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  • Optimization of protocols for derivation of mouse embryonic stem cell lines from refractory strains, including the non obese diabetic mouse.

Optimization of protocols for derivation of mouse embryonic stem cell lines from refractory strains, including the non obese diabetic mouse.

Stem cells and development (2011-09-22)
Timothy J Davies, Paul J Fairchild
ABSTRACT

The derivation of pluripotent embryonic stem cells (ESCs) from a variety of genetic backgrounds remains a desirable objective in the generation of mice functionally deficient in genes of interest and the modeling of human disease. Nevertheless, disparity in the ease with which different strains of mice yield ESC lines has long been acknowledged. Indeed, the generation of bona fide ESCs from the non obese diabetic (NOD) mouse, a well-characterized model of human type I diabetes, has historically proved especially difficult to achieve. Here, we report the development of protocols for the derivation of novel ESC lines from C57Bl/6 mice based on the combined use of high concentrations of leukemia inhibitory factor and serum-replacement, which is equally applicable to fresh and cryo-preserved embryos. Further, we demonstrate the success of this approach using Balb/K and CBA/Ca mice, widely considered to be refractory strains. CBA/Ca ESCs contributed to the somatic germ layers of chimeras and displayed a very high competence at germline transmission. Importantly, we were able to use the same protocol for the derivation of ESC lines from nonpermissive NOD mice. These ESCs displayed a normal karyotype that was robustly stable during long-term culture, were capable of forming teratomas in vivo and germline competent chimeras after injection into recipient blastocysts. Further, these novel ESC lines efficiently formed embryoid bodies in vitro and could be directed in their differentiation along the dendritic cell lineage, thus illustrating their potential application to the generation of cell types of relevance to the pathogenesis of type I diabetes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
RESGRO Culture Medium, 500ml, The RESGRO Culture Medium is a complete ready-to-use product that can be utilized to complement traditional murine Embryonic Stem (ES) cell culture media containing ESGRO murine LIF medium supplement. It is available in a 500mL format.
Sigma-Aldrich
RESGRO Culture Medium, 250ml, The RESGRO Culture Medium is a specialty media that can be utilized to complement traditional murine Embryonic Stem (ES) cell culture media containing ESGRO murine LIF medium supplement.
Sigma-Aldrich
ESGRO® Recombinant Mouse LIF Protein, ESGRO Leukemia Inhibitory Factor (LIF) supplement for mouse ES cell culture. Each vial contains 10^7 units/ml.
Sigma-Aldrich
ESGRO® Recombinant Mouse LIF Protein, ESGRO Leukemia Inhibitory Factor (LIF) supplement for mouse ES cell culture. Each vial contains 10^6 units/ml
Millipore
Millicell® EZ Slide, 4-well glass chamber slide, 1.7 cm2/well, 16/pk, sterile