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.

scholarly journals Streptolysin-O Treatment of Fetal Fibroblasts Improves Cell Fusion and In Vitro Development of Porcine Nuclear Transfer Embryos

2009 ◽  
Vol 55 (3) ◽  
pp. 236-239 ◽  
Author(s):  
Kenji NARUSE ◽  
Yan-Shi QUAN ◽  
Baek-Chul KIM ◽  
Su-Min CHOI ◽  
Chang-Sik PARK ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Nuclear Transfer ◽  
In Vitro Development ◽  
Streptolysin O ◽  
Fetal Fibroblasts ◽  
Vitro Development

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.

scholarly journals Effect of Fusion/Activation Protocol on In Vitro Development of Porcine Nuclear Transfer Embryos Constructed with Foreign Gene-Transfected Fetal Fibroblasts

2003 ◽  
Vol 65 (9) ◽  
pp. 989-994 ◽  
Author(s):  
Mario A. MARTINEZ DIAZ ◽  
Tadashi MORI ◽  
Masashi NAGANO ◽  
Seiji KATAGIRI ◽  
Yoshiyuki TAKAHASHI
Keyword(s):  
Nuclear Transfer ◽  
Foreign Gene ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Vitro Development

scholarly journals Metabolic programming of a Warburg effect-like phenotype in donor fibroblasts prior to somatic cell nuclear transfer

2017 ◽  
Author(s):  
◽  
Bethany Rae Mordhorst
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Nuclear Reprogramming ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Pharmaceutical Agents ◽  
Vitro Development

Gene edited pigs serve as excellent models for biomedicine and agriculture. Currently, the most efficient way to make a reliably-edited transgenic animal is through somatic cell nuclear transfer (SCNT) also known as cloning. This process involves using cells from a donor (which may have been gene edited) that are typically grown in culture and using their nuclear content to reconstruct a new zygote. To do this, the cell may be placed in the perivitelline space of an enucleated oocyte and activated artificially by a calcium-containing media and electrical pulse waves. While it is remarkable that this process works, it is highly inefficient. In pigs the success of transferred embryos becoming live born piglets is only 1-3%. The creation of more cloned pigs enables further study for the benefit of both A) biomedicine in the development of prognosis and treatments and B) agriculture, whether it be for disease resistance, feed efficiency, gas emissions, etc. Two decades of research has not drastically improved the cloning efficiency of most mammals. One of the main impediments to successful cloning is thought to be due to inefficient nuclear reprogramming and remodeling of the donor cell nucleus. In the following chapters we detail our efforts to improve nuclear reprogramming of porcine fetal fibroblasts by altering the metabolism to be more blastomere-like in nature. We used two methods to alter metabolism 1) pharmaceutical agents and 2) hypoxia. After treating donor cells both methods were used in nuclear transfer. Pharmaceutical agents did not improve in vitro development of gestational survival of clones. Hypoxia did improve in vitro development and we are currently awaiting results of gestation.

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts

2000 ◽  
Vol 57 (3) ◽  
pp. 262-269 ◽  
Author(s):  
Paul J. Verma ◽  
Zhong-Tao Du ◽  
Lesley Crocker ◽  
Renate Faast ◽  
Christopher G. Grupen ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Vitro Development

35 USE OF METAPHASE DONOR CELLS AND ACTIVATION WITH ROSCOVITINE FOR SOMATIC CELL NUCLEAR TRANSFER IN BOVINE

2017 ◽  
Vol 29 (1) ◽  
pp. 125
Author(s):  
G. V. Landschoot ◽  
V. Savy ◽  
N. Canel ◽  
S. Ferraris ◽  
D. Salamone
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
M Cells ◽  
In Vitro Development ◽  
Donor Cells ◽  
G1 Cells ◽  
Vitro Development

Cloning of domestic species by somatic cell nuclear transfer (SCNT) continues to be inefficient, probably due to an incomplete reprogramming of the reconstituted embryo. The ability of the embryonic cytoplasm to support reprogramming fluctuates within the cell cycle (Egli et al. 2007 Nature 447, 679–85). In this context, we compared the development capability and second polar body (2PB) extrusion of embryos produced by metaphase (M) cells, in comparison with G0/G1 cells, which are commonly used as nuclear donors. Because M cells have 2 sets of chromosomes (in contrast with G0/G1 cells, which have only 1 set), an activation protocol that impedes 2PB extrusion is required to produce reconstituted embryos with the correct ploidy. Therefore, we performed SCNT with M or G0/G1 cells, followed by different activation protocols, and evaluated in vitro development and 2PB extrusion of the reconstituted embryos. Cow oocytes were in vitro matured and enucleated as described by Gambini et al. (2014 PLoS One 14, 9). A group of cells at 70 to 80% confluence was synchronized in M stage using 0.05 μg mL−1 demecolcine for 3 to 4 h and used as nuclear donors for SCNT (M group). Another group of cells was induced into quiescence by serum starvation for 3 to 4 days before SCNT (G0/G1 group). For activation, reconstituted embryos were treated with 5 µM ionomycin (Io) for 4 min followed by 5-h incubation in 50 μM roscovitine for M group, or in 50 μM roscovitine and 5 μg mL−1 cytochalasin B for G0/G1 group. Parthenogenetic controls were activated with Io followed by 50 μM roscovitine alone (ROSCO) or with 5 μg mL−1 cytochalasin B (ROSCO/CB). Hoescht 33342 staining was performed 16 h post-Io to evaluate 2PB extrusion. Other activated oocytes were cultured in SOFaa medium and rates of cleavage, morulas, and blastocysts were evaluated at Days 2, 5 and 7 of in vitro development, respectively. Data were analysed by Fisher’s exact test (P < 0.05). Rates of 2PB extrusion were 72.72 (n = 33), 65.63 (n = 32), 80 (n = 15), and 42.86 (n = 14) for M, G0/G1, ROSCO, and ROSCO/CB, respectively. Results of in vitro development are shown in Table 1. In conclusion, somatic M cells can be used as donors to produce cloned embryos. The M and G0/G1 groups were able to induce cloned blastocysts, even though rates did not differed statistically from controls groups (ROSCO and ROSCO/CB). The M group was as effective as G0/G1. Although further analysis is required to establish the quality of the embryos, our results are encouraging for use in SCNT. Table 1.In vitro development of NT embryos produced with M and G0/G1 donor cells

Serum starvation of bovine fetal fibroblasts prior to nuclear transfer increases in vitro development rates

1999 ◽  
Vol 51 (1) ◽  
pp. 204
Author(s):  
J.R Hill ◽  
Q.A Winger ◽  
K.L Jones ◽  
J.A Thompson ◽  
R.C Burghardt ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Serum Starvation ◽  
In Vitro Development ◽  
Development Rates ◽  
Fetal Fibroblasts ◽  
Vitro Development
Sign in / Sign up

Export Citation Format

Share Document