Gene Targeting in Embryonic Stem Cells

Hughes, Elizabeth D.; Saunders, Thomas L.

doi:10.1007/978-3-642-20792-1_14

Cited by 7 publications

(5 citation statements)

References 21 publications

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“…With stable CIMR DNAme in hESCs at MLH1 carried through in vitro differentiation, novel DNAme mouse models can be developed through blastocyst complementation of DNAme-edited mESC lines or by direct CIMR DNA targeting prior to developmental transitions from naive to primed pluripotency. 51 , 52 , 53 , 54 …”

Section: Discussionmentioning

confidence: 99%

“…C57Bl/6N-PRX-B6N mESCs were maintained on irradiated mouse embryonic feeders according to standard protocols. 54 Feeders were plated on 0.1% Porcine gelatin/DPBS(−), and cultures were maintained in IMDM (HyClone SH30259.02) with 20% FBS (Hyclone SH30071.03 E), supplemented with 1000 U/mL LIF (ESGRO ESG1107), and 2βME at 1:2000 (Sigma M−7522), and Penicillin/Streptomycin at 0.2mM (ThermoFisher 15140122). Alkaline phosphatase staining was conducted according to manufacturer’s recommendations (Sigma SCR004).…”

Section: Methodsmentioning

confidence: 99%

“…In terms of GC content this entails finding a short region of lower GC density among a dense CGI. To easily visualize strong candidate regions, already available bisulfite PCR primer design software is helpful (e.g., methprimer 54 ), as candidate primers are designed specifically to bind more complex sequences of lower GC content and minimal CpGs. Extracted sequences can then be tested for sequence complexity as related to synthesis online (e.g., https://www.idtdna.com/site/order/megamerentry or similar service) and paired with candidate guide RNAs.…”

Section: Methodsmentioning

confidence: 99%

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Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

Tompkins,

Lizhar,

Shokrani

et al. 2023

Cell Reports Methods

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

Tompkins,

Lizhar,

Shokrani

et al. 2023

Cell Reports Methods

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“…Malic enzyme 1 mutant mice were generated by the Transgenic Animal Model Core at the University of Michigan using genetically engineered ES cells from EUCOMM (HEPD0722) [ 35 ]. ES cells were placed in culture as described [ 36 ]. ES cell clones were microinjected into albino C57BL/6 blastocysts to produce mouse ES cell-chimeras, as described [ 37 ].…”

Section: Methodsmentioning

confidence: 99%

Malic enzyme 1 knockout has no deleterious phenotype and is favored in the male germline under standard laboratory conditions

Alektiar,

Shan,

Radyk

et al. 2024

PLoS ONE

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Malic Enzyme 1 (ME1) plays an integral role in fatty acid synthesis and cellular energetics through its production of NADPH and pyruvate. As such, it has been identified as a gene of interest in obesity, type 2 diabetes, and an array of epithelial cancers, with most work being performed in vitro. The current standard model for ME1 loss in vivo is the spontaneous Mod-1 null allele, which produces a canonically inactive form of ME1. Herein, we describe two new genetically engineered mouse models exhibiting ME1 loss at dynamic timepoints. Using murine embryonic stem cells and Flp/FRT and Cre/loxP class switch recombination, we established a germline Me1 knockout model (Me1 KO) and an inducible conditional knockout model (Me1 cKO), activated upon tamoxifen treatment in adulthood. Collectively, neither the Me1 KO nor Me1 cKO models exhibited deleterious phenotype under standard laboratory conditions. Knockout of ME1 was validated by immunohistochemistry and genotype confirmed by PCR. Transmission patterns favor Me1 loss in Me1 KO mice when maternally transmitted to male progeny. Hematological examination of these models through complete blood count and serum chemistry panels revealed no discrepancy with their wild-type counterparts. Orthotopic pancreatic tumors in Me1 cKO mice grow similarly to Me1 expressing mice. Similarly, no behavioral phenotype was observed in Me1 cKO mice when aged for 52 weeks. Histological analysis of several tissues revealed no pathological phenotype. These models provide a more modern approach to ME1 knockout in vivo while opening the door for further study into the role of ME1 loss under more biologically relevant, stressful conditions.

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“…Knockout mice are generated by introducing a targeting vector encoding a modified, nonfunctional version of the gene of interest into mouse embryonic stem (ES) cells and selecting clones in which the targeting vector has integrated into the genome by homologous recombination, thereby replacing one of the wild-type alleles. The resulting ES cells are then used to generate chimeric mice, and eventually mice carrying the targeted allele in the germline in either a heterozygous or homozygous state (Hughes & Saunders, 2011). Global inactivation of tumor suppressor genes can in some cases model specific human hereditary tumor syndromes, for example, Li-Fraumeni syndrome, in which patients have inherited a mutant p53 allele and develop a range of tumor types, including breast cancer (Donehower et al, 1992; Malkin, 2011).…”

Section: Introductionmentioning

confidence: 99%

Genetically Engineered Mice as Experimental Tools to Dissect the Critical Events in Breast Cancer

Menezes

Das

Emdad

et al. 2014

Advances in Cancer Research

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Elucidating the mechanism of pathogenesis of breast cancer has greatly benefited from breakthrough advances in both genetically engineered mouse (GEM) models and xenograft transplantation technologies. The vast array of breast cancer mouse models currently available is testimony to the complexity of mammary tumorigenesis and attempts by investigators to accurately portray the heterogeneity and intricacies of this disease. Distinct molecular changes that drive various aspects of tumorigenesis, such as alterations in tumor cell proliferation and apoptosis, invasion and metastasis, angiogenesis, and drug resistance have been evaluated using the currently available GEM breast cancer models. GEM breast cancer models are also being exploited to evaluate and validate the efficacy of novel therapeutics, vaccines, and imaging modalities for potential use in the clinic. This review provides a synopsis of the various GEM models that are expanding our knowledge of the nuances of breast cancer development and progression and can be instrumental in the development of novel prevention and therapeutic approaches for this disease.

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Gene Targeting in Embryonic Stem Cells

Cited by 7 publications

References 21 publications

Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

Malic enzyme 1 knockout has no deleterious phenotype and is favored in the male germline under standard laboratory conditions

Genetically Engineered Mice as Experimental Tools to Dissect the Critical Events in Breast Cancer

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