Abstract

Somatic cell nuclear transfer is used to generate genetic models for research and new, genetically modified livestock varieties. Goat fetal fibroblast cells (gFFCs) are the predominant nuclear donors in Cashmere goat transgenic cloning, but have disadvantages. We evaluated the potential of goat adipose-derived mesenchymal stem cells (gADSCs) and goat skeletal muscle-derived satellite cells (gMDSCs) for somatic cell nuclear transfer, evaluating their proliferation, pluripotency, transfection efficiency and capacity to support full term development of embryos after additive gene transfer or homologous recombination. gADSCs and gMDSCs were isolated by enzyme digestion and differentiated into neurocytes, myotube cells and insulin-producing cells. Neuron-specific enolase, fast muscle myosin and insulin expression were determined by immunohistochemistry. Following somatic cell nuclear transfer with donor cells derived from gADSCs, gMDSCs and gFFCs, transfection and cloning efficiencies were compared. Red fluorescent protein levels were determined by quantitative PCR and western blotting. 5-Methylcytosine, H4K5, H4K12 and H3K18 were determined immunohistochemically. gADSCs and gMDSCs were maintained in culture for up to 65 passages, whereas gFFCs could be passaged barely more than 15 times. gADSCs and gMDSCs had higher fluorescent colony forming efficiency and greater convergence (20%) and cleavage (10%) rates than gFFCs, and exhibited differing H4K5 histone modification patterns after somatic cell nuclear transfer and in vitro cultivation. After transfection with a pDsRed2-1 expression plasmid, the integrated exogenous genes did not influence the pluripotency of gADSCs–pDsRed2-1 or gMDSCs–pDsRed2-1. DsRed2 mRNA expression by cloned embryos derived from gADSCs–pDsRed2-1 or gMDSCs–pDsRed2-1 was more than twice that of gFFCs–pDsRed2-1 embryos (P<0.01). Pregnancy rates of gADSCs–pDsRed2-1 and gMDSCs–pDsRed2-1 recipients were higher than those of gFFCs–pDsRed2-1 recipients (P<0.01). With their high proliferative capacity and transfection efficiency, gADSCs and gMDSCs are a valuable cell source for breeding new, genetically modified varieties of livestock by somatic cell nuclear transfer.

Highlights

  • In recent years genetically engineered pigs have been produced for xenotransplantation [1] and cattle have been successfully produced by somatic cell nuclear transfer (SCNT) [2]

  • Among the various parameters influencing the outcome of SCNT, including oocyte quality, embryo culture and recipient animal preparation, the type and quality of the nuclear donor cells are of vital importance [6]

  • Morphologic characteristic analysis combined with surface marker molecules confirmed that the cultured cells were goat skeletal muscle-derived satellite cells (gMDSCs) and were suitable for further research

Read more

Summary

Introduction

In recent years genetically engineered pigs have been produced for xenotransplantation [1] and cattle have been successfully produced by somatic cell nuclear transfer (SCNT) [2]. Accurate and efficient gene knockout and knock in are possible in mouse models. SCNT using pools of stable transfected cell clones is an efficient means of producing transgenic goats with appropriate expression patterns [3,4] and has facilitated the generation of goat models with inducible transgene expression [5]. Among the various parameters influencing the outcome of SCNT, including oocyte quality, embryo culture and recipient animal preparation, the type and quality of the nuclear donor cells are of vital importance [6]

Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call