Generating Embryonic Stem Cells from the Inbred Mouse Strain DBA/2J, a Model of Glaucoma and Other Complex Diseases

Reinholdt, Laura G.; Howell, Gareth R.; Czechanski, Anne; Macalinao, Danilo G.; MacNicoll, Katharine H.; Lin, Chyuan‐Sheng; Donahue, Leah Rae; John, Simon W. M.

doi:10.1371/journal.pone.0050081

Cited by 8 publications

(7 citation statements)

References 39 publications

(41 reference statements)

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“…Using this protocol, we have produced germline competent ES cell lines from a variety of mouse strains; including, those thought to be non-permissive, such as DBA 20 , PWD and NOD (our own unpublished data). Careful selection of healthy, blastocyst stage embryos, combined with minimal manipulations prior to plating promotes a high yield of ICM outgrowths from embryos that hatch naturally from the zona pellucida and adhere to MEF feeders.…”

Section: Anticipated Resultsmentioning

confidence: 99%

“…In incipient ES cell lines, we have found that serum-free 2i/LIF media (variant B) promotes robust NANOG expression and ultimately, higher yield of coat color chimeras through germline testing 20 . While serum-free culture conditions are typically employed in feeder-free culture systems, we have found that the use of feeders promotes more efficient derivation (# of embryos giving rise to ES cell lines / # embryos plated) and also yields a higher percentage of adherent ES cell lines, which are more favorable for gene targeting experiments.…”

Section: Anticipated Resultsmentioning

confidence: 99%

“…Proceed with ES cell lines that show maximum distribution of labeling as shown in Figure 6 (optimal vs. sub-optimal). If desired, quantitative assessment of labeling may also be performed using flow cytometry (see 20 ). CRITICAL: Even robust, germline competent mESCs are heterogenous in their pluripotency marker expression and in poorly performing mESC lines, there can still be some pluripotency marker expression.…”

Section: Methodsmentioning

confidence: 99%

“…We previously published efficient derivation of germ line competent mESC lines from the recalcitrant strain DBA/2J 20 . Crucial to the success of this protocol was the exclusion of serum during the outgrowth phase, combined with inhibition of MEK / ERK (1i: PD98059) signaling during the outgrowth phase and during subsequent culture of emergent ES cell lines (3i: CHIR99021, PD173074 and PD032901).…”

Section: Experimental Designmentioning

confidence: 99%

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Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains

et al. 2014

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Mouse embryonic stem cells (mESCs) are critical tools for genetic engineering, development of stem cell based therapies, and basic research on pluripotency and early lineage commitment. However, successful derivation of germline-competent embryonic stem cell lines has, until recently, been limited to a small number of inbred mouse strains. Recently, there have been significant advances in the field of embryonic stem cell biology, particularly in the area of pluripotency maintenance in the epiblast from which mESCs are derived. Here we describe a protocol for efficient derivation of germline competent mESCs from any mouse strain, including strains previously deemed non-permissive. We provide a primary method that is generally applicable to most inbred strains, as well as an alternative method for non-permissive strains. Using this protocol, mESCs can be derived in 3 weeks and fully characterized after an additional 12 weeks, at efficiencies as high as 90% and in any strain background.

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Section: Anticipated Resultsmentioning

confidence: 99%

Section: Anticipated Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Experimental Designmentioning

confidence: 99%

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Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains

et al. 2014

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“…C1qa +/− mice. Endothelin deficient mice for assessment of the Edn2 riboprobes were obtained from our colony (Reinholdt et al , 2012). …”

Section: Methodsmentioning

confidence: 99%

Combinatorial targeting of early pathways profoundly inhibits neurodegeneration in a mouse model of glaucoma

Howell

MacNicoll

Braine

et al. 2014

Neurobiology of Disease

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The endothelin system is implicated in various human and animal glaucomas. Targeting the endothelin system has great promise as a treatment for human glaucoma, but the cell types involved and the exact mechanisms of action are not clearly elucidated. Here, we report a detailed characterization of the endothelin system in specific cell types of the optic nerve head (ONH) during glaucoma in DBA/2J mice. First, we show that key components of the endothelin system are expressed in multiple cell types. We discover that endothelin 2 (EDN2) is expressed in astrocytes as well as microglia/monocytes in the ONH. The endothelin receptor type A (Ednra) is expressed in vascular endothelial cells, while the endothelin receptor type B (Ednrb) receptor is expressed in ONH astrocytes. Second, we show that Macitentan treatment protects from glaucoma. Macitentan is a novel, orally administered, dual endothelin receptor antagonist with greater affinity, efficacy and safety than previous antagonists. Finally, we test the combinatorial effect of targeting both the endothelin and complement systems as a treatment for glaucoma. Similar to endothelin, the complement system is implicated in a variety of human and animal glaucomas, and has great promise as a treatment target. We discovered that combined targeting of the endothelin (Bosentan) and complement (C1qa mutation) systems is profoundly protective. Remarkably, 80% of DBA/2J eyes subjected to this combined inhibition developed no detectable glaucoma. This opens an exciting new avenue for neuroprotection in glaucoma.

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Rapid target gene validation in complex cancer mouse models using re‐derived embryonic stem cells

et al. 2014

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Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

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Generating Embryonic Stem Cells from the Inbred Mouse Strain DBA/2J, a Model of Glaucoma and Other Complex Diseases

Cited by 8 publications

References 39 publications

Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains

Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains

Combinatorial targeting of early pathways profoundly inhibits neurodegeneration in a mouse model of glaucoma

Rapid target gene validation in complex cancer mouse models using re‐derived embryonic stem cells

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