Comparative analysis of various donor cell types for somatic cell nuclear transfer and its association with apoptosis and senescence

Abstract

Abstract. The aim of the present study was to characterize potential somatic cell nuclear transfer (SCNT) donor cells by comparing two lines of transfected cells with their non-modified parental controls in culture. Fetal fibroblasts used in the study originated from crossbred Landrace x Yorkshire x Duroc (LYD) or Yucatan mini-pigs. The LYD fibroblasts were modified by the transfection of a tetracycline on/off gene, whereas Yucatan fibroblasts were triple transfected with the complement regulatory factors, human decay-accelerating factor and human CD59, as well as H-transferase. At the 9th doubling passage, parameters associated with senescence and apoptosis, including morphology, mRNA expression (TP53, Bcl-2, Bax) and reactive oxygen species (ROS) levels, were evaluated. Population doubling (PD) time was calculated by assessing the time required for cell numbers to double by averaging the three cell passages. Quantitative polymerase chain reaction revealed that when comparing LYD with Yucatan fibroblasts, the latter exhibited a lower relative expression of TP53 and a higher relative expression of antiproliferative Bcl-2, which correlated with the PD time results (26 and 40 h, respectively). Tetracycline on/off transfected cell lines exhibited a lower relative expression of antiapoptotic Bcl-2 compared with their originating LYD cells. Similarly, triple transgenic cells exhibited higher TP53 and Bax mRNA expression levels than their non-transgenic counterparts. For ROS measurement, cells were incubated with 2',7'-dichlorofluorescin diacetate under the same conditions and were analyzed by flow cytometry. Yucatan fibroblasts exhibited higher ROS content than LYD cells. In addition, the two transgenic cell lines produced higher ROS levels than their corresponding non-transfected cell lines. In conclusion, these results indicate that characteristics associated with senescence and apoptosis in transfected cells during culture may affect the efficiency of SCNT when used as donor cells for the production of transgenic swine.

High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos

“…Despite notable successes, the e ciency of SCNT remains low [27][28][29][30] . Bovine broblasts are commonly used as donor cells, and their status can affect the success of nuclear transplantation [31] . Previous studies have linked Nacr to cell proliferation regulation by in uencing Cr-coA levels, but the speci c mechanisms underlying Kcr modi cation and cell growth remain insu ciently explored.…”

High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos

“…Fibroblasts from different sources are often used as donor cells during SCNT in domestic animals such as cattle and sheep 33 . Kim et al demonstrated that the ageing and apoptosis-related characteristics of donor cells during culture affect the developmental efficiency of porcine SCNT embryos 34 . Therefore, we investigated whether the proliferative activity of donor fibroblasts can similarly affect the developmental efficiency of bovine SCNT embryos.…”

Production of Transgenic Porcine Embryos Reconstructed with Induced Pluripotent Stem-Like Cells Derived from Porcine Endogenous Factors Using piggyBac System

“…Some of these studies have focused on donor cells to enhance the efficiency of SCNT (Tian et al, 2003). The strategies used in these studies include synchrony of the cell cycle stage (Campbell et al, 1994), age ( Jeon et al, 2012;Wang et al, 2015), origin (Li et al, 2014;Richter et al, 2012), passage (Li et al, 2014), and type (Kim and Hyun, 2014). However, the conditions for donor cells have not been optimized for high-efficiency production, the G 0 or G 1 phase of the donor cell cycle, long telomere lengths, or early passage, which would reduce damage or mutations in the chromosome of origin and cells (Wilmut et al, 2007).…”