The expression of β-galactosidase during long-term cultured goat skin fibroblasts and the effect of donor cell passage on in vitro development of nuclear transfer embryos

2016 ◽  
Vol 52 (5) ◽  
pp. 555-561 ◽  
Author(s):  
Haijun Liu ◽  
Hui Peng ◽  
Fang Liu ◽  
Qun Ma ◽  
Wenchang Zhang
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Cell Passage ◽  
In Vitro Development ◽  
Goat Skin ◽  
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.

scholarly journals 55IN VITRO DEVELOPMENT OF ENUCLEATED DOMESTIC CAT OOCYTES RECONSTRUCTED WITH SKIN FIBROBLASTS OF DOMESTIC AND LEOPARD CATS

2004 ◽  
Vol 16 (2) ◽  
pp. 149 ◽  
Author(s):  
C. Lorthongpanich ◽  
C. Laowtammathron ◽  
S. Muenthaisong ◽  
T. Vetchayan ◽  
M. Ketudat-Cairns ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Skin Fibroblasts ◽  
Domestic Cat ◽  
Developmental Potential ◽  
A Cell ◽  
Vitro Development

The domestic cat is a valuable model for studies in assisted reproductive technology in felid species. Therefore, in this experiment we evaluated the in vitro developmental potential of enucleated domestic cat oocytes reconstructed with somatic cells from domestic and leopard cats. Skin fibroblasts were isolated from female domestic and leopard cats. The oocytes were collected by aspiration of follicles from ovaries that were superovulated with 200IU PMSG. In vitro-matured oocytes were enucleated and individual donor cells (diameter 14–16μm) were inserted into the perivitelline space of the enucleated oocyte. Fusion was performed at 26–27h post-maturation by placing a cell-oocyte couplet between both tips of the needle electrode and electrostimulating with a 2-DC pulse (30V, 30μs) in fusion medium containing 0.3M Mannitol+0.1mM MgCl2. Activation was performed 1 to 2h post-fusion by incubation in 7% ethanol at room temperature for 5min followed by cultured in 10μgmL−1 cycloheximide and 1.25μgmL−1 cytochalasin D at 38°C in 5% O2, 5% CO2, 90% N2 conditions. After activation, the reconstructed embryos were cultured in 100-μL droplets of Tyrode’s medium (Gomez et al., 2003 Theriogenology 60, 239–251.) supplemented with 0.3% BSA at 38°C in a 5% O2, 5% CO2, 90% N2 environment for 2d. Then, 8-cell embryos were cultured in 100-μL droplets of Tyrode’s medium supplemented with 10% FCS at 38°C in a 5% O2, 5% CO2, 90% N2environment for 5d. The cleavage rates of oocytes reconstructed with either donor cell types were not different. The percentages of blastocyst formation from parthenogenotes and nuclear transfer embryos derived from domestic cat fibroblasts (8/56, 14.3% and 7/51, 13.7%, respectively) were significantly higher than that for nuclear transfer embryos constructed with leopard cat fibroblasts (3/45, 6.7%). These results indicate that enucleated domestic cat oocytes reconstructed with skin fibroblasts of leopard cats can develop to the blastocyst stage. This experiment was supported by Suranaree University of Technology. Table 1 In vitro development of domestic cat oocytes reconstructed with domestic and leopard skin fibroblasts and parthenogenetic activation

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<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<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<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 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

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

Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

2002 ◽  
Vol 14 (4) ◽  
pp. 191 ◽  
Author(s):  
M. A. Martinez-Diaz ◽  
K. Ikeda ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Kinase Activity ◽  
Short Interval ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
M Phase ◽  
Cytostatic Factor ◽  
Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

Roscovitine Treatment Improves Synchronization of Donor Cell Cycle in G0/G1 Stage and In Vitro Development of Handmade Cloned Buffalo (Bubalus bubalis) Embryos

2012 ◽  
Vol 14 (2) ◽  
pp. 146-154 ◽  
Author(s):  
Naresh L. Selokar ◽  
Monika Saini ◽  
Mushariffa Muzaffer ◽  
G. Krishnakanth ◽  
Ambika P. Saha ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Donor Cell ◽  
Bubalus Bubalis ◽  
In Vitro Development ◽  
Vitro Development

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

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