Production of Pigs by Hand-Made Cloning Using Mesenchymal Stem Cells and Fibroblasts

Abstract

Mesenchymal stem cells (MSCs) exhibited self-renewal and less differentiation, making the MSCs promising candidates for adult somatic cell nuclear transfer (SCNT). In this article, we tried to produce genome identical pigs through hand-made cloning (HMC), with MSCs and adult skin fibroblasts as donor cells. MSCs were derived from either adipose tissue or peripheral blood (aMSCs and bMSCs, respectively). MSCs usually showed the expression pattern of CD29, CD73, CD90, and CD105 together with lack of expression of the hematopoietic markers CD34and CD45. Flow cytometry results demonstrated high expression of CD29 and CD90 in both MSC lines, while CD73, CD34, and CD45 expression were not detected. In contrary, in reverse transcription-polymerase chain reaction (RT-PCR) analysis, CD73 and CD34 were detected indicating that human antibodies CD73 and CD34 were not suitable to identify porcine cell surface markers and porcine MSC cellular surface markers of CD34 might be different from other species. MSCs also had potential to differentiate successfully into chondrocytes, osteoblasts, and adipocytes. After HMC, embryos reconstructed with aMSCs had higher blastocyst rate on day 5 and 6 than those reconstructed with bMSCs and fibroblasts (29.6% ± 1.3% and 41.1% ± 1.4% for aMSCs vs. 23.9% ± 1.2% and 35.5% ± 1.6% for bMSCs and 22.1% ± 0.9% and 33.3% ± 1.1% for fibroblasts, respectively). Live birth rate per transferred blastocyst achieved with bMSCs (1.59%) was the highest among the three groups. This article was the first report to compare the efficiency among bMSCs, aMSCs, and fibroblasts for boar cloning, which offered a realistic perspective to use the HMC technology for commercial breeding.

Bone marrow mesenchymal stem cells as nuclear donors improve viability and health of cloned horses

“…As previously reported, higher in vitro preimplantation development was observed in bovine, 12 porcine 38 and goat 14 with MSCs as nuclear donors compared to fibroblasts. In the porcine, embryos reconstructed with adipose tissue MSCs (aMSCs) resulted in higher blastocyst rates compared to peripheral blood MSCs or fibroblast-reconstructed embryos, 11 which reflects the variability among different MSC sources. On the other hand, another report in the same species showed no differences in in vitro embryo development, but higher quality blastocysts were obtained when MSCs were used instead of fibroblasts.…”

“… 16 Although higher blastocyst rates were observed by using MSCs instead of fibroblasts in most reports, only porcine and bovine offspring has been obtained from MSC cloning procedures. 6 , 7 , 11 , 13 The developmental ability of embryos reconstructed using MSCs has been associated with their epigenetic, imprinting and pluripotency gene expression profile, which were similar to in vitro fertilized embryos. 7 , 12 In the horse, MSC-derived embryos also showed higher blastocyst rates than fibroblast-derived embryos, but no offspring was obtained.…”

“…Skin fibroblasts are the most common cells used, but cells derived from liver, 2 kidney, 3 granulosa, 4 , 5 and mesenchymal stem cells (MSCs) have also been tested. In the last years, cloning with MSCs has been evaluated in different farm animal species, such as porcine, 6 – 11 bovine, 12 , 13 caprine, 14 ovine, 15 and equine. 16 Although higher blastocyst rates were observed by using MSCs instead of fibroblasts in most reports, only porcine and bovine offspring has been obtained from MSC cloning procedures.…”

Designing and Reconstruction of pcDNA 3.0 Mammalian Expression Vector with its Multiple Cloning Sites by Directional Cloning Method

“…The present study was aimed to generate a suitable mammalian expression vector in order to exploit it in future for further cloning of the Hepatitis B virus X gene [16]. Our initial aim was to restructured the pcDNA availabe in our lab, which is a mammalian expression vector [17,18].…”

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

“…The cell type, cycle synchronization, lineage and time in culture, among other factors, are known to be crucial for the cloning outcome (Dominko et al, 1999;Yang et al, 2016b). Keefer et al (2001) used three different GFP transgenic cell lines to clone goats, and found that only one line was capable of producing viable animals.…”