The effect of Arbas Cashmere goat bone marrow stromal cells on production of transgenic cloned embryos

The aim of this study was to develop a method for the in vitro separation and culture of Arbas Cashmere goat bone marrow stromal cells (gBMSCs). Arbas Cashmere gBMSCs were isolated and cultured in vitro, and cell surface markers were identified immunohistochemically. The gBMSCs were differentiated into neurocytes and osteoblasts, and the expression of neuron-specific enolase and osteocalcin was identified by immunohistochemistry. The gBMSCs and goat fetal fibroblast cells (gFFCs) were compared for transient transfection efficiency and fluorescent colony-forming efficiency with Arbas Cashmere gFFCs as a control. pDsRed2-1 encodes DsRed2, a variant of the Discosoma sp. red fluorescent protein (DsRed). In addition, the coding sequence for DsRed2 contains a series of silent base-pair changes for higher expression in mammalian cells. Of the gBMSCs-pDsRed2-1, one fraction was tested for pluripotency, whereas the other fraction was manipulated using somatic cell nuclear transfer, and the in vitro growth status of transgenic embryos derived from gBMSCs-pDsRed2-1 and gFFCs-pDsRed2-1 was compared. The findings showed that gBMSCs were isolated and amplified to express CD29, CD44, and CD90 through adherent culture, with no marked signs of aging after multiple passages. Expression of neuron-specific enolase and osteocalcin by gBMSCs and gBMSCs-pDsRed2-1 was strongly induced by neuronal and osteogenic differentiation, whereas the integrated exogenous genes did not influence pluripotency (P > 0.05). The transient transfection efficiencies of gBMSCs and gFFCs after 48 hours were not significantly different; however, the fluorescent colony-forming efficiency of gBMSCs-pDsRed2-1 after G418 screening was approximately 13% higher than that of gFFCs-pDsRed2-1. The convergence and cleavage rates of cloned embryos derived from gBMSCs-pDsRed2-1 were higher than those derived from gFFCs-pDsRed2-1, whereas their eight-cell and blastocyst rates were similar. The red fluorescent protein expression levels were higher in transgenic embryos derived from gBMSCs-pDsRed2-1 compared with those derived from gFFCs-pDsRed2-1 (48.8% vs. 31.1%, respectively) (P < 0.01). Real-time quantitative Polymerase Chain Reaction analysis showed that DsRed2-1 messenger RNA expression of cloned embryos derived from gBMSCs was 2.24 greater than that of embryos derived from gFFCs-pDsRed2-1 (P < 0.01). Similarly, Western blot analysis showed that DsRed2 protein expression of cloned embryos derived from gBMSCs-pDsRed2-1 was 1.29 greater than that of embryos derived from gFFCs-pDsRed2-1 (P < 0.01). In this study, gBMSCs were also used for somatic cell nuclear transfer and shown to provide effective nuclear donor cells for breeding new genetically modified varieties of livestock.