The effect of Arbas Cashm cells on production of tr
Yu Ren 1, Haiqing Wu1, Hefei W
Research Center for Laboratory Animal Science, Inner a r t i c l e i n f o
Received 10 September 2013
Received in revised form 9 February 2014
Accepted 9 February 2014
Bone marrow stromal cell
Induced differentiation uantitative Polymerase pression of cloned emos derived from gFFCsd that DsRed2 protein 1.29 greater than that of
MSCs were also used for donor cells for breeding 1. Introduction
Breeding and application of new genetically modified varieties of Arbas Cashmere goats with a high economic * Corresponding author. Tel./fax: þ86 0471 4995071.
E-mail address: firstname.lastname@example.org (D. Liu). 1 These authors contributed equally to this work.
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Theriogenology 81 (2014) 1257–1267 2014 Elsevier Inc. All rights reserved.pDsRed2-1 (48.8% vs. 31.1%, respectively) (P < 0.01). Real-time q
Chain Reaction analysis showed that DsRed2-1 messenger RNA ex bryos derived from gBMSCs was 2.24 greater than that of embry pDsRed2-1 (P < 0.01). Similarly, Western blot analysis showe expressionof cloned embryosderived fromgBMSCs-pDsRed2-1was embryos derived from gFFCs-pDsRed2-1 (P< 0.01). In this study, gB somatic cell nuclear transfer and shown to provide effective nuclear new genetically modified varieties of livestock.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-Transfected cell
Somatic cell nuclear transfer
Embryonic development0093-691X/$ – see front matter 2014 Elsevier Inc http://dx.doi.org/10.1016/j.theriogenology.2014.02.0ere goat bone marrow stromal ansgenic cloned embryos ang, Xiao Wang, Hao Liang, Dongjun Liu*
Mongolia University, Huhhot, Inner Mongolia, China a b s t r a c t
The aim of this studywas to develop amethod for the in vitro separation and culture of Arbas
Cashmere goat bonemarrow stromal cells (gBMSCs). Arbas Cashmere gBMSCswere isolated and cultured in vitro, and cell surface markers were identified immunohistochemically. The gBMSCs were differentiated into neurocytes and osteoblasts, and the expression of neuronspecific 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 nomarked 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. All rights reserved. 07ology . ther io journal .com
Y. Ren et al. / Theriogenology 81 (2014) 1257–12671258value is strategically important for modern animal husbandry. Currently, the main existing technology for breeding new transgenic cloned varieties is transgenic cloning, which involves the transfer of exogenous genes to nuclear donor cells for somatic cell nuclear transfer (SCNT).
This technology generates individuals with specific genetic traits, and new varieties in which the exogenous genes are integrated and expressed stably. At present, fibroblasts are predominantly used as nuclear donor cells in Cashmere goat transgenic cloning [1–3]; these cells have the significant disadvantages of limited number of passages and low survival rate after transfection. These disadvantages have become a bottleneck in breeding new transgenic varieties of Cashmere goats. Bone marrow stromal cells (BMSCs) [4,5] are known to possess the capacity to differentiate into various tissue cells (osteoblasts, cartilage cells, tendon cells, fat cells, muscle cells, and so forth) after long-term culture in vitro. Bone marrow stromal cells are a reliable source of seed cells in tissue engineering than fibroblasts. The study of BMSCs has become a focus of research in recent years.
Therefore, our study aimed to develop an improved method for the in vitro separation and culture of Arbas
Cashmere goat BMSCs (gBMSCs). Furthermore, preliminary evaluation of the advantages and disadvantages of this technique for the generation of seed cells and to improve the low survival rate in genetic transfection of existing fibroblasts used as seed cells was conducted.
The present study aimed to establish a nuclear transfer technology system for Arbas Cashmere goats, and to investigate the use of gBMSCs as cell source for SCNT compared with goat fetal fibroblast cells (gFFCs), including its proliferation capacity, pluripotency, transfection efficiency, and capacity to support full-term development of
SCNT embryos after additive gene transfer or homologous recombination. The aim of gBMSCs for SCNT-mediated transgenesis in Arbas Cashmere embryos of the study was achieved. 2. Materials and methods 2.1. Females