Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle (original) (raw)
Related papers
Biology of Reproduction, 2002
This study examined bovine cloning strategies that may be used for gene targeting in animals of known phenotypic traits. Fibroblast cells derived from an adult and a fetus of the same genotype were transfected with a plasmid (pEGFP-N1) containing the enhanced green fluorescence protein and neomycin-resistant genes. After transfecting 2 ؋ 10 5 cells, 49 adult and 35 fetal cell colonies were obtained. Green fluorescence expression was observed in 35 out of 49 (71.4%) adult clones and in 30 out of 35 (85.7%) fetal clones. Developmental rates to the blastocyst stage following nuclear transfer (NT) did not differ among nontransfected cell lines (adult, 20.0%; NT fetal, 18.3%), whereas developmental rates were significantly lower for adult and fetal cell lines expressing enhanced green fluorescent protein (EGFP; 11.3% and 6.4%, respectively, P Ͻ 0.05). However, there was no decrease in NT developmental rates (19.8%) when donor nuclei from EGFP-transfected cell lines not expressing EGFP but retaining neomycin-resistant gene expression were used as donor nuclei. NT embryos from adult and fetal cell lines had similar morphology, cell number, and ploidy. The results indicated that adult and NT fetal cells (identical genotype) can complete clonal propagation, including transfection and selection, and can be used to produce transgenic NT embryos; however, a possible deleterious effect of EGFP on embryo development should be considered in future gene targeting studies.
Molecular Reproduction and Development, 2004
The present study examined effects of genetic manipulation and serum starvation on in vitro developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos and vitrification on in vivo developmental competence of transgenic SCNT blastocysts. Fetal oviduct epithelial cells (FOECs) were isolated from the oviduct of a Day 147 bovine fetus and transfected with a plasmid (pCE-EGFP-IRES-NEO) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes. There were no significant differences (P > 0.05) in cleavage rates or development rates to the blastocyst stage for SCNT embryos derived from FOECs (72.5 and 47.8%, respectively) or transfected FOECs (TFOECs, 73.8 and 47.7%, respectively); nor from serum-fed (73.6 and 47.2%, respectively) or serum-starved (72.7 and 48.3%, respectively) cells. Seventeen of Day 7 GFPembryos (eight fresh blastocysts and nine vitrified/ thawed blastocysts ) were transferred to recipients with one embryo per recipient. Two (25%) recipients were confirmed pregnant at Day 60 in fresh blastocysts group, and three recipients (33%) were confirmed pregnant at Day 60 in vitrified/thawed blastocysts group. Two healthy calves (25%) were obtained from fresh blastocysts and one (11%) from vitrified/thawed blastocysts. Microsatellite analysis confirmed that the three clones were genetically identical to the donor cells. Moreover, PCR and Southern blot demonstrated integration of transgene in genomic DNA of all three cloned calves. Expression of GFP in skin biopsies isolated from transgenic cloned calves and fibroblasts derived from the skin biopsies revealed the activity of EGFP gene, and G418 resistance in vitro of these fibroblasts confirmed the activity of Neor gene. Our results show that genetic manipulation and serum starvation of donor cells (FOECs) do not affect in vitro developmental competence of bovine SCNT embryos, and vitrified transgenic SCNT blastocysts can develop to term successfully. Mol. Reprod. Dev. 69: 278-288, 2004. ß 2004 Wiley-Liss, Inc.
Electronic Journal of Biotechnology, 2011
Since the first successful somatic cell nuclear transfer (SCNT) experiments were carried out, a number of domestic and agriculture species have been cloned using donor cells derived from different sources and origin. However, differences in nuclear transfer efficiency both in vitro and in vivo have been generally observed. These differences may be accentuated when transgenic cell lines are used as nuclear donors in an attempt to generate transgenic cloned offspring. The present study examined the suitability of cell lines derived from 3 different fetal sources and the effects of genetic manipulation of donor fetal fibroblasts with a red fluorescent plasmid, on the in vitro developmental potential and quality of nuclear transfer derived bovine embryos. We observed no differences in the cleavage rate of nuclear transfer embryos generated with any of the cell lines evaluated. However, the blastocyst rate was significantly affected when cell lines were derived from the 3 different fetal sources (21, 18 and 11%, respectively) or from 2 transgenic clonal cell lines that had originated from the same primary fetal cell (18 and 10%, respectively). Despite this difference, quality of embryos as measured by the total number of cells and by assessing some morphology aspects of their appearance was not different. Together these results indicate that fetal fibroblast cell lines derived from different fetal sources and transgenic clonal cell lines that had originated from the same fetus results in different in vitro developmental potential when used as donors for nuclear transfer experiments. Further studies, including evaluation of pregnancy rates, development to term, and epigenetic modifications of these cell lines will be necessary to better understand the differences observed in nuclear transfer efficiency.
Molecular Reproduction and Development, 2001
Adult granulosa donor cells used in the nuclear transfer (NT) procedure can result in cloned cattle. Subsequently, it may be possible to use the same cell type to produce cloned transgenic cattle. Therefore, this study examined the effect of genetic manipulation and serum levels in culture of donor granulosa cells on developmental rates and cell number of bovine NT embryos. A primary cell line was established from granulosa cells collected by aspirating ovarian follicles. Cells transfected with a plasmid containing the enhanced green fluorescence protein (EGFP) gene, and non-transfected cells were used for cloning between passage 10 and 15 as serumstarved and serum-fed donor cells. There were no significant differences (P b 0.1) in cleavage rates or development to the blastocyst stage for NT embryos from transfected (60.4 and 13.5%, respectively) or non-transfected (61.9 and 14.1%, respectively) and serum-starved (60.6 and 13.4%, respectively) or serum-fed (61.3 and 14%, respectively) cells. Development rates to blastocyst stage of embryos produced using cells at passage 15 (27.1%) were significantly higher than those produced with cells at passage 10, 11, and 13 (7, 11.5, and 14%, respectively, P`0.05). Green fluorescence was observed at different intensity levels in all blastocyst stage embryos resulting from transfected donor cells. The results of the present study indicated that genetically modified granulosa cells can be used to produce transgenic NT embryos and primary transgenic adult cells at late passage may be more effective donor cells than earlier passaged cells. Mol. Reprod. Dev. 60: 20±26,
Production of transgenic calves by somatic cell nuclear transfer
Chinese Science Bulletin, 2004
Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector (pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neo r ) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was carried out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves, and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves. PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.
Genetics and molecular research : GMR, 2005
An association of two techniques, nuclear transfer (NT), and transfection of somatic animal cells, has numerous potential applications and considerable impact, mainly in agriculture, medicine, pharmacy, and fundamental biology. In addition, somatic cell nuclear transfer is the most efficient alternative to produce large transgenic animals. We compared in vitro and in vivo developmental capacities of NT using fibroblast cells isolated from a 14-month-old cloned Simmental heifer (FCE) vs the same line transfected with a plasmid containing neomycin-resistant genes (TFCE). There were no significant differences (P > 0.5) in either fusion (116/149 = 78% vs 216/301 = 72%), cleavage (78/116 = 67% vs 141/216 = 65%) and blastocyst (35/116 = 30% vs 52/216 = 24%) rates or in pregnancy rate at 30 to 35 days after embryo transfer (2/17 vs 3/17) between NT using FCE and TFCE, respectively. Transfection and long-term in vitro culture of transfected cells did not affect developmental capacity of ...
Bovine nuclear transfer embryo development using cells derived from a cloned fetus
Animal Reproduction Science, 2001
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 * Corresponding author. Present address: Section : S 0 3 7 8 -4 3 2 0 ( 0 1 ) 0 0 1 0 6 -3 J.R. Hill et al. / Animal Reproduction Science 67 (2001) 17-26 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.
Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture
Theriogenology, 1995
We described an exclusively in vitro procedure for cloning and recloning bovine embryos. Embryos obtained by IVM/IVF/IVC developed to the morula stage were used as blastomere donors in cunjunction with IVM recipient oocytes. Reconstructed embryos were developed in vitro in co-culture using bovine oviductal epithelial cells. The resulting morulae were used as donors for recloning under the same experimental conditions. No significant difference was observed between cloning and recloning in terms of development (rates of blastocysts: 12.9 versus 14.9%), in the number of nuclei per blastocyst (63.8 versus 49.1), or in pregnancy rates (35.7 versus 33.3%). The high variability observed between replicates and the correlation between results in first and second cycle nuclear transfer may suggest an inherant potential of individual donor embryos to support development by cloning.
Cloning, 2000
We have examined the in vitro and in vivo development of cloned embryos produced by incorporation of fetal fibroblast into in vitro matured and enucleated cow oocytes by direct injection and by fusion. For injection, nuclei were either mechanically isolated using the microinjection needle or chemically isolated by treatment with NP-40 lysis buffer. Fetal fibroblasts were serum starved and treated with calcium ionophore before injection to induce chromatin condensation. A range of 8% to 16% of successfully injected oocytes developed to blastocysts in culture and a total of nine pregnancies resulted from transfer of cloned embryos produced by this method. Nuclear transfer by fusion resulted in 22% development to blastocysts. Unlike in mice, the embryos derived from injection did not result in viable pregnancies, which may suggest species differences. All pregnancies were terminated after 45 to 150 days from transfer. Two pregnancies resulted from transfer of cloned embryos obtained by fusion which produced two healthy female calves. The study proposes an alternative method for the production of cow cloned embryos. Further research, however, is required to optimize bovine cloning by injection.
Production of transgenic porcine blastocysts by hand-made cloning
Reproduction, Fertility and Development, 2004
Recently, a zona-free technique for bovine somatic cell nuclear transfer (NT) with no requirement for micromanipulation (i.e. hand-made cloning (HMC)) has been described. The present study demonstrates the application of the HMC technique in the production of transgenic porcine blastocysts. In vitro-matured zona-free porcine oocytes were bisected manually using a microblade and halves containing no chromatin (i.e. the cytoplasts) were selected. Two cytoplasts were electrofused with one transgenic fibroblast expressing enhanced green fluorescent protein and reconstructed embryos were activated in calcium ionophore (A23187) followed by 6-dimethylaminopurine. Subsequently, embryos were cultured in NCSU-23 medium supplemented with 4 mg mL–1 bovine serum albumin for 7 days. In five replicates, 93.0 ± 7.0% (mean ± s.e.m.) of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, 37/37 and 28/28). On Day 7 after activation, the respective blastocyst rates (per ...