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

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

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.

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

78 EFFECTS OF DIFFERENT ACTIVATION PROTOCOLS ON ACTIN FILAMENT DISTRIBUTION AND IN VITRO DEVELOPMENT OF MINIATURE PIG NT EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 147
Author(s):  
K. Yamanaka ◽  
S. Sugimura ◽  
T. Wakai ◽  
T. Shoji ◽  
H. Sasada ◽  
...  
Keyword(s):  
Actin Filament ◽  
Nuclear Transfer ◽  
Subsequent Development ◽  
Miniature Pig ◽  
Physical Damage ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Developmental Rates ◽  
Vitro Development

In the process of producing reconstructed oocytes nuclear transfer (NT) embryos by somatic cell nuclear transfer, in vitro-matured oocytes can be used as recipient ones. It, however, has been well documented that after IVF porcine embryos derived from in vitro-matured oocytes have a small number of cells and low viability compared from those in vivo. As one possible reason, abnormal actin filament distribution has been detected in abnormal embryo cleavage and small cell numbers (Wang et al. 1999 Biol. Reprod. 60, 1020-1028). Artificial activation, which is necessary for development of NT embryos, can affect actin filament distribution of porcine oocytes matured in vitro, resulting in fragmentation (Kawahara et al. 2002 Theriogenology 58, 1081-1095). In the present study, we investigated effects of different activation protocols on actin filament distribution and in vitro development of miniature pig NT embryos. Porcine oocytes collected from ovaries were matured in vitro for 40 to 44 h in NCSU-23. First, we compared different activation protocols in development rates to blastocysts of oocytes activated. We used three activation methods (15 �M ionomycin treatment for 20 min (I), double DC pulses of 1.2 kV/cm for 60 ms in intervals of 5 s (E), and 5 mg/mL cycloheximide treatment for 5 h (C)) to prepare seven activation protocols (I, E, C, I + C, I + E, E + C, and I + E + C). Second, we examined effects of different activation protocols on actin filament distribution and subsequent development of NT embryos activated by the different activation protocols. Matured oocytes were enucleated, and fused with miniature pig fetal fibroblasts in calcium-free medium; approximately 3 h later, the resultant NT embryos were activated with three activation protocols (E, I + C, or I + E + C). All data were analyzed by chi-square test. The developmental rates to blastocysts in the I, E, C, I + C, I + E, E + C, and I + E + C groups were 5.6, 11.1, 0.0, 36.1, 20.7, 14.6, and 24.7%, respectively, showing that the rate in oocytes activated with I + C was significantly higher (P < 0.05) than the rates in oocytes activated by other treatments. In NT embryos, the developmental rates to blastocysts in the E, I + C, or I + E + C groups were 4.1, 14.3, and 4.6%, respectively, showing that the rate in NT embryos activated with I + C was significantly higher (P < 0.05) than the rate in NT embryos activated with other treatments. The abnormal rate of actin filament distribution in NT embryos activated with E or I + E + C was significantly higher (P < 0.05) than that in NT embryos activated with I + C (26.7% or 33.3% vs. 6.7%). The present results suggest that in miniature pig NT embryos an activation protocol by ionomycin combined with cycloheximide treatments may avoid physical damage to actin filaments with the resultant improvement of subsequent development.

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P&lt;0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P&lt;0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P&lt;0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

scholarly journals 28 SIMPLIFIED ACTIVATION METHOD TO IMPROVE THE IN VITRO DEVELOPMENT OF HANDMADE CLONED (HMC) PORCINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 114
Author(s):  
Y. Du ◽  
Z. Yang ◽  
B. Lv ◽  
L. Lin ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Cell Number ◽  
Activation Method ◽  
The Other ◽  
Electrical Activation ◽  
In Vitro Development ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Group 2 ◽  
Vitro Development

Delayed activation is commonly used in pig somatic cell nuclear transfer (SCNT) where electrical activation is followed by chemical activation. However, chemical incubation of several hours (up to 4 or 6) is logistically not very convenient even though handmade cloning (HMC) could improve the overall efficiency of pig cloning (Du et al. 2007 Theriogenology 68, 1104–1110). It was reported that a brief exposure of cycloheximide (CX) before electrical activation could significantly increase developmental rate and total blastocyst cell number when simultaneous activation was performed in micromanipulator-based pig cloning (Naruse et al. 2007 Theriogenology 68, 709–716). The purpose of our present work is to investigate whether such activation method is also applicable for pig HMC. Data were analyzed by t-test using SPSS (11.0, SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. In vitro-cultured porcine fetal fibroblasts were used as donor cells. Cytoplast-fibroblast pairing, electrical fusion and activation of fused cytoplast-fibroblast pairs were performed as described previously (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205). Three groups were compared due to different activation protocol. In Group 1 (control), reconstructed embryos were cultured in porcine zygote medium 3 (PZM3) supplemented with 4 mg mL–1 BSA, 5 μg mL–1 cytochalasin B (CB), and 10 μg mL–1 CX for 4 h. In Group 2 (CX priming), fused pairs and the other halves of cytoplasts were incubated in HEPES-buffered TCM-199 medium supplemented with 10% calf serum, 10 μg mL–1 CX for 10 min just before the second fusion or electrical activation. In Group 3 (CB + CX priming), treatment similar to Group 2 was performed except that additional 5 μg mL–1 CB was added for the 10-min incubation. Reconstructed embryos were in vitro cultured in the well of the well (WOW) system for 6 days. Blastocyst rates and total cell numbers of Day 6 blastocysts were evaluated. As illustrated in Table 1, embryos pretreated with both CB and CX gave the best results, with better blastocyst formation (53.8 ± 4.8%; mean ± SEM) and higher cell number (77.2 ± 5.4) compared to the other 2 groups. Our data suggested that CX and CB priming could be used as a solution to the long chemical incubation in porcine SCNT by HMC, making the embryos more receptive to electrical activation. Table 1.In vitro development of HMC reconstructed embryos with different activation protocols

scholarly journals 71EFFECT OF CALCIUM IONOPHORE AND CYTOCHALASIN D ON ACTIVATION AND IN VITRO DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 157 ◽  
Author(s):  
S.J. Rzucidlo ◽  
S. Arat ◽  
S.L. Stice
Keyword(s):  
Nuclear Transfer ◽  
Calcium Ionophore ◽  
Cytochalasin D ◽  
Small Cells ◽  
In Vitro Development ◽  
Group Iii ◽  
Group I ◽  
Fetal Bovine ◽  
Vitro Development

The activation of oocytes is one of the most important steps for a successful cloning, and chemicals used for activation can affect the viability of cloned offspring. Therefore, some of them may be omitted for activation to eliminate their possible detrimental effect on nuclear transfer (NT) embryos. The objective of this study was to examine the effect of calcium ionophore (CaI, A23187, Sigma, St. Louis, MO, USA) and cytochalasin D (CD) on activation and in vitro development of nuclear transfer units derived from bovine granulosa cells (GCs) treated with the cell cycle inhibitor, roscovitine. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin, rIGF-1, bFSH, and bLH. GCs were isolated from ovarian follicles and cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Prior to NT, donor cells were exposed to 15mM roscovitine for 24 hours and small cells were used for NT. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused using a 20μs DC pulse of 40V/150μm. Two hours after fusion, NT units were assigned into four groups and activated by CaI (5μM for 10min.), and then incubated with cycloheximide (CHX, 10μgmL−1)+CD (2.5μgmL−1) for 1h, followed by CHX alone (without CD) for 5h (Group I) or CaI (for 10min.), followed by CHX alone for 6h (Group II). In the Group III, NT units were activated by CHX (10μgmL−1)+CD (2.5μgmL−1) for 1h, followed by CHX for 5h, and Group IV, CHX alone for 6h. The base activation medium was TCM199 with 1% FBS for CaI and 10% FBS for CHX and CD. After activation, NT units were cultured for 7 days in BARC medium. Differences in activation, cleavage and blastocyst formation rates among treatments were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. Our data showed that CaI and CD did not affect the activation and in vitro development of NT embryos derived from roscovitine-treated GCs. It suggests that both chemicals may be redundant during cloning procedure. This study was supported by a grant from ProLinia, Inc and TUBITAK, Turkey (VHAG-1908-102V048). Table 1 In vitro development of NT embryos in different activation treatments

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