Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells

2002 ◽  
Vol 62 (3) ◽  
pp. 300-306 ◽  
Author(s):  
Liangxue Lai ◽  
Kwang-Wook Park ◽  
Hee-Tae Cheong ◽  
Birgit Kühholzer ◽  
Melissa Samuel ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transgenic Pig ◽  
Donor Cells ◽  
Green Fluorescent

Production of transgenic porcine blastocysts by hand-made cloning

2004 ◽  
Vol 16 (3) ◽  
pp. 315 ◽  
Author(s):  
P. M. Kragh ◽  
G. Vajta ◽  
T. J. Corydon ◽  
S. Purup ◽  
L. Bolund ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Reconstructed Embryos ◽  
Green Fluorescent

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 successfully reconstructed embryos) were 6% (2/31), 7% (1/15), 7% (2/28), 3% (1/37) and 7% (2/28), resulting in an average of 6.0 ± 0.8%. Enhanced green fluorescent protein was expressed in all cells of all eight developing blastocysts. Efforts are now directed towards the production of offspring from such transgenic NT blastocysts.

PRODUCTION OF NUCLEAR TRANSFER-DERIVED SWINE THAT EXPRESS THE ENHANCED GREEN FLUORESCENT PROTEIN

2001 ◽  
Vol 12 (2) ◽  
pp. 173-181 ◽  
Author(s):  
Kwang-Wook Park ◽  
Hee-Tae Cheong ◽  
Liangxue Lai ◽  
Gi-Sun Im ◽  
Birgit Kühholzer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts

2000 ◽  
Vol 12 (2) ◽  
pp. 1 ◽  
Author(s):  
Sangho Roh ◽  
Hosup Shim ◽  
Woo-suk Hwang ◽  
Jong-taek Yoon
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Green Fluorescent ◽  
Vitro Development

Nuclear transfer using transfected donor cells provides an efficient new strategy for the production of transgenic farm animals. The present study assessed in vitro development of nuclear transfer embryos using green fluorescent protein (GFP) gene-transfected bovine fetal fibroblasts. In experiment 1, bovine fetal fibroblasts (BFF) were transfected with linearized pEGFP-N1 by electroporation, and the enucleated oocytes were reconstructed by nuclear transfer of transfected cells (BFF-GFP). The rates of blastocyst formation did not differ significantly between BFF and BFF-GFP (18.2% v. 15.6%). In experiment 2, before nuclear transfer, the donor cell stage was synchronized by serum deprivation or forming a confluent monolayer. The rates of cleavage (67.1% v. 71.8%) and blastocyst formation (15.8% v. 15.5%) did not differ between confluent and serum-starved cells after nuclear transfer. In experiment 3, the effects of different passages of donor fibroblast cells on the development of nuclear transfer embryos were investigated. Donor cells from ‘early’ (at passage 8–16) showed better blastocyst development (18.9%) than those from ‘late’ (at passage 17–32; 10.5%). In conclusion, this study suggests that transgenic somatic cell nuclei from early passages can be reprogrammed more effectively than those from late passages. In addition, GFP, a non-invasive selection marker, can be used to select transgenic nuclear transfer embryos.

Green fluorescent protein (GFP) transgenic pig produced by somatic cell nuclear transfer

2008 ◽  
Vol 53 (7) ◽  
pp. 1035-1039 ◽  
Author(s):  
ZhongHua Liu ◽  
Jun Song ◽  
ZhenKun Wang ◽  
JiangTian Tian ◽  
QingRan Kong ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Transgenic Pig ◽  
Green Fluorescent
Sign in / Sign up

Export Citation Format

Share Document