Production of cloned goats by nuclear transfer of cumulus cells and long-term cultured fetal fibroblast cells into abattoir-derived oocytes

2006 ◽  
Vol 73 (7) ◽  
pp. 834-840 ◽  
Author(s):  
Guo-Cheng Lan ◽  
Zhong-Le Chang ◽  
Ming-Jiu Luo ◽  
Yun-Liang Jiang ◽  
Dong Han ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Fibroblast Cells ◽  
Fetal Fibroblast

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

2012 ◽  
Vol 22 (1) ◽  
pp. 131-142 ◽  
Author(s):  
Amir Amiri Yekta ◽  
Azam Dalman ◽  
Poopak Eftekhari-Yazdi ◽  
Mohammad Hossein Sanati ◽  
Abdol Hossein Shahverdi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Coagulation Factor ◽  
Mammary Glands ◽  
Factor Ix ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Coagulation Factor Ix ◽  
Transgenic Goats

20 PRODUCTION OF NUCLEAR TRANSFER EMBRYOS FROM PORCINE FETAL FIBROBLAST CELLS CARRYING A TETRACYCLINE-INDUCIBLE TRANSGENE

2006 ◽  
Vol 18 (2) ◽  
pp. 118
Author(s):  
K. S. Ahn ◽  
M. Kwon ◽  
B. C. Koo ◽  
J. Y. Won ◽  
S. Y. Heo ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Transgenic Animals ◽  
Transformed Cells ◽  
Fibroblast Cells ◽  
Transgenic Pigs ◽  
Blastocyst Development ◽  
Fetal Fibroblast ◽  
Gfp Gene ◽  
Retrovirus Vector ◽  
Porcine Fetal Fibroblast

Constitutive expression of A transgene often results in serious physiological disturbances in transgenic animals. For instance, systemic overexpression of human growth hormone in transgenic pigs has resulted in detrimental side effects in general health and reproductive performance. One of the solutions to such problem would be inducible expression of a transgene that may restrict production of foreign proteins from transgenic animals only when needed. In this study, a retrovirus vector was designed to express the green fluorescent protein (GFP) gene under the control of the tetracycline-inducible promoter. Transformation of porcine fetal fibroblast cells was achieved by infection of the cells with the vector and subsequent antibiotic selection. To induce transgene expression, transformed porcine fetal fibroblast cells were cultured in medium supplemented with doxycycline for 48 h. Induction of the GFP gene was verified by the emission of fluorescence from transformed cells. Nuclei of transformed cells with or without doxycycline treatment were transferred into enucleated oocytes, and the induction efficiency was analyzed by monitoring fluorescent emission during development of reconstituted embryos to the blastocyst stage. In addition, differences in the rates of blastocyst development between experimental groups were analyzed by Student's t-test. Blastocyst formation of nuclear transfer embryos using transformed cells with tetracycline-inducible retrovirus vector (12.0%, 128/1072) was not significantly different (P > 0.05) from that with non-inducible control vectors (13.7%, 41/300), suggesting that an introduction of tetracycline-inducible retrovirus vector was not particularly harmful to the development of nuclear transfer embryos. Also, the blastocyst development rate of nuclear transfer embryos after induction of transgene by doxycycline (12.1%, 99/815) was not significantly different (P > 0.05) from that of the non-induced counterparts (11.3%, 29/257), suggesting that the induction of transgene did not affect the development of transgenic clone embryos. In a majority of embryos, high expression of the GFP gene was observed in cloned embryos with transgene induction, whereas poor or no GFP expression was detected in non-induced controls. The results from this study suggest that tetracycline-inducible expression of transgenes in nuclear transfer embryos may be used for production of foreign proteins in transgenic animals in a more controlled manner than with conventional procedures. Further experiments on transfer of cloned embryos carrying such an inducible transgene to recipients may enable production of transgenic pigs with fewer side effects from unregulated expression of the transgene.

scholarly journals Cats cloned from fetal and adult somatic cells by nuclear transfer

Reproduction ◽  
2005 ◽  
Vol 129 (2) ◽  
pp. 245-249 ◽  
Author(s):  
X J Yin ◽  
H S Lee ◽  
Y H Lee ◽  
Y I Seo ◽  
S J Jeon ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Nuclear Transfer ◽  
Cell Mass ◽  
Somatic Cells ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Skin Cells ◽  
Fetal Fibroblasts ◽  
Adult Skin

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.

scholarly journals 74PREGNANCY ESTABLISHMENT OF NUCLEAR TRANSFERRED EMBRYOS AFTER VITRIFICATION AND EMBRYO TRANSFER IN CATTLE

2004 ◽  
Vol 16 (2) ◽  
pp. 158
Author(s):  
P.C. Shen ◽  
J.C. Huang ◽  
C.H. Wang ◽  
W.T.K. Cheng ◽  
L.Y. Sung ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Commercial Application ◽  
High Survival ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Rehydration Solutions ◽  
Vitrification Solution

The success of cryopreservation of bovine cloned embryos is important to commercial application of somatic nuclear transfer (NT) in the dairy and beef industry, especially for solving the problem of embryo transfer (ET) between fresh NT embryos and recipients. This experiment was conducted to test the possibility of establishing pregnancy by embryo transfer of vitrified bovine NT embryos. Oocytes were aspirated from antral follicles of slaughterhouse ovaries, and subsequently cultured in maturation medium for 20h. Cumulus cells were then denuded from the oocytes by vigorous vortexing for later enucleation and donor nuclear transfer. Skin fibroblast cells used for NT were derived from cultured ear explants taken from an elite dairy cow, and cumulus cells were cultured from the cumulus-oocytes complexes collected by ultrasound-guided transvaginal retrieval. Fibroblasts and cumulus cells were cultured at passage 5 or 6 in 10% FBS DMEM at 37°C in 5% CO2 humidified air, and used as nuclear donors. After donor cell transfer, somatic cell-cytoplast pairs were then fused by applying two direct current pulses at 2.0kVcm−1 for a duration of 10μs/pulse. Fused embryos were activated in 10mgmL−1 cycloheximide in M199+7.5% FBS for 4h. Embryos were cultured in CR1aa plus 3mgmL−1 BSA for 2 days (initiation of activation=Day 0) at 39°C, 5% CO2, 5% O2 and 90% N2, and then cultured on bovine cumulus monolayers in CR1aa medium supplemented with 7.5% FBS for 5 successive days. Expanding and hatching blastocysts on Day 7 were selected for cyropreservation via liquid nitrogen surface vitrification (LNSV). Vitrification solution contained HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol and dimethylsulphoxide. A droplet of 1–2μL vitrification solution containing two blastocysts was directly dropped onto a cooled surface within 30s after 3min incubation in equilibration solution. Vitrified NT embryos were kept at −150°C vapor phase until recipients were synchronized to Day 7 for ET. Embryos are warmed and subsequently washed several times in rehydration solutions and M199+7.5% FBS medium. The warmed embryos from initial trials were cultured for 2h to evaluate their viability after cryopreservation. Non-surgical transfer was carried out to transfer two embryos to a synchronous recipient, and pregnancy was determined by palpation via rectum around Day 70 of transfer. After warming of vitrified embryos, similar high survival rates were achieved in NT embryos derived from either cumulus (93.8%, n=16), or fibroblast cells (95.8%, n=48) as nuclear donors, respectively (P&gt;0.05). The pregnancy data (Table 1) on Day 70 of gestation indicated that there were no significant differences among ET groups with fresh NT blastocysts, vitrified cumulus NT and fibroblast NT embryos (P&gt;0.05). This study demonstrates that cryopreserved bovine NT embryos via LNSV vitrification can maintain an in vivo developmental viability comparable to freshly produced NT counterparts. Further developmental potential of vitrified NT embryos to term is under investigation. Table 1 Pregnancy outcomes of vitrifield bovine NT embryos after embryo transfer

scholarly journals 65 PRODUCTION OF PORCINE NUCLEAR TRANSFER EMBRYOS USING FETAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS

2005 ◽  
Vol 17 (2) ◽  
pp. 182 ◽  
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Nuclear Transfer ◽  
Fibroblast Cell ◽  
State Committee ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Functional Quality ◽  
Donor Cells ◽  
Morphological Transformations

One of the most important factors that determine the developmental potential of mammalian cloned embryos is the structuro-functional quality of nuclear donor cells. Biochemical changes that are some of the earliest symptoms of apoptosis signal transduction are not reflected in the morphological features of somatic cells. Therefore, an appropriate system of cell selection would enable the sorting of donor nuclei with high morphological and biochemical susceptibility to somatic cloning. The aim of our study was to examine the in vitro developmental competencies of porcine nuclear transfer (NT) embryos reconstructed with fetal fibroblast cells that had been analyzed for apoptosis by live-fluorescent labelling. Frozen/thawed fetal fibroblast cells, which had been in vitro-cultured to a confluent state, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, a single cell suspension of nuclear donor cells was subjected to dyeing with live-DNA green fluorochrome YO-PRO-1. The recipient cells were in vitro-matured oocytes. Maternal chromosomes were removed by a chemically assisted microsurgical technique. Then, single nuclear donor cells were inserted into the perivitelline space of enucleated oocytes. Fibroblast cell-ooplast couplets were simultaneously fused and activated with two consecutive DC pulses of 1.2 kV/cm for 60 μs. Reconstructed embryos were in vitro cultured in 50-μL drops of NCSU-23 medium supplemented with 0.4% BSA-V for 6 to 7 days at 38.5°C in a humidified atmosphere of 5% CO2 and 95% air. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of approximately 50 different random samples collected from the population of fetal fibroblast cells, that had been labelled with YO-PRO-1 dye, it was found that a relatively high proportion of donor cells revealed ultrastructural apoptotic changes. The percentage of late apoptotic cells with advanced morphological transformations was about 40% of the total pool of the fibroblast cells. A total of 262/270 (97.0%) enucleated oocytes were subjected to reconstruction and 141/262 (53.8%) were successfully fused with non-apoptotic nuclear donor cells. Following the simultaneous fusion/activation protocol, reconstituted oocytes were selected for in vitro culture. Out of 262, 133 (50.8%) cultured NT embryos cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/133 (36.0%) and 10/133 (7.5%), respectively. In conclusion, morphology is a sufficient selection factor for detection of apoptosis in the cultured (confluent) fetal fibroblast cells to be used for cloning. Moreover, it was found that YO-PRO-1 fluorochrome may be not able to detect the early phases of apoptosis, because only the morphologically abnormal cells emitted the YO-PRO-1-derived fluorescence. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-KBN-084/P06/2002/4.2 from years 2003 to 2005.

36 TREATMENT OF PORCINE FETAL FIBROBLASTS WITH DNA METHYLATION INHIBITORS OR HISTONE DEACETYLATION INHIBITORS IMPROVES DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 118 ◽  
Author(s):  
D. I. Jin ◽  
N. Kenji ◽  
R. X. Han ◽  
S. M. Choi ◽  
M. Y. Kim ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Histone Deacetylation ◽  
Control Group ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Dna Methylation Inhibitors ◽  
Porcine Fetal Fibroblast

Epigenetic status of the genome of a donor nucleus has an important effect on the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). DNA methylation inhibitors [such as 5-aza-2′-deoxyctidine (5-aza-dC), zebularine, and RG108] and histone deacetylase inhibitors [such as trichostatin A (TSA), sodium butyrate (NaBu), and scriptaid (SCR)] have been widely used for the alteration of the levels of the epigenetic modification of somatic cells. This study was designed to investigate the DNA methylation status of porcine fetal fibroblast cells treated with TSA or 5-aza-dC and to determine whether treatments with DNA methylation inhibitors or histone deacetylation inhibitors could improve the in vitro development of porcine reconstructed embryos. When the levels of DNA methylation in the PRE-1 sequence (repeat sequence in a euchromatic region) were examined by bisulfite sequencing following treatment of porcine fetal fibroblast cells with TSA or 5-aza-dC for 1 h, DNA methylation was decreased in 5-nm or 50-nm concentrations even if they were not significantly different. To evaluate the effect of DNA methylation inhibitors and histone deacetylation inhibitors on development of porcine nuclear transfer embryos, porcine fetal fibroblast cells were treated with 5 nm of 5-aza-dC, zebularine, or RG108 for 1 h, or with 50 nm of TSA, NaBu, or SCR for 1 h, or treated with both 50 nm TSA and 5 nm 5-aza-dC for 1 h before NT. The reconstructed embryos were electrically fused and cultured in PZM-3 for 6 days. Developmental rates of the reconstructed embryos from donor cells treated with 5-aza-dC, zebularine, or RG-108 to blastocysts significantly increased compared to the control group (21.4, 23.3, and 22.1 v. 12.3%). Blastocyst rates of the reconstructed embryos from donor cells treated with TSA, SCR, and NaBu also were significantly improved compared to the control group (30.0, 23.9, and 22.4 v. 14.5%), and TSA treatment was the highest in blastocyst rates among the treated groups. However, the development rate to the blastocyst stage was not affected when the combination of TSA and 5-aza-dC was treated. In conclusion, treatment of donor cells with DNA methylation inhibitors or histone deacetylase inhibitors improved the subsequent blastocyst development of porcine reconstructed embryos even though combined treatment with both inhibitors had no beneficial effect.

scholarly journals In Vitro Development of Bovine Nuclear Transfer Embryos from Transgenic Clonal Lines of Adult and Fetal Fibroblast Cells of the Same Genotype1

2002 ◽  
Vol 66 (6) ◽  
pp. 1768-1774 ◽  
Author(s):  
Sezen Arat ◽  
John Gibbons ◽  
S. Jacek Rzucidlo ◽  
Donald S. Respess ◽  
Monica Tumlin ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
In Vitro Development ◽  
Clonal Lines ◽  
Vitro Development

scholarly journals 33 PRODUCTION OF TRANSGENIC-CLONED PIGS CARRYING HDAF, GnT-III AND HETEROZYGOUSLY DISRUPTED ±-1,3-GALACTOSYLTRANSFERASE GENES

2005 ◽  
Vol 17 (2) ◽  
pp. 166
Author(s):  
T. Fujimura ◽  
Y. Takahagi ◽  
H. Nagashima ◽  
S. Miyagawa ◽  
T. Shigehisa ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Nuclear Transfer ◽  
Regulatory Protein ◽  
Pcr Analysis ◽  
Fibroblast Cells ◽  
Drug Resistant ◽  
Transgenic Pigs ◽  
Sugar Chain ◽  
Fetal Fibroblast ◽  
Targeting Vector

In pig-to-human xenotransplantation, transplants are rapidly rejected by binding of human natural antibodies to porcine xenoantigen, mostly Gal α-1-3Gal oligosaccharides, and subsequent complement attack. To overcome this rejection, we so far have produced transgenic pigs expressing both human CD55/DAF (decay-accelerating factor, a complement-regulatory protein) and GnT-III (N-acetylglucosaminyltransferase III, a sugar chain modifying enzyme). In the present study, we heterozygously disrupted the α-1,3-galactosyltransferase (GT) gene, which catalyses the biosynthesis of Gal α-1-3Gal epitopes, in the fetal fibroblast cells from the DAF/GnT-III transgenic pigs by homologous recombination, and successfully produced GT-knockout pigs by nuclear transfer. Fibroblast cells isolated from Day 30 fetuses of DAF/GnT-III transgenic pigs were transfected with a GT-targeting vector. The targeting event in drug-resistant colonies was confirmed by PCR analysis, and targeted cells were used as nuclear donors. The reconstructed embryos were electrically activated and transferred to estrus-synchronized recipient pigs. At pregnancy Day 27 of gestation, fetuses were collected and their fibroblast cells were isolated for secondary nuclear transfer. The genomic DNA of live-born piglets produced by the secondary nuclear transfer were analyzed for the presence of DAF and GnT-III genes as well as the heterozygous disruption of the GT gene. From a total of 5.5 × 107 cells transfected with the GT-targeting vector, 2,749 drug-resistant colonies were obtained. Eighteen colonies were judged positive for targeting events by PCR analysis. After transfer of 321 cloned embryos reconstructed with the knockout cells to three recipients, four knockout fetuses were obtained from one recipient. Transfer of 633 cloned embryos reconstructed with the knockout fibroblast cells from one knockout fetus to six recipients gave rise to two live knockout piglets. PCR analysis of genomic DNA confirmed that the cloned piglets carried both DAF and GnT-III transgenes as well as the heterozygously disrupted GT gene.

80 EFFECT OF MEDIA, SYNCHRONIZATION OF FIBROBLAST CELLS, CULTURE TIME, OXYGEN CONCENTRATION, AND ACTIVATION ON NUCLEAR-TRANSFERRED PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 157
Author(s):  
J. H. Quan ◽  
H. B. Seok ◽  
S. K. Kim
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Atmospheric Condition ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Donor Cells ◽  
The Impact ◽  
Cells Cultured

The purpose of this study was to investigate the impact of culture medium, culture duration, and atmospheric condition on the fusion and in vitro development rates of nuclear transfer porcine embryos constructed by the microinjection of fetal fibroblast cells into in vitro-matured oocytes. Single fetal donor cells were deposited into the perivitelline space of enucleated oocytes, followed by electrical fusion and activation. Activated embryos were cultured in NCSU-23 medium supplemented with 5% FBS, at 38.5�C for 6 to 8 days in 5% CO2 and air. In Experiment 1, the fusion rates of nuclear transfer embryos did not differ from those of fetal fibroblast cells incubated in 5% FBS + NCSU-23 or 5% FBS + TL-HEPES medium, nor did fusion rates of donor cells differ among 1–8-h incubation durations. Fusion rates for the 4 treatment subclasses ranged from 72.1% to 78.0%. In Experiment 2, pre-synchronization in medium containing 0.1 �g mL-1 Hoechst(H) 33342 increased during the period from 0 and 8 h of culture up to 15 h, the end of the synchronization period, at which time there was a significantly increased percentage of porcine fibroblast cells at the G2/M stage (12.4%, 17.5%, and 47.6%; P &lt; 0.01). Neither an increase in the concentration of H 33342 (0.2–1.6 �g mL-1) nor a longer exposure time (12 h, 18 h, and 24 h) increased the proportion of porcine G2/M fibroblasts. In Experiment 3, fusion rates did not differ significantly between nuclear transfer embryos constructed using donor cells cultured in 5% FBS + NCSU-23 medium for 1–2, 6–8, or 12–14 days (60.0%, 73.3%, and 62.5%, respectively). The cleavage rate for nuclear transplant embryos using fetal fibroblast cells cultured for 1–2 days was 44.0%, which was significantly less than the 56.7% and 50.0% for 6–8 or 12–14 days of culture, respectively (P &lt; 0.05). In Experiment 4, the proportions of nuclear transfer embryos that developed to the e2 cell and to the blastocyst stage were not affected significantly by culture medium (5% FBS + NCSU-23 or 5% FBS + TL-HEPES) or by O2 concentration during culture (5% vs. 10%). The developmental rates to the e2 cell stage ranged from 65.9% to 70.1%, and those to the blastocyst stage ranged from 9.8% to 12.5%, for the 4 treatment subclasses. Blastocyst rate was highest for embryos cultured in 5% FBS + NCSU-23 under a gas atmosphere of 5% O2 in air.

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