Abstract
Chinese Kunming mice (Mus musculus Km), widely used as laboratory animals throughout China, remain very refractory for embryonic stem (ES) cell isolation. The present study was aimed to evaluate the effects of hybridization with 129/Sv mice, and culture media containing fetal bovine serum (FBS) or Knockout serum replacement (KSR) on ES cell isolation from Kunming mice. The results demonstrated that ES cells had been effectively isolated from the hybrid embryos of Kunming and 129/Sv mice using all three media containing 15% FBS, 15% KSR and their mixture of 14% KSR and 1% FBS, individually. These isolated ES cells had maintained in vitro undifferentiated for a long time, exhibiting all features specific for mouse ES cells. In addition, the rates of ES cell isolation in the medium containing 14% KSR and 1% FBS, was 46.67% and significantly higher than those in another two media containing only FBS or KSR (p < 0.05). Contrarily, no ES cell line had been established from Kunming mouse inbred embryos using the same protocols. These results suggested that ES cells with long-term self-renewal ability could be efficiently generated from hybrid embryos of Kunming and 129/Sv mice, and a small volume of FBS was necessary to isolate ES cells in the KSR medium when embryos and early ES cells cultured.
Highlights
Mouse Embryonic stem cells, which are pluripotential cells from early pre-implantation embryos and have the ability to generate all somatic cells and functional gametes [1,2], are used to explore expression and function of genes in vivo by genetic modification [3]
Hybrid embryos for embryonic stem (ES) cell isolation, which were produced from the pregnant Kunming females mated with 129/Sv males, were cultured on the inactivated mouse embryonic fibroblasts (MEF) feeder layers in the ES media supplemented with 15% Knockout Serum Repacement (KSR), 1% fetal bovine serum (FBS) + 14% Knockout serum replacement (KSR) and 15% FBS, individually
All primary ES cells produced from the picked ICM outgrowths, persisted the undifferentiated state and generated the ES cell lines in the two media containing 15% KSR and the mixture of 1% FBS + 14% KSR
Summary
Mouse Embryonic stem (mES) cells, which are pluripotential cells from early pre-implantation embryos and have the ability to generate all somatic cells and functional gametes [1,2], are used to explore expression and function of genes in vivo by genetic modification [3]. The Chinese Kunming mouse strain (Mus musculus Km, KM), a outbreed mouse strain originating from the Swiss albino mouse, are widely used in pharmacology and genetically related studies throughout China. It exhibits many advantages such as high disease resistance, large and frequent litters and rapid growth rates. We had established the parthenogenetical ES cell lines from the hybrid offspring oocytes of Kunming and 129/Sv mice using feeder cells, LIF, SR and FBS [24]. We investigated effects of hybridization with 129/Sv male mice on ES cell isolation from KM mice using in vivo fertilized embryos, in order to explore the genetic/epigenetic mechanism of KM mice which hamper ES cell isolation, and apply the mouse strain for targeted genetic manipulation
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