Establishment and genetic characteristics analysis of in vitro culture a fibroblast cell line derived from Wuzhishan miniature pig

SummaryIn evolution, the red panda (Ailurus fulgens) plays a pivotal role in the higher level phylogeny of arctoides carnivore mammals. The red panda inhabits certain Asian countries only and its numbers are decreasing. Therefore, the development of feasible ways to preserve this species is necessary. Genetic resource cryopreservation and somatic cell nuclear transfer (SCNT) have been used extensively to rescue this endangered species. The present study describes the establishment, for the first time, of a red panda ear fibroblast cell line, which was then cryopreserved, thawed and cultured. Through micromanipulation, interspecies embryos were reconstructed using the cryopreserved–thawed fibroblasts of the red panda as the donor and rabbit oocytes as recipients. A total of 194 enucleated rabbit oocytes were reconstructed with red panda ear fibroblasts; enucleated oocytes were activated without fusion as the control. The results show that the fibroblast cell line was established successfully by tissue culture and then cryopreserved in liquid nitrogen. Supplementation with 20% fetal bovine serum and 8% dimethyl sulphoxide in basic medium facilitated the cryopreservation. The interspecies embryos were successfully reconstructed. The cleavage, morulae and blastocyst rates after in vitro culture were 71, 47 and 23% (31/194), respectively. This study indicated that a somatic cell line could be established and cryopreserved from red panda and that rabbit cytoplast supports mitotic cleavage of the red panda karyoplasts and is capable of reprogramming the nucleus to achieve blastocysts.

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29 SCRIPTAID IMPROVES SOMATIC NUCLEAR TRANSFER EFFICIENCY DURING IN VITRO CULTURE OF PORCINE EMBRYOS DERIVED FROM INBRED MINIATURE PIG FETAL FIBROBLASTS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab29 ◽

2015 ◽

Vol 27 (1) ◽

pp. 107

Author(s):

R. Koppang ◽

N. R. Mtango ◽

M. Barcelo-Fimbres ◽

J. P. Verstegen

Keyword(s):

In Vitro Culture ◽

Nuclear Transfer ◽

Fibroblast Cell ◽

Treated Group ◽

Culture Conditions ◽

Control Group ◽

Miniature Pig ◽

Fetal Fibroblasts ◽

Blastocyst Rate

Porcine somatic cell nuclear transfer (SCNT) is limited to the same or next day surgical embryo transfer due to poor culture conditions in vitro. In this study, we aimed to overcome this problem by treating SCNT embryos with scriptaid, an inhibitor of histone deacetylase (HDACi) that helps with epigenetic reprogramming of the somatic nuclei. Scriptaid was chosen over other HDACi because it has been shown to improve histone acetylation in the same pattern as that of IVF embryos as well as its low toxicity characteristic (Zhao et al. 2009 Biol. Reprod. 81, 525–530; Zhao et al. 2010 Cell Reprogram. 12, 75–78). An inbred miniature pig fetal fibroblast cell line that is known to give low blastocyst rate in culture was used as a source of donor cells transferred into enucleated oocytes. Traditional SCNT was performed; embryos were fused and chemically activated in 10 µM ionomycin for 5 min and 2 mM DMAP for 3 to 4 h before being transferred into scriptaid. Embryos were treated with 500 nM scriptaid (Zhao et al. 2010) for 18 h and the untreated group was used as control. A total of 806 oocytes were used in 8 replicates. The constructed embryos were cultured in modified porcine zygote medium 5 (mPZM-5) for 7 days at 39°C in 5% O2, 5% CO2, 90% N2 atmosphere. Cleavage rates were assessed at 2.5 days and blastocyst rates at Day 7 after activation. Data were analysed by ANOVA using GLM, and percentages were transformed using arcsin square root using Statistix 10 software (Tallahassee, FL, USA). There were no differences in cleavage rates for control group v. scriptaid (55.3 v. 49.9%; P > 0.1; Table 1). The blastocyst rate per construct showed remarkable increase in the scriptaid treated group compared with the control group (12.8 v. 2.2%; P < 0.01; Table 1). Similarly, a significant effect was observed for blastocyst per embryos cleaved where scriptaid had higher rates compared with control (25.8 v. 5.8%; P < 0.01). These results indicated that improving nuclear reprogramming of miniature porcine SCNT clones by scriptaid treatment enhanced blastocyst production during the in vitro culture of porcine embryos. Table 1.Mean (± s.e.m.) measures of embryonic development of SCNT embryos

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