DIAMOND - Diabetic Stem Cell Modeling of Human Disease
A BCBC American Recovery and Reinvestment Act (ARRA) Signature Project Program
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Derivation of ES cell lines from three subtypes of NOD mice
To derive ES cell lines from NOD, NSG and NRG mice, embryos were harvested at 3.5 dpc and expanded blasts were cultured on inactivated mouse embryonic fibroblasts. If needed, morula or early blasts were cultured overnight in KSOM prior to plating for derivation. Standard mouse ES cell culture medium was used (DMEM, Lif, 1 mM glutamine, 1% nonessential amino acids, 0.1 mM b-mercaptoethanol), however 15% knockout serum replacement (KOSR) was used in place of FCS and the derivation medium was supplemented with 2i (1 µM PD0325901 and 2 µM CHIR99021) (Nichols et al., 2009, Nature Medicine 15, 814 - 818). After 8-10 days, ICM outgrowths were mechanically disaggregated and plated onto new feeder layers in 2i medium (standard mouse ES cell culture medium supplemented with Lif and 2i). After 2 days, the emergent ES cell lines were passaged by enzymatic treatment with 0.25% trypsin/EDTA and subsequently expanded to P3-P4 prior to freezing. Inactivated mouse embryonic fibroblasts from C57BL/6J mice were used throughout the derivation process. In some cases, serum free 3i medium was used for derivation and expansion as described by Ying et al. 2008 (Nature 453, 519-523).
Results to Date
| Currently derived cell lines | ||||||
|---|---|---|---|---|---|---|
| Strain | Common Name | Total | Male | Best karyotype | Status | Recommendation |
| NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ | NSG | 23 | 8 | 69% normal | on hold | re-derive |
| NOD.Cg-Rag1tm1MomIl2rgtm1Wjl/SzJ | NRG | 35 | 18 | 89% normal | testing germline contribution | continue testing |
| NOD.CBA/Ls-Tyr+/LtJ | NOD agouti | 5 | 3 | >70% normal | testing germline contribution | continue testing |
We included an Agouti congenic NOD+/+ stock that was prepared at the same time. We found that the initial ES cell lines from the NSG mice had poor karyotypes, possibly associated with the DNA repair defect caused by the scid mutation and we are remaking those lines. The other lines are under testing for germ-line competence
The ES cell lines were derived at Jackson Lab by Laura Reinholdt and Steve Murray with great help from our colleague Rene Maehr from Doug Melton's lab.
