Effect of elevated Ca2+ concentration in fusion/activation medium on the fusion and development of porcine fetal fibroblast nuclear transfer embryos

2002 ◽  
Vol 61 (4) ◽  
pp. 488-492 ◽  
Author(s):  
Hee-Tae Cheong ◽  
Kwang-Wook Park ◽  
Gi-Sun Im ◽  
Liangxue Lai ◽  
Qing-Yuan Sun ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Fetal Fibroblast ◽  
Porcine Fetal Fibroblast

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.

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.

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

34 PRODUCTION OF TRANSGENIC CLONED BUFFALO EMBRYOS CONTAINING OVEREXPRESSED STEAROYL Co-A DESATURASE GENE FOLLOWING EFFICIENT TRANSFECTION

2017 ◽  
Vol 29 (1) ◽  
pp. 124
Author(s):  
T. Sharma ◽  
D. Dua ◽  
N. Saini ◽  
M. K. Singh ◽  
S. K. Singla ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Nuclear Transfer ◽  
Cultured Cells ◽  
Saturated Fatty Acids ◽  
Phospholipid Composition ◽  
Calcium Ionophore ◽  
Basal Medium ◽  
Developmental Competence ◽  
Fetal Fibroblast ◽  
Gene Level

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyses the synthesis of monounsaturated fatty acids and polyunsaturated fatty acids from saturated fatty acids, which are components of triglycerides, wax esters, cholesteryl esters, and membrane phospholipids. Alterations in phospholipid composition have been implicated in a variety of diseases including obesity and the associated metabolic syndrome. SCD also magnifies the conjugated linoleic acid (CLA) content in milk; CLA is a natural fat element, having reputed therapeutic health values including anti-carcinogenic properties. In light of this fact, this study was designed to amplify the levels of SCD at the gene level. In order to achieve the enhanced expression of SCD gene, combination of techniques were used. The somatic cells (fetal fibroblast) culture were established from ear pinnae obtained from bovine fetus procured from the abattoir and were cultured in basal medium, comprising DMEM with 10% fetal bovine serum, 1X NEAA and 1X PS antibiotics. These isolated cultured cells were transfected with a gene construct carrying buSCD gene (pAcGFPN1-buSCD) as BLGP-buSCD-BLG3′UTR-CMVP-EGFP-SV40. The buffalo fetal fibroblast cells were transfected using 3 methods: Nucleofection, Fugene and Lipofection. The successful transfection, as confirmed by PCR and Southern hybridisation, proved Nucleofection to be more efficient in transfecting cells among the techniques used, which were further maintained and selected by Geneticin (G418). These selected transfected cells were then used for nuclear transfer. Somatic cell nuclear transfer (SCNT) has provided an efficient pathway for the production of transgenic animals. Buffalo cumulus–oocyte complexes (COCs) were collected from ovaries collected from abattoir and matured in TCM-199 supplemented with 10% FBS, 5 µg mL−1 FSH, and 1 µg mL−1 β-oestradiol for 21 h in CO2 incubator at 5% CO2 in air and 38.5°C temperature with >95% relative humidity. After 21 h, these COCs were denuded and subjected to zona removal. These zona-free oocytes were manually enucleated using microsurgical blades and 2 enucleated oocytes were fused with a transgenic cell using electro cell manipulator. Further, these reconstructed embryos were activated using calcium ionophore and cultured in IVC media thereafter for 8 days. The developmental competence rate as recorded on Day 8 was 53.26 ± 1.73%, 69.87 ± 6.24%, 62.99 ± 7.15%, 42.71 ± 5.05% and 28.00 ± 3.33% for 2-cell, 4-cell, 8–16 cell, morula, and blastocyst, respectively. When observed under fluorescence microscope, the embryos showed successful expression of GFP, which can be further used for animal production or further research analysis. In conclusion, amplified SCD at gene level will result in a boost to the dairy sector as well ameliorating human health due to its crucial role in anti-cancer, anti-diabetic, reduced cardio-vascular disease, and improved immune responses.

Developmental Competence of Nuclear Transfer Cow Oocytes after Direct Injection of Fetal Fibroblast Nuclei

Cloning ◽  
2000 ◽  
Vol 2 (2) ◽  
pp. 55-62 ◽  
Author(s):  
Orly Lacham-Kaplan ◽  
Maria Diamente ◽  
David Pushett ◽  
Ian Lewis ◽  
Alan Trounson
Keyword(s):  
Nuclear Transfer ◽  
Direct Injection ◽  
Developmental Competence ◽  
Fetal Fibroblast
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