Clonal Lines of Transgenic Fibroblast Cells Derived from the Same Fetus Result in Different Development When Used for Nuclear Transfer in Pigs1

Abstract

Different factors are believed to influence the outcome of nuclear transfer (NT) experiments. Besides the cell cycle stage of both recipient cytoplast and donor karyoplast, the origin of the donor cells (embryonic, fetal, and adult) is of interest. We compared in vitro development of NT embryos derived from small serum-starved (G0) or small cycling (G1) porcine fetal fibroblast cells. Serum starvation did not have a positive effect on cleavage rate or the percentage of embryos that developed to the morula and blastocyst stages. Next, we investigated the development of porcine NT embryos derived from different transgenic clonal cell lines that had originated from the same fetus. When different clonal lines of fetal fibroblasts were fused to enucleated metaphase II oocytes, differences in fusion rates as well as in development to the morula and blastocyst stages were observed (P < 0.05). When oocytes derived from sow ovaries were used as recipient cytoplasts, significantly better cleavage (P = 0.03) and blastocyst formation (P < 0.014) was obtained when compared with oocytes derived from gilts. Our data indicate that not only different cell lines, but also different clones derived from one primary cell line, result in different development when used for NT. In addition, the use of sow oocytes as a cytoplast source also improves the efficiency of NT experiments.

Developmental Ability of Somatic Cell Nuclear Transferred Embryos Aggregated at the 8-cell Stage or 16- to 32-cell Stage in Cattle

“…However, since there are some differences between our present and previous studies (e.g., donor cell types, treatment of donor cells and origin of recipient oocytes), it is difficult to compare these two studies. Cloning efficiency is reported to be different between the donor cell lines used [21,29,30]. In the present study, two of the three cloned calves from 3X embryos were stillborn.…”

Donor Cell Lines Considerably Affect the Outcome of Somatic Nuclear Transfer in the Case of Bovines

“…In this context, it is important to note that we measured whole DNA methylation and not the methylation level of single relevant genes. It is possible that the small differences discovered in passage 1 reflect more important epigenetic deviations than are assumed [25]. This could be a reason for the better suitability for NT of the appropriate line in increasing passages.…”

“…Therefore, some of the work presented in the literature deals with the question of whether certain cell types are more suitable for somatic cloning than the others [4,7,14]. Recently, some papers have hinted at the possibility that different lines of the same cell type could result in different success rates after NT (horse [12], pig [24,25]). …”

Effect of cytochalasin types on the production of heterozygous parthenogenetic porcine embryos and the isolation of putative parthenogenetic embryonic stem cells

“…Ovaries were transported from a local abattoir to the laboratory in 0.9% sodium chloride solution at between 33°C and 37°C. Cumulus-oocyte complexes (COCs) were matured for 46 h in BOMED medium [15] under mineral oil in a humidified atmosphere of 5% CO 2 , 5% O 2 and 90% N 2 at 38.5°C. BOMED medium consisted of Medium 199 (Life technologies, USA) supplemented with 0.5 mM cysteamine, 0.1 mg/ml sodium pyruvate, 75 μg/ml penicillin G, 50 μg/ml streptomycin sulphate, 5 μg/ml insulin, 5 μg/ml follicle stimulating hormone (FSH), 10 ng/ml recombinant human epidermal growth factor (EGF) and 10% sow follicular fluid.…”