-
J Liu,
L L Li,
S Du,
X Y Bai,
H D Zhang,
S Tang,
M T Zhao,
B H Ma,
F S Quan,
X E Zhao, Y Zhang
[show abstract]
[hide abstract]
ABSTRACT: To improve the efficiency of somatic cell nuclear transfer (SCNT) in goats, we evaluated the effects of the interval between fusion and activation (1 to 5 h), cytochalasin B (CB) treatment after electrofusion, and the number of transferred embryos on the in vivo and in vitro development of cloned caprine embryos. The majority of the reconstructed embryos had condensed chromosomes and metaphase-like chromosomes at 2 and 3 h after fusion; cleavage and blastocyst rates from those two groups were higher (P < 0.05) than those of embryos activated 1, 4, or 5 h after fusion. Treatment with CB between fusion and activation improved in vitro and in vivo development of nuclear transfer (NT) goat embryos by reducing the fragmentation rate (P < 0.05). Although there were no significant differences in NT efficiency, pregnancy rate and kids born per recipient were increased by transfer of 20 or 30 embryos per recipient compared with 10 embryos. We concluded that CB treatment for 2 to 3 h between fusion and activation was an efficient method for generating cloned goats by somatic cell NT. In addition, increasing the number of embryos transferred to each recipient resulted in more live offspring from fewer recipients.
Theriogenology 07/2011; 76(6):1076-83. · 1.96 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The objective of this study was to compare the effect of two culture media: modified synthetic oviductal fluid (mSOF) and G1.2/G2.2, on the developmental competence of bovine somatic cell-cloned embryos. Cloned embryos were produced by transferring adult skin fibroblasts into enucleated MII oocytes. After activation, the reconstructed embryos were randomly allotted to either mSOF or G1.2/G2.2 for culture (the embryos were transferred from G1.2 to G2.2 on days 3 of culture). The development competence of cloned embryos in these two culture systems was compared in terms of cleavage rate, blastocyst formation rate and apoptosis cell number in day 7 blastocyts. To investigate the in vivo developmental competence of cloned embryos in the two culture systems, a total of 87 and 104 blastocysts derived from mSOF and G1.2/G2.2 medium groups were transferred individually to recipient Angus cows, respectively. No differences were observed in terms of cleavage rate, day 7 blastocyst rate and blastocyst cell number between these two culture systems. However, the day 6 blastocyst formation rate was significantly higher in G1.2/G2.2 than that in mSOF. In addition, blastocysts cultured in mSOF have a higher percentage of apoptotic blastomeres compared to those in G1.2/G2.2 (8.5 ± 1.2 vs 16.8 ± 1.5, p<0.05). Although difference in pregnancy rate was not observed 40 days after embryo transfer, significantly higher pregnancy rate was observed in G1.2/G2.2 group after 90 days of embryo transfer (12.4% vs 37.5%, p<0.05). Moreover, calving rate was significantly improved in G1.2/G2.2 group compared to mSOF group (27.9% vs 6.7%, p<0.05). In conclusion, our results indicate that G1.2/G2.2 can improve developmental competence of bovine SCNT embryos both in vitro and in vivo, which is more suitable for culture of bovine SCNT embryos than mSOF medium.
Reproduction in Domestic Animals 06/2011; 46(3):404-9. · 1.36 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Although mammary epithelial cell lines can provide a rapid and reliable indicator of gene expression efficiency of transgenic animals, their short lifespan greatly limits this application. To provide stable and long lifespan cells, goat mammary epithelial cells (GMECs) were transduced with pLNCX2-hTERT by retrovirus-mediated gene transfer. Transduced GMECs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), proliferation assays, karyotype analysis, telomerase activity assay, western blotting, soft agar assay, and injection into nude mice. Non-transduced GMECs were used as a control. The hTERT-GMECs had higher telomerase activity and extended proliferative lifespan compared to non-transfected GMECs; even after Passage 50, hTERT-GMECs had a near diploid complement of chromosomes. Furthermore, they did not gain the anchorage-independent growth property and were not associated with a malignant phenotype in vitro or in vivo.
Theriogenology 04/2009; 71(9):1417-24. · 1.96 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method with high specificity and rapidity under isothermal conditions. According to the LAMP method, a rapid and simple detection system was established for bovine embryo sexing. Two sets of primers were designed by targeting the bovine male-specific sequence and bovine common sequence respectively. The reaction condition of the detection system was optimized within 60 min under isothermal conditions of 65 degrees C by detection of the reaction mixture on agarose gel. Especially, the primers F2 and B2 could replace the F3 and B3 as outer primers, making the primer design simpler and the amplification efficiency higher. Additionally, codeposition of dNTPs was firstly performed to detect the reaction products by addition of 1 microl 0.1 mM CuSO(4), the visible ring-shaped deposit was found in the middle of the reaction tube with negative mixture. It could be employed as an alternative method in the detection of the reaction products in place of the time-consuming electrophoresis or the turbidity meter. Furthermore, the embryo sexing system was carried out in the embryo transfer and achieved 98% of efficiency and 99.5% of accuracy.
Reproduction in Domestic Animals 12/2008; 44(1):116-21. · 1.36 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The combination of somatic cell nuclear transfer (SCNT) and transgenic technology leads to the production of transgenic cloned animals, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. The purpose of this study was to establish an efficient procedure to prepare human lactoferrin (hLTF) transgenic donor cells for SCNT. Thus, two cell culture systems were employed: caprine mammary epithelial cells (for evaluation of the hTLF transgenic expression in vitro), and fetal-derived fibroblast cells (for identification of competent transgenic donor cells). Induced by hormonal signals, recombinant hLTF was detected in the supernatant of transfected mammary epithelial cells by Western blot. Reliable hLTF transgenic fibroblast cell clones were identified by screening with multiple PCR amplification, EGFP fluorescence, and chromosomal counting (32.5+/-2.3%). This study may provide an effective upstream system to prepare SCNT donor cells for the production of human recombinant pharmaceuticals from the milk of transgenic animals.
Theriogenology 10/2008; 71(2):376-84. · 1.96 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The objective was to investigate the effect of phytohemagglutinin (PHA) on the fusion of mammary gland epithelial (MGE) cells into enucleated oocytes in goats. The toxicity of PHA was evaluated by testing its effect on the development of parthenogenetic caprine oocytes. The effective dose and duration of PHA treatment (100 microg/mL, 20 min incubation) was selected and used to compare fusion efficiency and embryo development following nuclear transfer. Two electrofusion protocols, chamber fusion (CF) and pressurized microelectrode fusion (pMEF), were also compared, when couplets were treated with and without PHA (100 microg/mL, 20 min). Fusion rate of couplets increased from 52.8 to 74.0% for the CF protocol (P<0.05), but was not significantly different for the pMEF protocol (72.7% vs. 78.1%) after PHA treatment. There were no significant differences between treated group and control in rates of subsequent cleavage or blastocyst development. Following transfer of the cloned blastocysts derived from the PHA-treated group and the control group into synchronized recipients, pregnancy rates (Day 30) were not significantly different between treated group and control (28.6% vs. 25.0%). However, all recipients aborted within 120d, microsatellite DNA analyses confirmed that the aborted fetuses were genetically identical to the donor goat. In conclusion, the fusion rate of caprine MGE cell couplets was improved by pre-incubating couplets in medium containing 100 microg/mL PHA prior to electrical pulsing, and embryos derived from PHA treatment established early pregnancies.
Theriogenology 06/2008; 69(9):1165-71. · 1.96 Impact Factor
