsequent mitotic divisions. If reprogramming is not carried out precisely, then some genes may not be correctly expressed in NT animals. The purpose of this study was to determine if differential gene expression between the livers of NT fetuses when compared to an embryo transfer (ET) derived fetus could be detected and the genes identified. An Angus fetus at 45 d of gestation was collected and a non-clonal cell line established for use as NT donor cells. Two NT fetuses were propagated and compared to the original. Differential Display Reverse Transcription Polymerase Chain Reaction (ddRT-PCR) was used to identify genes that were differentially expressed. Differentially abundant cDNAs were subcloned, sequenced and their corresponding mRNAs were verified by semi-quantitative RT-PCR. Twenty-three Expressed Sequence Tags (ESTs) were sequenced in Bos taurus and submitted to GenBank. The results of ddRT-PCR identified 39 genes/ESTs that were potentially differentially expressed. Fifteen of the genes were tested by semi-quantitative RT-PCR, but no significant differences were detected.

Nuclear transfer using a bovine mammary epithelial cell line (BMEC)

The developmental potential of nuclei from a bovine mammary epithelial cell line (BMEC) in nuclear transfer was investigated. For nuclear transfer donors, BMEC cells (passage 15) were cultured for 4–5 days after seeding at cell densities of 1.0 × 105 cells/cm2 (high-density group) or 0.8 × 104 cells/cm2 (low-density group). First, the effective electric stimulation for fusion of enucleated oocytes with BMEC cells was examined. Fusion rates reached maximum with two DC pulses of 30 V/150 µm for 20 µs in the high-density group and with two DC pulses of 25 V/150 µm for 10 µs in the low-density group. The fusion rate (37.5%) in the high-density group was significantly (P < 0.005) lower than in the low-density group (71.4%). Second, the in vitro developmental potential of nuclear transfer embryos derived from BMEC cells was examined. In the high-density and low-density groups, 18.8% and 24.1% of fused oocytes, respectively, developed to blastocyst stage. The results of this study indicate that nuclei from BMEC cells support the development of nuclear transfer embryos to the blastocyst stage and that the efficiency of oocyte–cell fusion is affected by the culture conditions of the donor BEMC cells before nuclear transfer.

Transgenic technology and applications in swine

The introduction of foreign DNA into the genome of livestock and its stable integration into the germ line has been a major technical advance in agriculture. Production of transgenic livestock provides a method to rapidly introduce “new” genes into cattle, swine, sheep and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Several recent developments will profoundly impact the use of transgenic technology in livestock production. These developments are: 1) the ability to isolate and maintain in vitro embryonic stem (ES) cells from preimplantation embryos, embryonic germ (EG) and somatic cells from fetuses; and somatic cells from adults, and 2) the ability to use these embryonic and somatic cells as nuclei donors in nuclear transfer or “cloning” strategies. Cell based (ES, EG, and somatic cells) strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. There are many potential applications of transgenic methodology to develop new and improved strains of livestock. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition and increased disease resistance. Development of transgenic farm animals will allow more flexibility in direct genetic manipulation of livestock.

Bovine nuclear transfer embryo development using cells derived from a cloned fetus

Many different cell types have been used to generate nuclear transfer embryos and fetuses. However, little is known about the potential of fibroblasts derived from a nuclear transfer fetus as donor cells for nuclear transfer. The ability of cloned fetuses or animals to be cloned themselves is of great interest in determining whether successive generations of clones remain normal or accumulate genetic or phenotypic abnormalities. We generated a bovine fibroblast cell line from a cloned fetus, that continued to divide beyond 120 days (94 doublings,18 passages) in continuous culture. As long-term survival of cells in culture is a desirable characteristic for use in transgenic cell production, passage 2 and 18 cells were compared as donor cells for nuclear transfer (NT). When cells from passage 2 (2 weeks in culture) and passage 18 (4 months in culture) were used for nuclear transfer, there was no significant difference in development rate to blastocyst (35.4 versus 44.6%, P=0.07). A greater proportion of late passage cells were in G0/G1 whether under serum-fed (64 versus 56%, P<0.01) or serum-starved (95 versus 88%, P<0.01) culture conditions. Following embryo transfer, equivalent day 30 pregnancy rates were observed for each group (P 2: 2/19 versus P 18: 2/13). A slightly retarded fetus was surgically removed at day 56 and the remaining three fetuses died in utero by day 60 of gestation. Our results show that fibroblast cells derived from regenerated cloned fetuses are capable of both in vitro and in vivo development. The longevity of this regenerated cell line would allow more time for genetic manipulations and then to identify stable transfected cells prior to their use as NT donor cells. Although no live fetuses were produced in this study the results provide encouraging data to show that a cloned fetus can itself be recloned to produce another identical cloned fetus. Further studies on this and other recloned fetuses are necessary to determine whether the failure to produce live offspring was a result of inadequate sample size or due to the cell type selected.

Developmental competence of bovine embryos reconstructed by the transfer of somatic cells derived from frozen tissues.

The ability of frozen-thawed fetal skin was examined to generate viable cell lines for nuclear transfer. Fetal skin frozen at -35 degrees C or -80 degrees C in the presence of 5% DMSO were used as tissue explants to generate somatic cells. The resultant confluent cells were then used as donors for nuclear transfer (NT). Of the bovine NT embryos reconstructed from the somatic cells, 78% to 81% showed cleavage, 43% to 48% reached the stage of morula formation and 34% to 35% reached blastocyst formation. There were no significant differences in development (P>0.05) when the NT embryos were compared with those reconstructed from fresh somatic-cell-derived skin tissues (75%, 45%, and 38%, for cleavage, and development to morula and blastocyst stages, respectively). The results indicated that cell lines derived from bovine fetal skin cryopreserved by a simple method could be used as donors in nuclear transfer. The resulting embryos showed similar development in vitro to those reconstructed from unfrozen fetal-skin-derived somatic cells.

Bovine Nuclear Transfer Using Cumulus Cells Derived from Serum-Starved and Confluent Cultures

Bovine cumulus cells after oocyte maturation were cultured under serum-starvation condition or grown to confluence. Their cell size, cell cycle phases, and developmental potential as donors for nuclear transfer (NT) were investigated. The majority of trypsinized cells of both cultures were found within the medium size range (15-20 μm). Flow cytometric analysis revealed that small- and medium-sized cells had significantly higher percentages of nuclei existing in the G0/G1 phase (95-98%) than large-sized cells (82%) regardless of the culture conditions. Serum-starved cells of different sizes (small, 9-14 μm; medium, 15-20 μm; large, 21-26 μm) had the same fusion rate, cleavage, development to blastocysts, and blastocyst cell number of NT embryos. NT embryos reconstituted from serum-starved and confluent medium-sized cells showed the same developmental rate. These results indicate that the most available medium-sized cells derived from both serum-starvation and confluent cultures can be used as a G0/G1-phase nuclei source.

Influence of the timing of blastomere isolation after the removal of nocodazole in bovine nuclear transfer

This study was conducted to investigate the influence of the timing of blastomere isolation after the removal of nocodazole on the subsequent division of blastomeres and developmental ability of reconstituted bovine embryos. The division rate of isolated blastomeres was examined at 3, 5 and 24 h of culture after nocodazole removal. Furthermore, isolated blastomeres and those of whole embryos were used as donors in nuclear transfer to determine the development of reconstituted embryos. The division rate of isolated blastomeres at 3 h was significantly lower than the presumptive division rate of blastomeres from whole embryos (P<0.05). When these blastomeres were used as donor nuclei, the dividing blastomeres yielded a significantly higher development rate than blastomeres from whole embryos (P<0.05). These results confirm that the timing of blastomere isolation influences the subsequent division of blastomeres and the developmental ability of the reconstituted embryos.

Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts: Work in progress

Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 μM, 5 min) and cycloheximide (10 μg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified, in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% ± SEM) were not affected (83.7 ± 9.2 vs 79.8 ± 4.6; P>0.05), but cleavage (55.7 ± 2.9 vs 92.8 ± 3.9; P=0.0002) and blastocyst rates (7.2 ± 5.0 vs 32.6 ± 7.8; P=0.0025, vitrified vs nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos.

Cultured bovine mammary gland cells as donors for nuclear transfer

Cultured bovine mammary gland cells as donors for nuclear transfer

The use of vitrified multigenerational cloned cattle embryos as donors in nuclear transfer

The use of vitrified multigenerational cloned cattle embryos as donors in nuclear transfer

Human Factor IX Transgenic Sheep Produced by Transfer of Nuclei from Transfected Fetal Fibroblasts

Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.

Blastocysts as donors for nuclear transfer in cattle

Blastocysts as donors for nuclear transfer in cattle

Nuclear transfer in the bovine embryo: A comparison of 5‐day, 6‐day, frozen‐thawed, and nuclear transfer donor embryos

Micromanipulation and electrofusion were utilized for nuclear transfer in bovine embryos. Embryonic blastomeres from 5-day (estrus = day 0), 6-day, frozen-thawed 5-day, and first-generation nuclear transfer embryos (embryos were themselves a product of nuclear transfer with the original donor being a 5-day embryo) were transferred into bisected bovine oocytes by electrofusion. The percentage of donor cells fusing with the recipient oocytes was compared between different types of donor embryos. The percentage of embryos developing normally into morula or blastocysts following 6 days culture in the sheep oviduct was also recorded and compared between different donor embryo types. No significant differences were found between donor blastomeres for the percent successfully fused to oocytes: 5-day, 294 of 513 (57.3%); 6-day, 252 of 405 (62.2%); frozen-thawed 5-day, 111 of 144 (77.1%); nuclear transfer, 142 of 223 (63.7%); or the percent developing normally following nuclear transfer: 5-day, 92 of 444 (20.7%); 6-day, 84 of 357 (23.5%); frozen-thawed 5-day, 32 of 127 (25.2%); nuclear transfer, 31 of 199 (15.6%). These data suggest that a variety of donor embryos can successfully be utilized for bovine embryo cloning. Also, development of blastomeres from frozen-thawed 5-day donors and from donors that are themselves the product of nuclear transfer suggest that the production of multiple identical offspring is possible by frozen storage of seed stock and serial recloning.