scholarly journals Production of Boer goat (Capra hircus) by nuclear transfer of cultured and cryopreserved fibroblast cells into slaughterhouse-derived oocytes

2009 ◽  
Vol 54 (No. 10) ◽  
pp. 448-460 ◽  
Author(s):  
Y. Tao ◽  
W. Han ◽  
M. Zhang ◽  
Y. Zhang ◽  
J. Fang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Microsatellite Analysis ◽  
Capra Hircus ◽  
Fibroblast Cells ◽  
Tissue Slices ◽  
Normal Paraffin ◽  
Boer Goat ◽  
Reconstructed Embryos ◽  
Paraffin Tissue

The present study reports a birth of goat clone produced by nuclear transfer of cultured and cryopreserved fibroblast cells into slaughterhouse-derived oocytes. The donors of somatic cells were fibroblasts derived from the ear skin of a Boer goat while the recipient ooplasm was <I>in vitro</I> matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were cultured <I>in vitro</I> and then the morphologically normal embryos were transferred to the surrogates. The reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural oestrus. Five of them were treated with hCG after transfer. Among them, one was pregnant and gave birth to a live kid. Due to the improper delivery aid, the cloned kid died accidentally after birth. The cloned kid was then anatomised to observe the viscera development, and the results showed that the organs were normal. Paraffin tissue slices were prepared and stained to ensure the viscera development further, and the results suggested that the organs also developed well in spite of incipient hydropericardium. The microsatellite analysis identified the cloning. It is suggested that the optimised nuclear transfer protocol and proper hCG treatment lead to the successful birth of a goat clone.

91 PRODUCTION OF A CLONED BOER GOAT (CAPRA HIRCUS) BY SOMATIC CELL NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 163
Author(s):  
Y. Tao ◽  
W. Han ◽  
M. Zhang ◽  
J. Ding ◽  
X. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Capra Hircus ◽  
Blastocyst Development ◽  
Tissue Slices ◽  
Boer Goat ◽  
Reconstructed Embryos ◽  
Significant Difference

We reported the birth of a goat clone produced by somatic cell nuclear transfer. The fusion and activation protocols of reconstructed oocytes and embryo transfer procedure were optimized. The donors of somatic cells were fibroblasts derived from ear skin of a Boer goat while the recipient ooplasm was in vitro-matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were activated by ionomycin, 6-dimethylaminopurine (6-DMAP), and cytochalasin B (CB) singly or simultaneously (termed as Ionomycin, Ionomycin+6-DMAP, and Ionomycin+6-DMAP+CB). The result showed that the cleavage rate in single ionomycin was significantly lower than that in Ionomycin+6-DMAP and 6-DMAP+CB (34.38% vs. 69.85% and 72.02%; P &lt; 0.05). However, the cleavage rates and blastocyst rates had no significant difference after in vitro culture (P &gt; 0.05). When the cloned embryos were co-cultured with fetal mouse fibroblast monolayer, the blastocyst development rate increased. The reconstructed embryos were equilibrated 1–3 h, 3–6 h, and 6–9 h after fusion, and then activation was undertaken by ionomycin+6-DMAP. We found that the cleavage rates had no significant difference during 1–3 h and 3–6 h (72.58% vs. 72.97%; P &gt; 0.05), but both were significantly higher than during 6–9 h (64.40%) (P &lt; 0.05). A total of 491 reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural estrus. One of those who were treated with hCG after transfer was pregnant and gave birth to a live kid on 153 days. The lamb died accidentally 8 h after birth. The cloned offspring was then dissected and proved well in all organs. Staining of paraffin tissue slices of the viscera suggested that the organs developed well. Microsatellite analysis indicated that the lamb was derived from the somatic cell donor doe genetically.

scholarly journals Generation of Dwarf Goat (Capra hircus) Clones Following Nuclear Transfer with Transfected and Nontransfected Fetal Fibroblasts and In Vitro-Matured Oocytes1

2001 ◽  
Vol 64 (3) ◽  
pp. 849-856 ◽  
Author(s):  
C.L. Keefer ◽  
H. Baldassarre ◽  
R. Keyston ◽  
B. Wang ◽  
B. Bhatia ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Capra Hircus ◽  
Fetal Fibroblasts

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

In vitro development of reconstructed ibex (Capra ibex) embryos by nuclear transfer using goat (Capra hircus) oocytes

2007 ◽  
Vol 73 (1-3) ◽  
pp. 135-141 ◽  
Author(s):  
Liang Wang ◽  
Tao Peng ◽  
Hai Zhu ◽  
Zili Lv ◽  
Tingting Liu ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Capra Hircus ◽  
Capra Ibex ◽  
In Vitro Development ◽  
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.

TRIM28 down-regulation on methylation imprints in bovine preimplantation embryos

Zygote ◽  
2018 ◽  
Vol 26 (6) ◽  
pp. 449-456 ◽  
Author(s):  
Xin Ma ◽  
Sheng Zhang ◽  
Meiling Zhang ◽  
Yiran Zhu ◽  
Panpan Ma ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Preimplantation Embryos ◽  
Differentially Methylated Region ◽  
Fibroblast Cells ◽  
Mrna Sequence ◽  
Cell Stage ◽  
H19 Gene

SummaryTRIM28/KAP1/TIF1β was identified as a universal transcriptional co-repressor and is critical for regulating post-fertilization methylation reprogramming in preimplantation embryos. In this study, three siRNAs (si647, si742, and si1153) were designed to target the TRIM28 mRNA sequence. After transfection of the mixture of the three siRNA (siMix) into bovine fibroblast cells, the most effective one for TRIM28 knockdown was selected. By injecting RNAi directed against TRIM28 mRNA, we found that TRIM28 knockdown in oocytes had the most effect on the H19 gene, in which differentially methylated region (DMR) methylation was almost completely absent at the 2-cell stage (1.4%), while control embryos showed 74% methylation. In addition, global H3K9me3 levels at the 2-cell stage were significantly higher in the in vitro fertilization (IVF) group than in the TRIM28 knockdown group (P<0.05). We further show that TRIM28 is highly expressed during oocyte maturation and reaches peak levels at the 2-cell stage. In contrast, at this stage, TRIM28 expression in somatic cell nuclear transfer (SCNT) embryos decreased significantly (P<0.05), suggesting that Trim28 transcripts are lost during SCNT. TRIM28 is required for the maintenance of methylation imprints in bovine preimplantation embryos, and the loss of TRIM28 during SCNT may contribute to the unfaithful maintenance of imprints in cloned embryos.

56CARDIOMYOCYTES FOR THE STUDY OF DEDIFFERENTIATION IN BOVINE NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 150
Author(s):  
A. Lucas-Hahn ◽  
M. Schwarzer ◽  
E. Lemme ◽  
L. Schindler ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Fusion Rate ◽  
Marker Genes ◽  
Cardiac Marker ◽  
Reconstructed Embryos

Nuclear transfer facilitates the study of the dedifferentiation process of differentiated somatic cells. Cardiomyocytes are a good model of terminally differentiated cells showing a unique gene expression pattern of cardiac marker genes. The purpose of this study was to test bovine cardiomyocytes as donor cells in nuclear transfer. Cardiomyocytes were isolated from fetal heart muscle (3–5 months of gestation), which were obtained at the abbatoir and immediately perfused with cold Custodiol (Dr. Franz Köhler Chemie, Germany) to reduce metabolism and protect the cells against ischaemia. Subsequently, hearts were perfused with collagenase in Krebs-Henseleit buffer (KHB) to dissociate the tissue and isolate single elongated, contractile cells. For nuclear transfer and fusion the cardiomyocytes were rounded up by exposure to increasing calcium concentrations (2.5–200μM) in the culture medium before the cells were incubated in suspension for 46–48 hours in MEM medium plus 10% FCS. Nuclear transfer was performed as described earlier (Lucas-Hahn et al., 2002, Theriogenology 57, 433). As a control, adult female fibroblasts were employed. Fusion rate, cleavage (day 3 of in vitro culture) and development up to the morula/blastocyst (day 7 of in vitro culture) were recorded and statistically analysed with Student’s t-test. A total of 243 nuclear transfer complexes with cardiomyocytes and 127 with fibroblasts were produced. Fusion rates for cardiomyocyte complexes were significantly (P&lt;0.001) lower (28.8%) compared to fibroblasts (84.3%). Cleavage rates were 48.1% for cardiomyocytes and 62.8% for the fibroblast-derived embryos. The developmental capacity to the morula/blastocyst was significantly (P&lt;0.01) reduced for cardiomyocyte (9.4%) compared with the fibroblast-derived (32.4%) reconstructed embryos. Most of the Day 7 embryos were frozen for investigation of gene expression patterns of cardiac marker genes. Staining with Hoechst 33342 for counting total cell numbers revealed that 87.3±20.9 blastocysts were derived from fibroblasts and 100 blastocysts from cardiomyocytes. These results indicate that nuclear transfer with terminally differentiated cardiomyocytes is possible, although with reduced rates. Studies are underway to analyze the gene expression of cardiac marker genes in reconstructed embryos to gain insight into dedifferentiation after nuclear transfer using cardiomyocytes as a model. This study was supported by Deutsche Forschungsgemeinschaft (DFG; Ni 256/16-1)

70 FETAL FIBROBLAST CELLS PERMEABILIZED WITH STREPTOLYSIN O IMPROVE THE RATES OF FUSION AND IN VITRO DEVELOPMENT OF PORCINE RECONSTRUCTED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 152
Author(s):  
K. Naruse ◽  
Y. M. Shin ◽  
Y. S. Quan ◽  
C. S. Park ◽  
D. I. Jin
Keyword(s):  
Cell Number ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
In Vitro Development ◽  
Reconstructed Embryos ◽  
Streptolysin O ◽  
Donor Cells ◽  
Developmental Rates ◽  
Vitro Development

Streptolysin O (SLO) is known to bacterial proteins that form very large pores in the plasma membrane of mammalian cells. SLO has been used in the delivery of proteins into living cells following permeabilization. The objective of this study was to investigate the effect of permeabilization of donor cells using SLO on in vitro development of porcine reconstructed embryos. Porcine fetal fibroblast cells were treated with Ca2+-free DMEM medium containing 200 ng mL−1 of SLO for 50 min before or after trypsinization. Those SLO-treated donor cells were injected into enucleated oocytes, fused with 2 DC pulses (1.2 kV cm−1, 30 µs) and cultured in procine zygote medium-3 (PZM-3) for 6 days. In vitro development of the reconstructed embryos was examined. SLO treatment after trypsinzation significantly increased (P &lt; 0.05) the percentage of fusion rates and blastocyst developmental rates compared with that before trypsinization or in the nontreated group. Additionally there were no significant differences in fusion rates, cleavage rates, blastocyst developmental rates, and total cell number of blastocysts between the SLO-treated group before trypsinzation and the nontreated group. Next, after the trypsinzation treatment, fetal fibroblast cells were incubated in Ca2+-free DMEM containing 200 ng mL−1 of SLO for 0, 30, 50, and 70 min and SLO-treated donor cells were also tested for fusion rate and developmental capability following reconstruction. The 50-min group of SLO-treated cells significantly increased (P &lt; 0.05) the percentage of fusion rates (90.6 vs. 77.6, 85.4, and 78.5%) and blastocyst developmental rates (24.7 vs. 13.5, 11.2, and 13.5%) compared with the other groups (Table 1). However, there was no significant difference in the total cell number of blastocysts among SLO-treated groups. Although cleavage rates the in SLO-treated groups were not significantly different from those of the nontreated group, there the cleavage rates were slightly in SLO-treated groups. In conclusion, permeabilization of porcine fetal fibroblast cells with SLO improves the fusion rates and in vitro development of porcine reconstructed embryos. Table 1.Effects of SLO treatment of fetal fibroblasts by different exposure times on in vitro development of porcine reconstructed embryos

scholarly journals Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 295-302 ◽  
Author(s):  
Walt Yamazaki ◽  
Christina Ramires Ferreira ◽  
Simone Cristina Méo ◽  
Cláudia Lima Verde Leal ◽  
Flávio Vieira Meirelles ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Standard Treatment ◽  
Oocyte Activation ◽  
Blastocyst Development ◽  
Reconstructed Embryos ◽  
Developmental Capacity ◽  
Bovine Oocytes

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p <0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.

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